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AC Burton Vascular Biology Laboratory, Division of Respiratory Medicine, London Health Sciences Centre, London, Ontario, Canada N6A 4G5
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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We investigated the
role of K+ channels in the attenuated pulmonary artery (PA)
contractility characteristic of acute Pseudomonas pneumonia. Contractility of PA rings from the lungs of control or pneumonia rats was assessed in vitro by obtaining cumulative concentration-response curves to the contractile agonists KCl, phenylephrine, or PGF2
on PA rings before and after
treatment with K+ channel blockers. In rings from pneumonia
rats, paxilline (10 µM), tetraethylammonium (2 mM) (blockers of
large-conductance Ca2+-activated K+ channels),
and glybenclamide (ATP-sensitive K+ channel blocker, 80 µM) had no significant effect on the attenuated contractile responses
to KCl, phenylephrine, and PGF2. However, 4-aminopyridine (2 mM), a blocker of voltage-gated K+
channels (delayed rectifier K+ channel) reversed this
depressed contractility. Therefore, large-conductance Ca2+-activated K+ and ATP-sensitive
K+ channels do not contribute to the attenuated PA
contractility observed in this model of acute pneumonia. In contrast,
4-aminopyridine enhances contraction in PA rings from pneumonia lungs,
consistent with involvement of a voltage-gated K+ channel
in the depressed PA contractility in acute pneumonia. Unraveling the
precise mechanism of attenuated contractility in pneumonia could lead
to innovative therapies for the pulmonary vascular abnormalities
associated with this disease.
4-aminopyridine; glybenclamide; levcromakalim; rat intrapulmonary artery; Pseudomonas pneumonia
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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OUR LABORATORY HAS PREVIOUSLY demonstrated, both in vitro (55) and in vivo (19), depressed contractility of the pulmonary vasculature in rats with Pseudomonas pneumonia. Excess nitric oxide (NO) (produced by inducible NO synthase) partly contributes, but does not fully explain, the depressed pulmonary vascular contractility observed in rats with acute pneumonia (55). In addition, our laboratory has suggested a possible role for cytochrome P-450 (CYP) metabolites [epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE)] of arachidonic acid (AA) in this phenomenon (57). Therefore, the mechanism of action involved in this phenomenon is not yet completely understood, although K+ channel activation may contribute to endotoxin-mediated hyporeactivity to vasoconstrictor agents in some vascular beds (9, 22, 35).
Many endogenous and synthetic agents can modulate the contractility or
relaxation of vascular beds and are used to investigate vascular
reactivity in vivo and in vitro. Some of these agents are constrictors,
such as phenylephrine (PE) and PGF2, whereas others are
dilators, such as acetylcholine, sodium nitroprusside, and
PGI2 (17, 45, 51). In acute lung injury (such
as occurs in pneumonia or systemic sepsis), attenuated pulmonary
vascular contractility has been demonstrated both in vivo
(18-20) and in vitro (31, 53, 56) to a
number of stimuli, including hypoxia, angiotensin II, norepinephrine,
and potassium chloride (KCl). Furthermore, sepsis and pneumonia impair
hypoxic pulmonary vasoconstriction, thereby reducing arterial
oxygenation and enhancing hypoxemia, both in animal models (19,
20, 50) and in humans (3, 13, 30). The mechanism of
this loss of contractility in response to environmental and
pharmacological stimuli is not yet fully understood. How inflammation
associated with infection affects pulmonary vascular responses is
important, because understanding this phenomenon could at least
partially explain the abnormal gas-exchange characteristic of patients
with pneumonia.
Various types of K+ channels contribute to vessel tone in many vascular beds (10, 25, 37). Opening of K+ channels causes hyperpolarization of the smooth muscle cell membrane. This hyperpolarization closes L-type and T-type Ca2+ channels and reduces Ca2+ entry into the cell, resulting in lower levels of cytosolic free Ca2+ and reduced vascular tone (38, 43). NO activates large-conductance Ca2+-activated K+ (BKCa) channels, resulting in hyperpolarization of vascular smooth muscle cells, thus attenuating contractility (35, 48). In addition, although no known receptors have yet been identified, EETs and 20-HETE modulate the function of Ca2+-activated K+ (KCa) channels in vascular smooth muscle cells, thus influencing vascular contractility (24, 48). Therefore, we investigated the role of K+ channels in the attenuated pulmonary artery (PA) contractility characteristic of acute Pseudomonas pneumonia.
