© 2000 American Heart Association, Inc.
Vascular Biology |
Shear-Induced Increase in Hydraulic Conductivity in Endothelial Cells Is Mediated by a Nitric Oxide–Dependent Mechanism
From the Departments of Physiology (Y.S.C., J.A.Y.) and Chemical Engineering (S.L., J.M.T.), Biomolecular Transport Dynamics Laboratory, The Pennsylvania State University, University Park, Pa, and the Department of Bioengineering (J.A.F.), University of California, San Diego, La Jolla, Calif.
Correspondence to Dr John M. Tarbell, 155 Fenske Laboratory, Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802. E-mail jmt{at}psu.edu
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Abstract—This study addresses the role of nitric oxide (NO) and its downstream mechanism in mediating the shear-induced increase in hydraulic conductivity (Lp) of bovine aortic endothelial cell monolayers grown on porous polycarbonate filters. Direct exposure of endothelial monolayers to 20-dyne/cm2 shear stress induced a 4.70±0.20-fold increase in Lp at the end of 3 hours. Shear stress (20 dyne/cm2) also elicited a multiphasic NO production pattern in which a rapid initial production was followed by a less rapid, sustained production. In the absence of shear stress, an exogenous NO donor, S-nitroso-N-acetylpenicillamine, increased endothelial Lp 2.23±0.14-fold (100 µmol/L) and 4.8±0.66-fold (500 µmol/L) at the end of 3 hours. In separate experiments, bovine aortic endothelial cells exposed to NO synthase inhibitors, NG-monomethyl-L-arginine and NG-nitro-L-arginine methyl ester, exhibited significant attenuation of shear-induced increase in Lp in a dose-dependent manner. Inhibition of guanylate cyclase (GC) with LY-83,583 (1 µmol/L) or protein kinase G (PKG) with KT5823 (1 µmol/L) failed to attenuate the shear-induced increase in Lp. Furthermore, direct addition of a stable cGMP analogue, 8-bromo-cGMP, had no effect in altering baseline Lp, indicating that the GC/cGMP/PKG pathway is not involved in shear stress–NO–Lp response. Incubation with iodoacetate (IAA), a putative inhibitor of glycolysis, dose-dependently increased Lp. Addition of IAA at levels that did not affect baseline Lp greatly potentiated the response of Lp to 20-dyne/cm2 shear stress. Finally, both shear stress–induced and IAA-induced increases in Lp could be reversed with the addition of dibutyryl cAMP. However, additional metabolic inhibitors, 2 deoxyglucose (10 mmol/L) and oligomycin (1 µmol/L), or reactive oxygen species scavengers, deferoxamine (1 mmol/L) and ascorbate (10 mmol/L), failed to alter shear-induced increases in Lp. Our results show that neither the NO/cGMP/PKG pathway nor a metabolic pathway mediates the shear stress–Lp response. An alternate mechanism downstream from NO that is sensitive to IAA must mediate this response.
Key Words: shear stress • nitric oxide • hydraulic conductivity • endothelial cells
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Endothelial cells provide the principal barrier to transport of fluids and solutes between blood and underlying tissue. In arteries, shear-dependent permeability has been hypothesized to play a role in the localization of atherosclerotic lesions in regions of vessel branching and curvatures.1 The first clear demonstration of a direct effect of shear stress on endothelial permeability was reported by Jo et al.2 Other investigators3 4 5 have provided supporting evidence for flow- or shear-dependent endothelial solute permeability. More recently, Sill et al,6 using the same in vitro model as Jo et al, demonstrated that endothelial monolayer hydraulic conductivity (Lp) is also sensitive to acute changes in shear stress level. This finding is consistent with an earlier whole-artery study reported by Lever et al7 and with a study involving frog mesenteric venular capillary measurements performed by Williams and Huxley.4 The only insight into the cell-signaling mechanism underlying the shear stress response of Lp was provided by Sill et al, who demonstrated that the response could be blocked by addition of a cAMP analogue or a phosphodiesterase inhibitor, suggesting a role for cAMP in the pathway.
It is well established that shear stress stimulates the release of nitric oxide (NO) from cultured endothelial cells.8 9 Direct infusion of NO synthase (NOS) inhibitors into intact vessels alters endothelial cell transport properties from feline10 and rat11 mesenteric microvessels. Moreover, superfusion of an NO donor into frog mesenteric microvessels elevates capillary Lp,12 whereas superfusion with a NOS inhibitor reduces capillary Lp.13 Several studies that used intact vessels from a variety of species14 15 16 17 have also demonstrated that agonist-meditated alteration of transport properties can be attenuated by inhibiting NOS. Yuan et al5 have reported that flow modulates albumin permeability in isolated porcine coronary venules by a NO-related mechanism. These studies indicate that NO can alter endothelial transport properties in a variety of blood vessels.
