Impairment of endothelium-dependent pulmonary vasodilation has been implicated in the development of pulmonary hypertension. Pulmonary vascular smooth muscle cells and endothelial cells communicate electrically through gap junctions; thus, membrane depolarization in smooth muscle cells would depolarize endothelial cells. In this study, we examined the effect of prolonged membrane depolarization induced by high K+ on the endothelium-dependent pulmonary vasodilation. Isometric tension was measured in isolated pulmonary arteries (PA) from Sprague-Dawley rats, and membrane potential was measured in single PA smooth muscle cells. Increase in extracellular K+ concentration from 4.7 to 25 mM significantly depolarized PA smooth muscle cells. The 25 mM K+-mediated depolarization was characterized by an initial transient depolarization (5–15 s) followed by a sustained depolarization that could last for up to 3 h. In endothelium-intact PA rings, ACh (2 μM), levcromakalim (10 μM), and nitroprusside (10 μM) reversibly inhibited the 25 mM K+-mediated contraction. Functional removal of endothelium abolished the ACh-mediated relaxation but had no effect on the levcromakalim- or the nitroprusside-mediated pulmonary vasodilation. Prolonged (∼3 h) membrane depolarization by 25 mM K+ significantly inhibited the ACh-mediated PA relaxation (−55 ± 4 vs. −29 ± 2%, P < 0.001), negligibly affected the levcromakalim-mediated pulmonary vasodilation (−92 ± 4 vs. −95 ± 5%), and slightly but significantly increased the nitroprusside-mediated PA relaxation (−80 ± 2 vs. 90 ± 3%, P < 0.05). These data indicate that membrane depolarization by prolonged exposure to high K+ concentration selectively inhibited endothelium-dependent pulmonary vasodilation, suggesting that membrane depolarization plays a role in the impairment of pulmonary endothelial function in pulmonary hypertension.
- membrane potential
- pulmonary circulation
endothelium-dependent regulation of pulmonary vascular tone plays an important role in maintaining low pulmonary arterial pressure (37). Endothelium-derived relaxing factors (EDRF), such as nitric oxide (NO), and endothelium-derived hyperpolarizing factors (EDHF; see Refs. 11, 27) cause pulmonary vasodilation by increasing cGMP production (3, 17), decreasing cytosolic free Ca2+ concentration ([Ca2+]cyt; see Refs. 40, 46), and inducing membrane hyperpolarization (2, 11, 34,39, 46) in vascular smooth muscle cells. Dysfunctional endothelium has been implicated in the development of pulmonary hypertension (10, 13). The cellular mechanisms involved in the impairment of endothelium-dependent pulmonary vasodilation in patients with pulmonary hypertension have been demonstrated to include vascular injury (induced by shear stress, ischemia, and inflammation; see Ref. 41), inhibition of NO synthase (12), and decrease of endothelial capacity for prostacyclin synthesis (41).
It has been demonstrated that membrane depolarization in vascular smooth muscle cells is associated with pulmonary (2, 8, 15, 22, 30, 36,44) and systemic (24) arterial hypertension in animals. Furthermore, membrane depolarization due to inhibited K+ channels has also been observed in pulmonary artery smooth muscle cells (PASMC) from patients with primary pulmonary hypertension (PPH; see Refs. 43, 47).
Vascular smooth muscle cells and endothelial cells are coupled electrically and pharmacologically by gap junctions (6). Because of the high permeability of junction channels to ions like K+ and Ca2+ (unitary conductance ranges from 30 to 100 pS; see Ref. 6), electrical changes occurring in smooth muscle cells can be quickly transferred to endothelial cells. In other words, membrane depolarization in PASMC (e.g., due to inhibited K+ channel function) can be passively spread over a long distance to depolarize endothelial cells and affect endothelial function. Experiments were therefore designed to determine if membrane depolarization by prolonged exposure to high K+ concentration ([K+]) affects the endothelium-dependent pulmonary vasodilation in isolated pulmonary arterial rings.