More than one type of K+ channels may be involved in the
relaxation of vascular smooth muscle, and this is dependent on the species and vascular bed studied (5, 6, 54). Nevertheless, the physiological role of ATP-sensitive K+
(KATP) channels in regulating membrane potential in PAs is
not certain. Thus the opening of even a few KATP channels
by various endogenous vasodilators and metabolic inhibitors would have
substantial effects on membrane potential and vascular tone (14,
37, 39). Furthermore, levcromakalim (which opens
KATP channels, Ref. 8) attenuates the
contractile response of small rat PAs to hypoxia, KCl, and
PGF2 (60). Glybenclamide closes
KATP channels in vascular smooth muscle (11).
Administration of glybenclamide to animals with endotoxin-induced
hypotension improves blood pressure (29, 52). This has led
investigators to suggest that activation of KATP channels
contributes to the loss of vascular tone associated with sepsis.
Voltage-gated, delayed rectifier K+ (KV) current that is sensitive to blockade with 4-aminopyridine (4-AP) has been identified in vascular smooth muscle tissues (12). Specifically, a number of investigators have demonstrated a critical role for KV channels in setting the resting membrane potential in PA smooth muscle cells (2, 42, 58). In addition, inhibition of KV channels and increase in intracellular Ca2+ have been demonstrated to play a role in the initiation of hypoxic pulmonary vasoconstriction (42, 59).
BKCa, KATP, and KV channels have all been identified in arterial smooth muscle cells (39, 54). Nevertheless, their role in regulation of vascular tone in disease has not been established. Therefore, we tested the hypothesis that K+ channels contribute to the depressed PA contractility observed in the acute pneumonia model of the rat. We provide evidence for the selective involvement of KV channels in this phenomenon, suggesting new therapeutic approaches for treating the attenuated PA contractility associated with pneumonia.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Acute Pneumonia Model
A bacterial culture (Pseudomonas aeruginosa) was prepared fresh weekly and was suspended in phosphate-buffered saline solution. Male Sprague-Dawley rats (330-350 g) were randomized to the pneumonia group or control group. All rats had a jugular venous line placed for fluid administration. Rats were anesthetized with halothane (1-2%) and a balance of pure oxygen. A catheter was tunneled subcutaneously from the intracapsular region to a midline incision in the neck. The Silastic venous catheter (0.023 in. ID, Clay Adams) attached to PE-50 (Clay Adams) was advanced into the superior vena cava via the right external jugular vein. The catheter was attached to a harness-swivel device (Harvard Instruments, St. Laurent, PQ) to protect the catheter and allow the rats to move freely about the cage. The incision was closed with 2-0 silk.Animals in the pneumonia group were injected intratracheally with 0.15 ml of saline containing 3×108 colony-forming units/ml. A small midline incision was made to expose the trachea. A catheter was advanced through a tracheostomy into a distal bronchus for injection of the solution containing bacteria. Within 36 h, this instillation of bacteria produced an acute localized pneumonia in the left lung, with the remainder of the lung appearing grossly normal. Animals in the control group had a tracheostomy but did not have an intratracheal injection, as clinical evidence in patients has demonstrated that a febrile response with a possibility of an infection may result after bronchoalveolar lavage (44).
Postoperatively, the rats were housed separately and allowed free access to standard rat chow and water. Fluid was administered by a continuous infusion of heparinized saline (1 U/ml) at 2-3 ml/h. Fentanyl (1.0 µg/ml) was added to the intravenous infusion for analgesia.