It is widely believed that NO affects transport through a downstream pathway involving cGMP.18 Another downstream effect of NO that could impact the endothelial transport barriers is its alteration of cellular energy metabolism. For example, Salzman et al19 demonstrated that NO reduces the transepithelial resistance of cultured Caco-2BBe intestinal epithelial monolayers by a mechanism involving ATP depletion, not the alteration of cGMP. In endothelial cells, Padgett and Whorton20 21 have shown that NO inhibits a key glycolytic enzyme, GAPDH. However, to date, the role of NO as a mediator of shear-induced increase in endothelial monolayer Lp has not been evaluated. Therefore, the objective of the present study was to elucidate the role of NO and its downstream effectors in mediating the response of Lp to acute changes in shear stress.
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Chemicals
The following chemicals were obtained from Sigma Chemical Co: Hanks’ balanced salt solution, BSA (fraction V, 30% solution), minimal essential medium (MEM), FBS, glutamic acid, sodium bicarbonate, gelatin, fibronectin, penicillin G, streptomycin sulfate, NG-monomethyl-L-arginine (L-NMMA), NG-nitro-L-arginine methyl ester (L-NAME), ß-nicotinamide adenine dinucleotide phosphate, N-(1-naphthyl)ethylenediamine, flavin adenine dinucleotide, sulfanilamide, nitrate reductase, dibutyryl cAMP (DBcAMP), 8-bromo-cGMP, 2-deoxyglucose (DOG), oligomycin, iodoacetate (IAA), deferoxamine, and ascorbate. NG-Monomethyl-D-arginine (D-NMMA), LY-83,583, and KT5823 were purchased from Calbiochem. S-Nitroso-N-acetylpenicillamine (SNAP) was obtained from Research Biochemicals Intl. 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate–acetylated LDL was obtained from Biomedical Technologies. Polycarbonate filters (Transwell chambers, 0.4-µm pore size, 24.5-mm diameter) were obtained from Corning Costar. Trypsin was obtained from GIBCO-BRL. Finally, high vacuum grease was obtained from Dow Corning.
Cell Culture
Primary bovine aortic endothelial cells (BAECs)
were harvested from bovine thoracic aortas and subsequently maintained
in MEM–10% FBS as described previously.22 Cells were
plated at a density of 2.5x105
cells/cm2 on polycarbonate membrane Transwell
filters pretreated with gelatin (5 mg/L, type A from porcine skin) and
fibronectin (30 µg/mL). Cells between passages 6 and 12 were used in
experiments.
Experimental Protocol
Measurement of Water Flux Under Shear Stress
A detailed description of the experimental apparatus
used to measure water flux under shear stress has been
presented by Sill et al.6 Briefly, a polycarbonate
membrane Transwell filter containing the
endothelial monolayer was sealed between 2 pieces of
polycarbonate assembly separating the luminal and abluminal
compartments. The compartments were continuously provided with
positive-pressure gassing of 5% CO2/95%
balanced air to maintain the pH of the medium. The abluminal chamber
was connected to a reservoir via Tygon tubing and borosilicate glass
tubing. The reservoir could be lowered to a desired height to create
the hydrostatic pressure gradient required to drive water flux across
the cell monolayer. To eliminate the oncotic pressure gradient, the
same medium (MEM–1% BSA) was added to both the luminal and the
abluminal compartments. The luminal compartment contained a cylindrical
disk, which was rotated by a motor drive to produce a defined shear
stress that varied linearly from zero at the center to a maximum value
at the edge (mean value was two thirds of the maximum value). All
subsequent values of shear stress will be given as the maximum shear
value. It should be noted, however, that if the dependence of
Lp on shear stress is nonlinear, the spatial
variation of shear stress in the apparatus will provide
some distortion in the measured shear dependence based on the maximum
(or mean) value.
To measure fluid flux across the monolayer, a bubble was inserted into
the borosilicate glass tubing and tracked with a spectrophotometer
mounted on a screw rod, which was driven by a stepper motor. This
traveling spectrophotometer was interfaced to a computer, and the
bubble position was displayed as a function of time on the computer
screen. The bubble displacement was then converted to fluid volume flux
(Jv) through a calibration equation.
