High K+-induced membrane depolarization attenuates endothelium-dependent pulmonary vasodilation

Jan E. Seiden1, Oleksandr Platoshyn2, Alan E. Bakst1, Sharon S. McDaniel2, and Jason Xiao-Jian Yuan2

1 Department of Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201; and 2 Department of Medicine, University of California, San Diego, California 92103-8382


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -actin antibody, indicating that the cultures were all smooth muscle cells.

Measurement of membrane potential. Membrane potential (Em) in single PASMC was measured using an intracellular electrode (30-100 MOmega ) 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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of 25 mM K+ on Em. The resting Em 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 on Em was characterized by a transient depolarization (due apparently to Ca2+-dependent action potentials) followed by a steady-state depolarization (Fig. 1A) that was maintained for at least 180 min (the longest time tested; Fig. 1B). 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 (EK).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of 25 mM K+ on membrane potential (Em) in pulmonary artery smooth muscle cells (PASMC). A: representative record of Em in PASMC superfused with 25 mM K+. Extracellular application of 25 mM K+ induced both transient (spike) and sustained (plateau) depolarization (from -46 ± 2 to -25 ± 2 mV, P < 0.001, and -36 ± 2 mV, P < 0.01, respectively, n = 15). B: summarized data showing Em before (control, open bar) and during incubation of the cells in modified Krebs solution containing 25 mM K+ (filled bars) for 10, 30, 60, 120, and 180 min, respectively. Data are expressed as means ± SE with the no. of experiments shown in parentheses. *** P < 0.001 vs. control.

ACh-mediated pulmonary vasodilation depends on intact endothelium. Increase in [K+]o from 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. 2A, left, and B). ACh (2 µM) reversibly relaxed the endothelium-intact PA rings precontracted with 25 mM K+ (by -63 ± 5%, n = 12; Fig. 2A, left, and C). 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. 2A, right, and C). These results indicated that the ACh-mediated pulmonary vasodilation completely depends on intact endothelium (5).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2.   Endothelial dependence of ACh-induced relaxation in isolated pulmonary artery (PA) rings. A: PA rings with (+Endo, left) and without (-Endo, right) endothelium were precontracted with 25 mM K+. ACh (2 µM) was applied after 20 min when the 25 mM K+-induced tension reached plateau. B: summarized data showing active tension (mg tension/mg tissue wt) induced by 25 mM K+ in endothelium-intact (open bar) and endothelium-denuded (filled bar) PA rings. C: summarized data showing ACh-induced relaxation in endothelium-intact (open bar) and endothelium-denuded (filled bar) PA rings preconstricted with 25 mM K+. Data are expressed as means ± SE with no. of experiments shown in parentheses. *** P < 0.001 vs. endothelium intact.

Inhibitory effect of membrane depolarization on ACh-mediated pulmonary vasodilation. Increasing [K+]o from 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. 3A). 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 and C).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3.   Inhibitory effect of prolonged membrane depolarization on ACh-induced relaxation in isolated PA rings. A and B: endothelium-intact PA rings were preconstricted with 25 mM K+. ACh (2 µM) was applied when the vessels had been superfused with the 25 mM K+-containing solution for ~20 min (A) or ~3 h (B). Tension amplitude (mg) was normalized to the tissue weight (mg) and is depicted as mg/mg. C: summarized data showing ACh-induced relaxation in PA rings preconstricted with 25 mM K+ for ~20 min (short, open bar) and ~3 h (long, filled bar). Data are expressed as means ± SE with no. of experiments shown in parentheses. *** P < 0.001 vs. short.

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. 4A). Increasing [K+]o from 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. 4A, bottom). Because 60 mM K+ in the bath solution shifted EK to -21 mV, opening of K+ channels under these conditions could not cause further hyperpolarization (i.e., Em would not become more negative than the EK) 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. 4B), 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).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4.   Levcromakalim (Crom)-induced pulmonary vasodilation is independent of intact endothelium. A: Crom (10 µM) was applied to the endothelium-intact PA ring when the vessel was preconstricted with 25 and 60 mM K+ (top). Bottom: summarized data showing comparison of Crom-induced relaxations in PA rings preconstricted with 20, 25, 30, 40, 60, and 80 mM K+. Data are expressed as means ± SE (n = 8). B: Crom (10 µM) was applied to the endothelium-intact (+Endo) and endothelium-denuded (-Endo) PA rings preconstricted with 25 mM K+ (top). Bottom: summarized data showing Crom-induced relaxation in PA rings with (+Endo) and without (-Endo) endothelium. Data are expressed as means ± SE with no. of experiments shown in parentheses.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of prolonged membrane depolarization on Crom-induced relaxation in isolated PA rings. A and B: endothelium-intact PA rings were constricted with 25 mM K+. Crom (2 µM) was applied when the vessels had been superfused with the 25 mM K+-containing solution for ~20 min (A) or ~3 h (B). Tension amplitude (mg) was normalized to the tissue weight (mg) and is depicted as mg/mg. C: summarized data showing Crom-induced relaxation in PA rings preconstricted with 25 mM K+ for ~20 min (short, open bar) and ~3 h (long, filled bar). Data are expressed as means ± SE with no. of experiments shown in parentheses.