Forty-four hours after surgery, rats were anesthetized with pentobarbital (20 mg iv), and the thorax was opened. The heart and lungs were removed en bloc, perfused through the PA with modified Krebs solution, and placed in Krebs buffer at 4°C (vide infra). Small intrapulmonary arteries were dissected out from the affected lobe in the pneumonia lung, or the corresponding lobe of lung from control rats, and studied as described below. All animals used in this study were cared for following the principles and guidelines of the Canadian Council on Animal Care and were supervised by a veterinarian. In addition, the ethics review committee at the University of Western Ontario (London, ON) approved all protocols.
In Vitro Vascular Reactivity
From each lung, small intrapulmonary arteries (200-400 µm diameter) were dissected out under a light microscope (Nikon Canada, Missisauga, Ontario) and were cut into 1.0- to 2.0-mm cylindrical segments. Each vessel segment was suspended on two stainless steel wires in 5-ml organ baths as described earlier (55). The two stainless steel wires were inserted through the lumen: one connected to a micromanipulator (Marzhauser MM33, Fine Science Tools, North Vancouver, BC) and the other to an FT03 force displacement transducer (Grass Instruments, Quincy, MA). Isometric tension recordings were obtained on a Grass Instruments polygraph (model 79D). The microvascular segment preparations were suspended for 30 min under an initial tension of 50 mg in jacketed 5-ml organ baths at 37 ± 0.5°C. The vessels were then allowed to equilibrate for 1 h under a previously determined resting wall tension (120 mg/mm). The effective lumen radius (ELR; ELR = L/2
, where
L is the internal circumference of the vessel) and the
optimal resting wall tension for the vessels were determined in pilot
experiments, as previously described, in our laboratory
(49). Briefly, vessel radius was incrementally increased
from the resting ELR, and, after each stepwise increase in ELR, the
baseline arterial tension and the contraction to KCl (40 mM) were
recorded, followed by repeated washing with Krebs solution, until the
contraction to KCl was maximal. The increase in ELR was then plotted
against change in wall tension (mg/mm), and the optimal resting tension
was determined. In all studies, vessels were tested for the presence of
a functional endothelium by precontracting with a submaximal
concentration of PE (2.4 µM) and relaxing with acetylcholine (10 µM) at the beginning of each experiment. All vessels that were used
in this study relaxed >50% to acetylcholine.
The vascular preparations were bathed in a modified Krebs bicarbonate buffer solution of the following composition (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4 · 7H2O, 1.2 KH2PO4, 11.1 dextrose, and 22.1 NaHCO3, (pH = 7.4). Organ baths were continuously gassed with 95% O2-5% CO2.
Determination of glybenclamide concentration required to inhibit levcromakalim (KATP opener) effect. The concentration of glybenclamide required to inhibit levcromakalim-induced relaxation was determined. Small PAs dissected from the left lungs of naive rats (n = 8) were used. Vessels were equilibrated with vehicle (5 mg/ml sodium hydroxide and 5% glucose) or glybenclamide (10, 20, 40, 80, 160, or 320 µM) for 30 min. Arteries were precontracted with PE (2.4 µM), and a concentration-relaxation curve was constructed for levcromakalim.
Assessment of Reactivity
Contractility of intrapulmonary arteries from pneumonia and control rats was studied by using three different agonists: KCl, PE, and PGF2. The order of application of the three agonists to each arterial ring was randomized. With each contractile agonist, arteries were allowed to contract until a plateau was obtained before
the next incremental concentration was added. After maximal contraction
with each agonist was obtained, the bath was washed three to four times
with fresh Krebs solution. When the vessel segments returned to the
initial resting tension (~15-20 min later), the next cumulative
concentration-response curve was obtained.
Role of K+ Channel Blockers
In arterial rings from control and pneumonia rats, log (concentration)-contraction curves were obtained to the contractile agonists KCl, PE, and PGF2 before and after treatment
with one of the K+ channel blockers [10 µM paxilline or
its inactive control, 10 µM paxillinol, 2 mM tetraethylammonium
(TEA), 80 µM glybenclamide, 2 mM 4-AP] or their vehicle controls
(DMSO for paxilline and paxillinol, and 5 mg/ml sodium hydroxide and
5% glucose for glybenclamide). Individual rings were used for each
blocker or vehicle.
In arteries treated with glybenclamide (80 µM) or vehicle, the log (concentration)-relaxation curves to levcromakalim (a selective KATP channel opener) were also determined.