MATERIALS AND METHODS
Tissue preparation. Pulmonary artery (PA) rings were dissected from male Sprague-Dawley rats (150–200 g; see Ref. 45). Animals were decapitated, and the lungs and heart were removed and placed in modified Krebs solution (MKS) at room temperature (24°C). Under a dissecting microscope, the right and left branches (2nd order) of the main PA were dissected free of lung tissue. Adipose and adventitial tissues were carefully removed, and the arterial segments were cut into 2-mm-long rings. In some of the experiments, endothelium of the PA rings was mechanically removed by gently rubbing the inner lumen of the rings with a rough-surfaced wooden stick. This procedure did not damage the rings because agonist-mediated contraction was actually greater in the PA rings with denuded endothelium compared with that in rings with intact endothelium. Functional removal of endothelium was confirmed by demonstrating a 90–100% loss of ACh (2 μM)-induced relaxation.
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Academy of Science, revised 1996).
Tension measurement. Isometric tension was measured using the method previously described (25). Two stainless steel hooks (0.1 mm diameter) were inserted through the lumen of the isolated PA rings. One hook was fixed to the bottom of the organ chamber (0.75 ml volume), and the other was connected to an isometric force transducer (model 52–9529; Harvard Apparatus, South Natick, MA) that was mounted directly above the tissue chamber. Isometric tension was continuously monitored and recorded digitally with IBM-compatible PC-based data-acquisition instrumentation (DATAQ) and software (WINDAQ; Dataq Instruments, Akron, OH). The vessels were superfused at a rate of 2.0–2.5 ml/min with 37°C solution. Resting passive tension was maintained throughout experiments at 600–625 mg, which offered the maximal tension when the rings were exposed to 40 mM K+-containing solution.
Cell preparation. The rat PA rings were incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml collagenase (Worthington Biochemical, Freehold, NJ). After the incubation, a thin layer of adventitia was carefully stripped off with fine forceps, and endothelium was removed by gently scratching the intimal surface with a surgical blade. The remaining pulmonary arterial smooth muscle was then digested with 1.5 mg/ml collagenase (Worthington), 0.5 mg/ml elastase (Sigma), and 1 mg/ml bovine albumin (Sigma) for 45 min at 37°C to create a single cell suspension of PASMC. The cells were then resuspended and plated on 25-mm coverslips and were incubated in a humidified atmosphere of 5% CO2 in air at 37°C in 10% FBS culture medium for 3–5 days. Before each experiment, the cells were incubated in 0.3% FBS culture medium for 12–24 h to stop cell growth. The primary cultured PASMC were stained with the membrane-permeable nucleic acid stain 4′,6′-diamidino-2-phenylindole (DAPI, 5 μM; Molecular Probes) to estimate total cell numbers in the cultures. The specific monoclonal antibody raised against smooth muscle α-actin (Boehringer Mannheim, Indianapolis, IN) was used to evaluate cellular purity of cultures. All of the DAPI-stained cells in the primary cultures also cross-reacted with the smooth muscle cell α-actin antibody, indicating that the cultures were all smooth muscle cells.
Measurement of membrane potential. Membrane potential (E m) in single PASMC was measured using an intracellular electrode (30–100 MΩ) filled with 3 M KCl. Data were acquired by an electrometer (Electro 705; World Precision Instruments, Sarasota, FL) coupled to an IBM-compatible computer and a chart recorder and were analyzed using the DATAQ data-acquisition software (WINDAQ; Dataq Instruments).
Reagents and solutions. The MKS consisted of (in mM) 138 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5 HEPES, 1.8 CaCl2, and 10 glucose buffered to pH 7.4 with 2 M Tris. In high-K+ (25 or 60 mM) solution, NaCl in the MKS was replaced, mole for mole, by KCl to maintain the solution's osmolarity. Sodium nitroprusside (SNP; Sigma) was directly dissolved in MKS on the day of use. Preweighed (150 mg) vials of ACh were reconstituted with distilled water to make a stock solution of 2 mM; aliquots of the stock solution were then diluted 1:1,000 in relevant solution for a final concentration of 2 μM. Levcromakalim (SmithKline Beecham) was dissolved in DMSO to make a stock solution of 100 mM; aliquots of the stock solution were then diluted 1:10,000 in MKS to make a final concentration of 10 μM levcromakalim. Vehicle controls were performed. DMSO alone negligibly affected the 25 mM K+-mediated contraction (by 5 ± 1%, P = 0.77), whereas 10 μM levcromakalim that was dissolved in the same amount of DMSO significantly decreased the tension by 81 ± 3% (P < 0.001). All solutions were exposed to room air and had an oxygen tension ranging from 120 to 130 Torr. The pH values of all solutions were checked after addition of the drugs and were readjusted to 7.4.