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. 6A). 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 and C).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of prolonged membrane depolarization on sodium nitroprusside (SNP)-induced relaxation in isolated PA rings. A and B: endothelium-denuded PA rings were constricted with 25 mM K+. SNP (10 µM) was applied when the vessels had been superfused with 25 mM K+-containing solution for ~20 min (A) or ~3 h (B). Tension amplitude (mg) was normalized to the tissue weight (mg) and is depicted as mg/mg. C: summarized data showing SNP-induced relaxation in PA rings preconstricted with 25 mM K+ for ~20 min (short, open bar) and ~3 h (long, filled bar). Data are expressed as means ± SE with no. of experiments shown in parentheses. * P < 0.05 vs. long.

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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, resting Em is mainly controlled by the K+ permeability and EK (determined by the ratio of extracellular and intracellular [K+]). An increase in [K+]o from 4.7 mM (in MKS) to 25 mM K+ shifts EK from -85 mV to about -43 mV (assuming intracellular [K+] is 141 mM), thereby causing membrane depolarization (Em 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 on Em in PASMC was characterized by an initial transient depolarization followed by a sustained membrane depolarization for up to 3 h. The initial Em transient was apparently due to the Ca2+-dependent action potential, since removal of extracellular Ca2+ abolished the transient decrease (depolarizing) of Em (data not shown). Because of the change in the EK, increasing [K+]o to 25 mM caused a sustained Em depolarization that could be maintained as long as the cells were perfused with 25 mM K+-containing solution (Fig. 1B).

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+]cyt in 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+]cyt derived from the extracellular space and intracellular stores (11, 27). In endothelial cells, the ACh-induced rise in [Ca2+]cyt is 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). Because Em 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+]cyt may 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+]cyt and 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+]o in 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+]o to 25 mM, which exceeds the [K+]o for 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.


    ACKNOWLEDGEMENTS

We thank Dr. J. Wang, Dr. C. L. Bailey, and R. L. Walker for technical assistance.


    FOOTNOTES

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.

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.

Address for reprint requests and other correspondence: J. Yuan, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.edu).

Received 5 August 1998; accepted in final form 13 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adams, D. J., J. Barakeh, R. Laskey, and C. van Breemen. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB J. 3: 2389-2400, 1989[Abstract/Free Full Text].

2.   Archer, S., J. M. C. Huang, V. Hampl, D. P. Nelson, P. J. Shultz, and E. K. Weir. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 91: 7583-7587, 1994[Abstract].

3.   Arnold, W. P., C. K. Mittal, S. Katsuki, and F. Murad. Nitric oxide activates guanylate cyclase and increases guanosine 3', 5'-cyclic monophosphate levels in various tissue preparations. Proc. Natl. Acad. Sci. USA 74: 3203-3207, 1977[Abstract].

4.   Berridge, M. J., M. D. Bootman, and P. Lipp. Calcium---a life and death signal. Nature 295: 645-648, 1998.

5.   Chand, N., and B. M. Altura. Acetylcholine and bradykinin relax intrapulmonary arteries by actin on endothelial cells: role in lung vascular diseases. Science 213: 1376-1379, 1981[ISI][Medline].

6.   Christ, G. J., D. C. Spray, M. El-Sabban, L. K. Moore, and P. R. Brink. Gap junctions in vascular tissues: evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ. Res. 79: 631-646, 1996[Abstract/Free Full Text].

7.   Clapp, L. H., and A. M. Gurney. ATP-sensitive K+ channels regulate resting potential of pulmonary arterial smooth muscle cells. Am. J. Physiol. Heart Circ. Physiol. 262: H916-H920, 1992[Abstract/Free Full Text].

8.   Cornfield, D. N., T. Stevens, I. F. McMurtry, S. H. Abman, and D. M. Rodman. Acute hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 265: L53-L56, 1993[Abstract/Free Full Text].

9.   Demirel, E., R. E. Laskey, S. Purkerson, and C. van Breemen. The passive calcium leak in cultured porcine aortic endothelial cells. Biochem. Biophys. Res. Commun. 191: 1197-1203, 1993[ISI][Medline].

10.   Dinh-Xuan, A. T., T. W. Higenbottam, C. A. Clelland, J. Pepke-Zaba, G. Cremona, A. Y. Butt, S. R. Large, F. C. Wells, and J. Wallwork. Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung disease. N. Engl. J. Med. 324: 1539-1547, 1991[Abstract].