Reagents
L-PE hydrochloride, acetylcholine iodide, glybenclamide, TEA, and 4-AP were purchased from Sigma Chemical (St. Louis, MO). Levcromakalim (BRL-38227) was a gift from SmithKline Beecham pharmaceuticals. KCl (BDH Chemicals) solution (2.0 M) was freshly prepared in distilled water when needed. PGF2
(dinoprost tromethamine, Upjohn) was purchased from the hospital
pharmacy. Stock solutions of PE (10 mM), PGF2 (10 mM),
acetylcholine (10 mM), and levcromakalim (10 mM) were prepared in
distilled water and frozen (
4°C) in aliquots until needed.
Glybenclamide (10 mM) was dissolved in 25 mg/ml sodium hydroxide,
rapidly diluted to 5 mg/ml with 5% glucose solution, and frozen in
aliquots until needed. Paxilline and its negative control paxillinol
were purchased from BIOMOL Research Laboratories, (Plymouth Meeting,
PA) and dissolved in DMSO. TEA (1.0 M) and 4-AP (1.0 M) were freshly
prepared in distilled water when needed.
Data Analysis
Figures 1-5 were plotted using Prism (GraphPad Software, San Diego, CA) and analyzed using the statistics package for Social Sciences (SPSS-PC, version 6.0; SPSS, Chicago, IL) and GraphPad Instat (GraphPad Software). Each log (concentration)-response curve was plotted using nonlinear regression analysis for dose-response curves, and the EC50 of agonist (where applicable) and maximal developed wall tension (Tmax) values were determined for comparison. To study the effect of pneumonia on responses of PAs to different contractile agonists and to determine whether the K+ channel blockers altered these responses, cumulative concentration-contraction curves were compared between pneumonia and control groups by using repeated-measures ANOVA. The rest of the data were analyzed by using other statistical tests, as reported in RESULTS. Results are expressed as means ± SE of n values, where n is the number of rats. A value of P < 0.05 was considered significant.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Role of BKCa Channels
Log (concentration)-contraction curves were obtained for the contractile agonists KCl, PE, and PGF2 on rings before
and after treatment with paxilline or with its negative control,
paxillinol (both at 10 µM with 10-min equilibration in organ bath).
Paxilline (10 µM) has been previously shown to act as a selective and
reversible blocker of large-conductance Ca2+-activated
channels, whereas the structural analog paxillinol had no effect on
these channels (28). Individual PA rings, from control and
pneumonia rats, were used for paxilline or paxillinol. DMSO was used as
vehicle control for paxilline and paxillinol with a final organ bath
concentration of 0.001%, which did not alter baseline tone or
contractile responses of arterial rings. Similarly, log
(concentration)-contraction curves were obtained to the contractile
agonists KCl, PE, and PGF2 on rings before and after
treatment with TEA (2 mM; 10-min equilibration in organ bath), a
blocker of large-conductance Ca2+-activated channels
(37). TEA (2 mM) did not modify baseline tone of arterial
rings. Arteries from pneumonia animals had significantly depressed
contractile responses to KCl, PE, and PGF2 compared with
control (Fig. 1). Neither of the
BKCa blockers (paxilline and TEA) had any significant
effect on the depressed contractility observed in PA rings from
pneumonia rats, because the log (concentration)-contraction curves to
KCl, PE, and PGF2 obtained before and after incubation with these blockers were not significantly different (Fig. 1). In
addition, contractile responses obtained in the presence of paxillinol
(the inactive structural control for paxilline) were similar to those
obtained with paxilline in Fig. 1. Therefore, paxilline and TEA, at
concentrations known to block BKCa channels, could not
reverse the attenuated PA contractility in pneumonia. This indicated
that KCa channels were not involved in this phenomenon and
led us to investigate the role of other K+ channels.