Statistics. Data are expressed as means ± SE. Statistical analysis was performed using the paired or unpaired Student's t-test. Differences were considered to be significant at P < 0.05.
Effects of 25 mM K+ on Em.
The resting E m in PASMC was −42 ± 1 mV (n = 56). Increasing extracellular K+concentration ([K+]o) from 4.7 to 25 mM significantly depolarized the cells (Fig.1). The 25 mM K+-mediated effect onE m was characterized by a transient depolarization (due apparently to Ca2+-dependent action potentials) followed by a steady-state depolarization (Fig.1 A) that was maintained for at least 180 min (the longest time tested; Fig.1 B). These results indicated that 25 mM K+ induces a transient depolarization that was due primarily to Ca2+-dependent action potentials and a steady-state membrane depolarization that was apparently due to the change of the K+ equilibrium potential (E K).
ACh-mediated pulmonary vasodilation depends on intact endothelium. Increase in [K+]ofrom 4.7 to 25 mM (25 K) caused a significant contraction (by 462 ± 27 mg/mg, n = 52) in isolated PA rings with intact endothelium (Fig.2 A,left, andB). ACh (2 μM) reversibly relaxed the endothelium-intact PA rings precontracted with 25 mM K+ (by −63 ± 5%,n = 12; Fig.2 A,left, andC). Functional removal of endothelium significantly enhanced the 25 mM K+-mediated PA contraction (from 462 ± 27 mg/mg, n = 52, to 963 ± 164 mg/mg, n = 9, P < 0.001) but abolished the ACh-induced PA relaxation (from −63 ± 5%, n = 12, to 0.3 ± 2.0%,n = 12,P < 0.001; Fig.2 A,right, andC). These results indicated that the ACh-mediated pulmonary vasodilation completely depends on intact endothelium (5).
Inhibitory effect of membrane depolarization on ACh-mediated pulmonary vasodilation.
Increasing [K+]ofrom 4.7 to 25 mM caused membrane depolarization in PASMC (Fig. 1) and endothelial cells (23). ACh caused a 55% relaxation in the PA rings precontracted by brief (∼20 min) application of 25 mM K+ (Fig.3 A). The ACh-mediated PA relaxation, however, was significantly reduced to 29% in the PA rings preconstricted by long-term (∼3 h) exposure to 25 mM K+ (Fig. 3,B andC).
Effect of membrane depolarization on levcromakalim- or nitroprusside-mediated pulmonary vasodilation.
Levcromakalim is a selective ATP-sensitive K+ channel opener and causes membrane hyperpolarization in PASMC (7, 28). Application of 10 μM levcromakalim significantly and reversibly caused relaxation in the endothelium-intact PA rings precontracted with 25 mM K+ (Fig.4 A). Increasing [K+]ofrom 25 to 60 mM, however, almost abolished the relaxant effect of levcromakalim (from −92 ± 4%,n = 8, to −6 ± 2%, n = 8;P < 0.001; Fig.4 A,bottom). Because 60 mM K+ in the bath solution shiftedE K to −21 mV, opening of K+ channels under these conditions could not cause further hyperpolarization (i.e.,E m would not become more negative than theE K) and thus could not exert any further relaxant effect on the rings. The relaxant effect of a 20-min application of levcromakalim was comparable in the endothelium-intact and -denuded PA rings (−78 ± 2 vs. −73 ± 2%, n = 16;P = 0.17; Fig.4 B), suggesting that the relaxant effect is independent of intact endothelium. Prolonged (∼3 h) membrane depolarization induced by 25 mM K+ had no effect on the levcromakalim-induced PA relaxation (−92 ± 4%,n = 8, vs. −95 ± 5%,n = 8;P = 0.69; Fig.5).