11.   Edwards, G., K. A. Dora, M. J. Gardener, C. J. Garland, and A. H. Weston. K+ is an endothelium-derived hyperpolarizing factor in rat arteries. Nature 396: 269-272, 1998[ISI][Medline].

12.   Ganitkevich, V. Y., and G. Isenberg. Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca2+ transients in guinea-pig coronary myocytes. J. Physiol. (Lond.) 470: 35-44, 1993[Abstract].

13.   Giaid, A., and D. Saleh. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N. Engl. J. Med. 333: 214-221, 1995[Abstract/Free Full Text].

14.   Griffith, T. M., D. H. Edwards, A. C. Newby, M. J. Lewis, and A. H. Henderson. Production of endothelium-derived relaxant factor is dependent on oxidative phosphorylation and extracellular calcium. Cardiovasc. Res. 20: 7-12, 1986[ISI][Medline].

15.   Harder, D. R., J. A. Madden, and C. Dawson. Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat. J. Appl. Physiol. 59: 1389-1393, 1985[Abstract/Free Full Text].

16.   Hirst, G. D. S., and F. R. Edwards. Sympathetic neuroeffector transmission in arteries and arterioles. Physiol. Rev. 69: 546-604, 1989[Free Full Text].

17.   Ignaro, L. J., T. M. Burke, K. S. Wood, M. S. Wolin, and P. J. Kadowitz. Association between cyclic GMP accumulation and acetylcholine-elicited relaxation of bovine intrapulmonary artery. J. Pharmacol. Exp. Ther. 228: 682-690, 1984[Abstract].

18.   Ignaro, L. J., R. E. Byrns, G. M. Buga, and K. S. Wood. Endothelium derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ. Res. 61: 866-879, 1987[Abstract].

19.   Inagami, T., M. Naruse, and R. Hoover. Endothelium as an endocrine organ. Annu. Rev. Physiol. 57: 171-189, 1995[ISI][Medline].

20.   Johns, A., T. W. Lategan, N. J. Lodge, U. S. Ryan, C. van Breemen, and D. J. Adams. Calcium entry through receptor operated channels in bovine pulmonary artery endothelial cells. Tissue Cell 19: 733-745, 1987[ISI][Medline].

21.   Lee, S.-D., K. R. Shroyer, N. E. Markham, C. D. Cool, N. F. Voelkel, and R. M. Tuder. Monoclonal endothelial cell proliferation is present in primary but not secondary pulmonary hypertension. J. Clin. Invest. 101: 927-934, 1998[Abstract/Free Full Text].

22.   Madden, J. A., M. S. Vadula, and V. P. Kurup. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 263: L384-L393, 1992[Abstract/Free Full Text].

23.   Marchenko, S. M., and S. O. Sage. Smooth muscle cells affect endothelial membrane potential in rat aorta. Am. J. Physiol. Heart Circ. Physiol. 267: H804-H811, 1994[Abstract/Free Full Text].

24.   Martens, J. R., and C. H. Gelband. Alterations in rat interlobar artery membrane potential and K+ channels in genetic and nongenetic hypertension. Circ. Res. 79: 295-301, 1996[Abstract/Free Full Text].

25.   Mayer, B., K. Schmidt, P. Humbert, and E. Bohme. Biosynthesis of endothelium-derived relaxing factor: a cytosolic enzyme in porcine aortic endothelial cells Ca2+-dependently converts L-arginine into an activator of soluble guanylyl cyclase. Biochem. Biophys. Res. Commun. 164: 678-685, 1989[ISI][Medline].

26.   McCormack, D. G., J. C. Mak, P. Minette, and P. J. Barnes. Muscarinic receptor subtypes mediating vasodilatation in the pulmonary artery. Eur. J. Pharmacol. 158: 293-297, 1988[ISI][Medline].

27.   Nagao, T., and P. M. Vanhoutte. Endothelium-derived hyperpolarizing factor and endothelium-dependent relaxations. Am. J. Respir. Cell Mol. Biol. 8: 1-6, 1993[ISI][Medline].

28.   Nelson, M. T., and J. M. Quayle. Physiological roles and properties of potassium channels in arterial smooth muscle. Am. J. Physiol. Cell Physiol. 268: C799-C822, 1995[Abstract/Free Full Text].

29.   Nilius, B., F. Viana, and G. Droogmans. Ion channels in vascular endothelium. Annu. Rev. Physiol. 59: 145-170, 1997[ISI][Medline].

30.   Osipenko, O. N., A. M. Evans, and A. M. Gurney. Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O2-sensitive potassium current. Br. J. Pharmacol. 120: 1461-1470, 1997[Abstract].