Role of KATP Channels
Preliminary experiments were performed to establish the concentration of glybenclamide required to fully block KATP channels in arterial rings in our experimental setup. To accomplish this, the concentration of glybenclamide required to inhibit levcromakalim-induced relaxation was determined in arteries from naive rats (Fig. 2). Arteries were precontracted with PE (2.4 µM), and a log (concentration)-relaxation curve was constructed for the KATP channel opener levcromakalim (see representative traces in Fig. 2A). A range of doses of glybenclamide (10-320 µM) was studied, as described in METHODS. Glybenclamide at the lower concentrations tested (10, 20, and 40 µM) blocked the levcromakalim relaxant response of the PE-contracted PA rings but not when higher concentrations of levcromakalim (>100 µM) were achieved in the organ bath. Glybenclamide at 80 µM fully blocked the relaxant responses to levcromakalim without significantly affecting the PE response of these arteries (control PE precontraction = 83.3 ± 11.5 mg/mm, n = 8, compared with PE precontraction in presence of 80 µM glybenclamide = 63.3 ± 11.3 mg/mm, n = 5, not significant). Glybenclamide at 160 and 320 µM also fully inhibited the levcromakalim relaxant response. However, these higher concentrations of glybenclamide also significantly attenuated the PE precontractile responses (28.6 ± 8.4 mg/mm, n = 3, and 32.7 ± 10.2 mg/mm, n = 4, respectively; ANOVA followed by Tukey's test, P < 0.05 compared with control). In the presence of higher concentrations of glybenclamide, it appeared as though levcromakalim caused a contraction of naive arteries. However, what really occurred was a restoration of the PE contraction in arterial rings treated simultaneously with levcromakalim and high concentrations of glybenclamide. Note that this response was variable and only significant for the 160 and 320 µM concentrations of glybenclamide and 10-100 µM levcromakalim. Thus glybenclamide at 80 µM was the lowest dose found to fully block the relaxant responses to levcromakalim in rat PAs without having significant effects on contractility of these arteries to PE (Fig. 2B).To assess the role of KATP channels in pneumonia, arterial
rings from pneumonia or control rats were equilibrated with vehicle (5 mg/ml sodium hydroxide and 5% glucose) or glybenclamide (80 µM) for
30 min before assessment of contractility to KCl, PE, and
PGF2 (Fig. 3). In
addition, log (concentration)-relaxation curves were constructed to
levcromakalim in these arterial rings (Fig. 3) to confirm blockade of
KATP channels by glybenclamide at 80 µM, the dose of
KATP blocker determined earlier to be effective in PA rings
from naive rats. Neither vehicle nor glybenclamide had any effect on
baseline tone in any of the PA rings studied. The KATP
blocker glybenclamide had no significant effect on the depressed
contractility observed in PA rings from pneumonia rats (Fig. 3).
Therefore, glybenclamide at a concentration (80 µM) that fully
blocked KATP channels in pulmonary vessels from control and
pneumonia rats (Fig. 3D) had no effect on the depressed
contractility of PA rings from pneumonia rats. This indicated that
KATP channels were not involved in this phenomenon.
Therefore, the role of KV channels was next evaluated.
Role of KV Channels
4-AP is a blocker of KV channels (delayed rectifier K+ channel) (12). On arterial rings from pneumonia and control rats, log (concentration)-contraction curves were obtained to the contractile agonists KCl, PE, and PGF2
before and after treatment with 4-AP (2 mM, 10-min equilibration in
organ bath). Similar to the other K+ blockers, 4-AP (2 mM)
did not modify baseline tone of arterial rings. Representative tracings
of the effect of 4-AP on PE and KCl responses in PA rings from
pneumonia rats are shown in Fig. 4. Note
that, in PA rings from pneumonia rats, treatment with 4-AP resulted in
a significant increase in contractility to cumulatively added KCl (Fig.
4A) and PE (Fig. 4B). In rings from pneumonia rats, 4-AP enhanced the contractile responses to KCl, PE, and PGF2, but this enhancement varied with the agonist
tested. In PA rings from pneumonia rats, 4-AP restored the KCl
contractile response to that obtained in PA rings from control rats
(overlapping curves for control and pneumonia + 4-AP) but only
partially restored the PE and PGF2 responses (Fig.