SNP, an NO donor, is a potent vasodilator that relaxes the PA by increasing intracellular cGMP (3, 17, 18), activating K+ channels (2, 34, 39, 46), and inducing membrane hyperpolarization (2, 46). SNP (10 μM) significantly relaxed the endothelium-denuded PA rings preconstricted by short-term (∼20 min) application of 25 mM K+ (Fig.6 A). The relaxant effect of SNP was slightly, but statistically significantly, enhanced in the vessels preconstricted by membrane depolarization due to prolonged (∼3 h) exposure to high [K+] (Fig. 6,B andC).
The results that prolonged membrane depolarization negligibly affected the levcromakalim- or nitroprusside-mediated pulmonary vasodilation, supporting the contention that the inhibitory effect of membrane depolarization by prolonged exposure to high [K+] on PA relaxation is selective to the endothelium-dependent pulmonary vasodilation.
Prolonged membrane depolarization inhibited the endothelium-dependent PA relaxation induced by ACh but had no effect on the endothelium-independent PA relaxation induced by levcromakalim and nitroprusside. The results indicate that prolonged membrane depolarization selectively inhibits endothelium-dependent pulmonary vasodilation. Given the fact that smooth muscle cells communicate electrically with the endothelial cell via gap junctions, the depolarization-mediated endothelial dysfunction may play a role in the impairment of endothelium-dependent pulmonary vasodilation observed in animals and patients with pulmonary hypertension.
In PA smooth muscle, restingE m is mainly controlled by the K+ permeability and E K(determined by the ratio of extracellular and intracellular [K+]). An increase in [K+]ofrom 4.7 mM (in MKS) to 25 mM K+shifts E K from −85 mV to about −43 mV (assuming intracellular [K+] is 141 mM), thereby causing membrane depolarization (E m becomes less negative). In vascular smooth muscle cells, membrane depolarization opens voltage-gated Ca2+ channels (a major pathway for Ca2+ entry), promotes Ca2+ influx, increases [Ca2+]cyt, and triggers vasoconstriction (4, 8, 31, 36). As shown in Fig. 1, the 25 mM K+-mediated effect onE m in PASMC was characterized by an initial transient depolarization followed by a sustained membrane depolarization for up to 3 h. The initialE m transient was apparently due to the Ca2+-dependent action potential, since removal of extracellular Ca2+ abolished the transient decrease (depolarizing) ofE m (data not shown). Because of the change in theE K, increasing [K+]oto 25 mM caused a sustainedE m depolarization that could be maintained as long as the cells were perfused with 25 mM K+-containing solution (Fig.1 B).
ACh causes pulmonary vasodilation by inducing production and release of NO, prostacyclin, and EDHF from endothelial cells (5, 17-19, 27). The response is initiated by an increase in [Ca2+]cytin endothelial cells due to activation of muscarinic receptors (e.g., M3 receptor in PA; see Ref. 26). The essential role of Ca2+ in the production and release of NO is due to the presence of constitutive Ca2+-sensitive NO synthase in endothelial cells (14, 25). The production and release of EDHF are also regulated by a change of [Ca2+]cytderived from the extracellular space and intracellular stores (11, 27). In endothelial cells, the ACh-induced rise in [Ca2+]cytis due to Ca2+ release from intracellular stores and Ca2+influx through sarcolemmal Ca2+-permeable channels (1, 26,42).