31.   Post, J. M., C. H. Gelband, and J. R. Hume. [Ca2+]i inhibition of K+ channels in canine pulmonary artery, novel mechanisms for hypoxia-induced membrane depolarization. Circ. Res. 77: 131-139, 1995[Abstract/Free Full Text].

32.   Post, J. M., J. R. Hume, S. L. Archer, and E. K. Weir. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am. J. Physiol. Cell Physiol. 262: C882-C890, 1992[Abstract/Free Full Text].

33.   Revest, P. A., and N. J. Abbott. Membrane ion channels of endothelial cells. Trends Pharmacol. Sci. 13: 404-407, 1992[ISI][Medline].

34.   Robertson, B. E., R. Schubert, J. Hescheler, and M. T. Nelson. cGMP-dependent protein kinase activates Ca-activated K channels in cerebral artery smooth muscle cells. Am. J. Physiol. Cell Physiol. 265: C299-C303, 1993[Abstract/Free Full Text].

35.   Rodman, D. M., T. Yamaguchi, K. Hasunuma, R. F. O'Brien, and I. F. McMurtry. Effect of hypoxia on endothelium-dependent relaxation of rat pulmonary artery. Am. J. Physiol. Lung Cell. Mol. Physiol. 258: L207-L214, 1990[Abstract/Free Full Text].

36.   Smirnov, S. V., T. P. Robertson, J. P. T. Ward, and P. L. Aaronson. Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells. Am. J. Physiol. Heart Circ. Physiol. 266: H365-H370, 1994[Abstract/Free Full Text].

37.   Stamler, J. S., E. Loh, M.-A. Roddy, K. E. Currie, and M. A. Creager. Nitric oxide regulates basal systemic and pulmonary vascular resistance in healthy humans. Circulation 89: 2035-2040, 1994[Abstract].

38.   Stevens, T., D. N. Cornfield, I. F. McMurtry, and D. M. Rodman. Acute reductions in PO2 depolarize pulmonary artery endothelial cells and decrease [Ca2+]i. Am. J. Physiol. Heart Circ. Physiol. 266: H1416-H1421, 1994[Abstract/Free Full Text].

39.   Tanaguchi, J., K.-I. Furukawa, and M. Shigekawa. Maxi K+ channels are stimulated by cyclic guanosine monophosphate-dependent protein kinase in canine coronary artery smooth muscle cells. Pflügers Arch. 423: 167-172, 1993[ISI][Medline].

40.   Twort, C. H. C., and C. van Breemen. Cyclic guanosine monophosphate-enhanced sequestration of Ca2+ by sarcoplasmic reticulum in vascular smooth muscle. Circ. Res. 62: 961-964, 1988[Abstract].

41.   Voelkel, N. F., R. M. Tuder, and E. K. Weir. Pathophysiology of primary pulmonary hypertension: from physiology to molecular mechanisms. In: Primary Pulmonary Hypertension, edited by L. J. Rubin, and S. Rich. New York: Dekker, 1997, p. 83-129.

42.   Wang, X., F. Lau, L. Li, A. Yoshikawa, and C. van Breemen. Acetylcholine-sensitive intracellular Ca2+ store in fresh endothelial cells and evidence for ryanodine receptors. Circ. Res. 77: 37-42, 1995[Abstract/Free Full Text].

43.   Yuan, J. X.-J., A. M. Aldinger, M. Juhaszova, J. Wang, J. V. Conte, Jr., S. P. Gaine, J. B. Orens, and L. J. Rubin. Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 98: 1400-1406, 1998[Abstract/Free Full Text].

44.   Yuan, J. X.-J., W. F. Goldman, M. L. Tod, L. J. Rubin, and M. P. Blaustein. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am. J. Physiol. Lung Cell. Mol. Physiol. 264: L116-L123, 1993[Abstract/Free Full Text].

45.   Yuan, J. X.-J., M. L. Tod, L. J. Rubin, and M. P. Blaustein. Contrasting effects of hypoxia on tension in rat pulmonary and mesenteric arteries. Am. J. Physiol. Heart Circ. Physiol. 259: H281-H289, 1990[Abstract/Free Full Text].

46.   Yuan, J. X.-J., M. L. Tod, L. J. Rubin, and M. P. Blaustein. NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels. Proc. Natl. Acad. Sci. USA 93: 10489-10494, 1996[Abstract/Free Full Text].

47.   Yuan, J. X.-J., J. Wang, M. Juhaszova, S. P. Gaine, and L. J. Rubin. Attenuated K+ channel gene transcription in primary pulmonary hypertension. Lancet 351: 726-727, 1998[ISI][Medline].


Am J Physiol Lung Cell Mol Physiol 278(2):L261-L267
1040-0605/00 $5.00 Copyright © 2000 the American Physiological Society