5). In PA rings from pneumonia rats, 4-AP
restored the KCl Tmax response to that in control arteries (Table 1, KCl). In contrast, 4-AP
significantly enhanced the PE Tmax response but did not
fully restore contractility to PE to the control level. The
EC50 for PE in PA rings from pneumonia rats was still
significantly different from that in PA rings from control rats (Table
1, PE). 4-AP significantly reduced the EC50 for
PGF2 but did not significantly alter the
Tmax in rings from pneumonia compared with control rats
(Table 1, PGF2). Note that 4-AP did not have any
significant effect on contractility of PA rings from control rats, as
shown in Fig. 5. On arterial rings from control rats, log
(concentration)-contraction curves to the contractile agonists KCl, PE,
and PGF2 before and after treatment with 4-AP were
overlapping (Fig. 5, control compared with control + 4-AP for each
agonist).
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None of the K+ channel blockers (paxilline, TEA, and glybenclamide) or their vehicle controls had any significant effect on contractility of PA rings from control rats (data not shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The results of the present study demonstrate that BKCa channels do not contribute to the depressed pulmonary vascular contractility observed in this model of acute Pseudomonas pneumonia. In addition, this study shows that, although the vasodilator KATP channels are present in small intrapulmonary arteries of the rat, these channels are not involved in this attenuated contractility. In contrast, 4-AP enhances contraction in PA rings from pneumonia lungs, consistent with involvement of a KV channel in the depressed PA contractility observed in this acute pneumonia model of the rat.
BKCa channels have been described in arteries of different vascular beds, including the pulmonary circulation (4, 34, 39, 41). In addition, slight changes in membrane potential of vascular smooth muscle have been associated with significant changes in vascular tone (26, 38, 39). Paxilline, an indole diterpene alkaloid, is a potent and selective blocker of BKCa channels (28). In addition, a structurally different blocker, TEA, is selective for BKCa channels at low concentrations (15). In our study, neither paxilline nor TEA had a significant effect on the depressed contractility observed in pneumonia, confirming that BKCa channels are not involved in this phenomenon.
Cromakalim, levcromakalim, and other K+ channel openers repolarize or hyperpolarize and relax vascular smooth muscle primarily via activation of KATP channels (38, 43, 47). These channels regulate the resting potential of PA smooth muscle cells and are closed by the KATP closer glybenclamide (10, 11, 43). Depending on the tissue or vascular bed studied, investigators have used low doses (10-30 µM) of glybenclamide to achieve blockade of KATP channels (11, 39, 40). In our study, lower concentrations of glybenclamide were not effective. Levcromakalim relaxed PE-contracted PA rings from control and pneumonia rats, an effect fully blocked by 80 µM glybenclamide. These data demonstrate that small intrapulmonary artery rings from rats have functional KATP channels. Pretreatment with glybenclamide at 160 and 320 µM caused depressed contractility of the PA rings to PE. The reason for this effect of high concentrations of glybenclamide is not clear but could reflect the ability of glybenclamide at high concentrations to act as a partial agonist of KATP channels, as suggested by Cai et al. (7).
The involvement of KATP channels in altered vascular tone
has been suggested. Landry and Oliver (29) showed that
KATP channels, at least in part, mediate the vasodilation
and hypotension observed in endotoxemic dogs (29). In
support of this, Vanelli et al. (52) reported that
glybenclamide restored the hypotension caused by endotoxin infusion
into pigs. In our rat model, as our laboratory has previously reported
(55), acute Pseudomonas pneumonia resulted in
depressed PA contractility to KCl, PE, and PGF2. In this paper, we have confirmed that, although small intrapulmonary arteries from animals with pneumonia have functional KATP channels,
pretreatment of PA rings from pneumonia rats with glybenclamide (80 µM) did not affect the depressed vascular contractility to KCl, PE,
and PGF2.
KATP and BKCa channels have been implicated in the depressed contractility of systemic vessels to different agonists in endotoxin animal models (22, 46). Our data suggest that these channels are not important in the attenuated pulmonary vascular contractility in this rat model of acute pneumonia. Possible reasons for these differences include the type of insult, such as lipopolysaccharide-induced vascular hyporeactivity (9, 22, 46), compared with the depressed contractility that we observed in the inflammation associated with acute pneumonia. In addition, we cannot rule out species-dependent variations (4, 23, 52) and differences in the regulation of vascular tone among different vascular beds (6, 25, 48). Indeed, different distributions of KCa channels between central and peripheral arteries at either the endothelial or smooth muscle levels or both have been suggested (5).