Endothelial cells are generally believed to lack (or express very little) voltage-gated Ca2+channels (1, 20, 29, 33, 42). Thus membrane depolarization per se in endothelial cells would be unable to cause Ca2+ influx. The major pathway for Ca2+ influx in endothelial cells is passive inward Ca2+ leakage (9,42), which depends mainly on the electrochemical gradient for Ca2+ (1, 20, 29, 33, 42). BecauseE m determines the electrochemical gradient of Ca2+(the driving force for Ca2+influx), membrane depolarization decreases the passive inward Ca2+ leakage and thus attenuates the evoked increase in [Ca2+]cyt(38). This may be a cellular mechanism by which membrane depolarization due to prolonged exposure to high [K+] attenuated the ACh-induced pulmonary vasodilation. Indeed, Stevens et al. (38) demonstrated that membrane depolarization (by acute hypoxia) inhibits Ca2+ influx by reducing the Ca2+ driving force in isolated pulmonary arterial endothelial cells. The resultant decrease in endothelial cell [Ca2+]cytmay contribute to decreased production of NO during acute hypoxia (19,35, 38).
Why the ACh-induced PA relaxation is attenuated in a time-dependent manner (i.e., significant difference between the responses elicited after 10 min and ∼3 h exposure to 25 mM K+) is unclear. Such a time-dependent effect suggests that secondary changes, in addition to membrane depolarization and the decreased Ca2+ driving force, occur in endothelial cells during the ∼3-h period of depolarization. It has been demonstrated that membrane depolarization facilitates inositol trisphosphate (IP3) production in vascular smooth muscle cells (12). An increase in cytosolic IP3 concentration in PASMC during prolonged membrane depolarization could increase IP3 content in endothelial cells through gap junctions and gradually deplete Ca2+ from the IP3-sensitive intracellular stores. Thus the ACh-mediated increase in [Ca2+]cytand NO syntheses would be attenuated, and the ACh-induced pulmonary vasodilation would be inhibited. This speculation provides an alternative explanation for why the ACh-induced relaxation in K+-pretreated PA rings is attenuated in a time-dependent manner.
Recently, Edwards et al. (11) demonstrated that ACh induces membrane hyperpolarization in rat artery in part by causing a small accumulation of K+ in the space between smooth muscle cells and endothelial cells. The small increase in [K+]oin the myo-endothelial space activates Na+-K+-ATPase and the inward rectifier K+channels in smooth muscle cells and thus causes vasodilation. This mechanism would be disrupted by increasing [K+]oto 25 mM, which exceeds the [K+]ofor activating the small outward K+ currents through the inward rectifier K+ channels (5–16 mM). We cannot rule out the possibility that the observed attenuation in the ACh-mediated relaxant response was due to the increase of [K+]o.
In summary, intercellular communication through gap junctions among vascular smooth muscle cells and between endothelial cells and smooth muscle cells provides the mechanistic basis for integrated electrical coupling in the vasculature (6). The gap junctions between smooth muscle cells and endothelial cells allow not only electrical currents (especially cationic currents) but also many intracellular second messengers (e.g., IP3) to pass through and among smooth muscle cells and endothelial cells. Thus a very small depolarization in smooth muscle cells can be passively spread over relatively large distances to other smooth muscle cells and endothelial cells. Such passive current and potential spread in blood vessels is an important mechanism in which smooth muscle cells and endothelial cells are integrated with each other in response to endogenous and exogenous stimuli (6, 16).
The impairment of endothelium-dependent pulmonary vasodilation (10, 13) and monoclonal endothelial cell proliferation (21) has recently been proposed to play an important role in the pathogenesis of PPH (41). In addition, membrane depolarization via dysfunctional K+ channels has been recently observed in pulmonary arterial smooth muscle cells from patients with PPH (43, 47). In acutely and chronically hypoxic animals, membrane depolarization has been shown to correlate with increased pulmonary arterial pressure (31, 32, 36, 44). The results from the present study demonstrate that membrane depolarization by prolonged exposure to high [K+] selectively inhibits the endothelium-dependent PA relaxation, suggesting that pulmonary endothelial function may be partially impaired by membrane depolarization in the smooth muscle and endothelial cells.
We thank Dr. J. Wang, Dr. C. L. Bailey, and R. L. Walker for technical assistance.
Address for reprint requests and other correspondence: J. Yuan, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail:).
This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan). J. X.-J. Yuan is an Established Investigator of the American Heart Association.
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