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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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
-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 M)
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
|
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).
|
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).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
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.
Calciuma 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
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
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
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
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
16.
Hirst, G. D. S.,
and
F. R. Edwards.
Sympathetic neuroeffector transmission in arteries and arterioles.
Physiol. Rev.
69:
546-604,
1989
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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].