KV currents have been described in human PA myocytes (27). In addition, Archer et al. (2) described electrophysiologically distinct myocytes isolated from conduit and resistance rat PAs, indicating differential distribution of KCa and KV channels along the vascular tree. This might explain why KV channels, but not other K+ channels, seem to play a role in the depressed contractility observed in pneumonia. In isolated, freshly dispersed smooth muscle cells from rabbit portal vein and coronary arteries, the activity of KV channels is enhanced by signal transduction mechanisms involving vasoactive agonists that activate cAMP-dependent protein kinase A or protein kinase C (1, 12). Further studies are needed to determine whether these signaling pathways play a role in our pneumonia model.
In this study, we showed that, in rings from pneumonia rats, 4-AP (a
selective KV blocker) reversed the attenuated contractile responses to KCl, PE, and PGF2, indicating a role for
the KV channel in the depressed PA contractility in acute
pneumonia. 4-AP fully restored the contractile response to KCl, whereas
it only partially restored the PE and PGF2 responses,
indicating the involvement of other factors or mechanisms in this
depressed contractility. Because PE and PGF2 are
receptor-dependent agonists, variation in restoration of contractility
to these agents with 4-AP treatment might reflect differences in
agonist-receptor interactions and signaling pathways between arteries
from control and pneumonia rats. In addition, our laboratory recently
demonstrated an attenuation in CYP metabolic activity of AA in lung
microsomes from rats with pneumonia (57). In pneumonia,
there is a reduction in the rate of formation of pulmonary EETs and
20-HETE, which are potent constrictors of PA rings in vitro
(57) and which have been suggested to act as intracellular
signaling molecules in vascular smooth muscle cells and endothelium
(24, 32). Our laboratory also demonstrated depressed
contractility to the CYP metabolites of AA, EETs, and 20-HETE in PA
rings from rats with pneumonia. NO mediates part of the observed
depressed PA contractility in pneumonia, although the mechanisms
underlying this phenomenon are not fully known (55, 57).
In many vascular beds, NO can activate KCa channels,
resulting in hyperpolarization of vascular smooth muscle cells, thus
affecting its contractility (16, 33). Although no known
receptors have yet been identified, both EETs and 20-HETE could exert
their contractile effects by modulating the function of K+
and Ca2+ channels in vascular smooth muscle cells and
modulating the effect of NO on these channels (21, 24, 36,
48). It remains to be established whether a link exists among
NO, EETs, 20-HETE, and KV channels in PAs of the rat.
In conclusion, our data reveal that KV channels, but not BKCa and KATP channels, contribute to the depressed PA contractility observed in this model of acute Pseudomonas pneumonia. This observation is important, because it might shed the light on better therapeutic strategies for the treatment of pneumonia.
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ACKNOWLEDGEMENTS |
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Appreciation is expressed to Viki Massey, Advanced Registered Technologist, and the Microbiology Staff at the London Health Sciences Centre for providing the Pseudomonas aeruginosa organisms. The authors thank Dr. Stephen Sims, Department of Physiology at the University of Western Ontario, for useful suggestions and for advice on which K+ channel blockers to use. The authors also thank Dr. Sims for useful discussion of the data and for providing guidance during the writing of this manuscript.
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FOOTNOTES |
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Grant support for this work was provided by the Canadian Institutes of Health Research. A scholarship from the Ontario Thoracic Society and an Ontario Graduate Scholarship in Science and Technology supported A. Yaghi.
Address for reprint requests and other correspondence: D. G. McCormack, London Health Sciences Centre, Victoria Hospital, 375 South St., London, Ontario, Canada N6A 4G5 (E-mail: dmccorm{at}uwo.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
June 7, 2002;10.1152/japplphysiol.01146.2001
Received 19 November 2001; accepted in final form 31 May 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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