Lp could be calculated by the following equation:
Lp=Jv/S · 
P,
where S is the surface area of the monolayer, and P is the
hydrostatic pressure differential across the monolayer (10
cm H2O). There may have been a slight excess of
protein near the luminal surface because of concentration polarization
driven by the volume flux, but this is expected to have a negligible
oncotic effect at a protein concentration of 1%.7
Response of Lp to SNAP
Experiments were conducted in the presence of an exogenous NO
donor, SNAP, at 100 and 500 µmol/L without shear stress. After
establishment of baseline Lp for 1 hour, SNAP was
added to the luminal compartment, and Lp was
measured for an additional 3 hours.
Nitrite/Nitrate Determination
Endothelial monolayers grown to confluence on
polycarbonate filters in Transwell chambers were rinsed twice with
MEM–1% BSA. Then 2 mL of MEM–1% BSA was pipetted into the luminal
side of the chamber, and the filter, while still attached to the
Transwell chamber, was mounted onto a glass slide and sealed with
high vacuum grease. Finally, the monolayer was exposed to defined shear
stress (20 dyne/cm2) with or without the NOS
inhibitor, L-NMMA, by using the rotating disk
apparatus. Cell perfusate samples (500 µL) were
taken and replaced with fresh experimental media at 0, 5, 30, 60, 120,
and 180 minutes after addition of shear stress. The concentrations of
the stable products, NO2-
and NO32-, were determined as
described previously.8
Effect of NOS Inhibitors on Shear-Induced Increase
in Lp
In separate experiments, the endothelial cell
monolayers were preincubated for 1 hour with L-NMMA (10, 50, and
100 µmol/L), L-NAME (100 µmol/L), or D-NMMA (100
µmol/L), then exposed to a pressure differential of 10
cm H2O without shear stress for 1 hour to
establish the baseline Lp, and subsequently
subjected to 20-dyne/cm2 shear stress for 3
hours. Lp was measured continuously during the
1-hour preshear period and the 3-hour shear period.
Mechanism Downstream From NO: cGMP/PKG Pathway
Endothelial cell monolayers were exposed to
20-dyne/cm2 shear stress in the presence of
inhibitors of guanylate cyclase (GC) and
protein kinase G (PKG). In separate experiments, monolayers were
incubated with LY-83,583 (10 µmol/L, a selective GC
inhibitor) or KT5823 (1 µmol/L, a PKG
inhibitor) for 30 minutes before the addition of shear
stress. In addition, the response of Lp after
direct exposure to the stable cGMP analogue, 8-bromo-cGMP, was
examined. After the establishment of baseline Lp,
8-bromo-cGMP (1 mmol/L) was added onto the monolayer, and
Lp was measured for 3 hours.
Mechanism Downstream From NO: Metabolic Pathway
First, either IAA (10 µmol/L, a putative GAPDH
inhibitor) or DOG (10 mmol/L, a selective
inhibitor of glycolysis) was added just before the onset of
20-dyne/cm2 shear stress.
Endothelial monolayer Lp was then
measured for 3 hours. In similar sets of experiments, monolayers were
preincubated with DOG (10 mmol/L) and/or oligomycin (1
µmol/L, an inhibitor of mitochondrial ATP synthesis) in a
glucose-free medium before the addition of
20-dyne/cm2 shear stress.
Because peroxynitrite (OONO-) can inhibit metabolic and other enzymes, experiments were conducted in which monolayers were preincubated with scavengers of reactive oxygen species, deferoxamine (1 mmol/L) or ascorbate (10 mmol/L), for 30 minutes before the addition of 20-dyne/cm2 shear stress. Endothelial monolayer Lp was then measured for 3 hours.
Data Presentation and Statistical Analysis
As described previously,6 on application of the
10-cm H2O pressure head, there was an initial
decrease in Lp, which stabilized over a period of
30 to 50 minutes. Therefore, a 1-hour period was allotted to establish
a baseline Lp before further intervention.
Experiments with baseline Lp values
<5.0x10-7 cm ·
s-1 ·
cm H2O-1 were used for
evaluation. About 10% of the experiments were rejected because of
elevated baseline values associated with incomplete monolayer
formation.
Five-minute average Lp values were calculated, normalized with respect to the established baseline Lp, and presented as mean±SEM. Significant differences between group means were performed every 30 minutes after establishment of baseline Lp and analyzed by a 2-way (time and treatment) ANOVA with the use of statistical analysis software (SAS). Time was the repeated factor, and planned pairwise comparisons were performed with the Bonferroni correction. A level of P<0.05 was considered significant for the statistical analysis.
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Results |
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Figure 1
illustrates the basic
response of cultured bovine aortic endothelial
monolayer Lp to a
physiological shear stress of 20
dyne/cm2. As reported previously in our
laboratory,6 shear stress induced a significant increase
in Lp: 4.70±0.20-fold after 3 hours of shear
stress.
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Shear stress of 20 dyne/cm2 alone (n=5) elicited
a time-dependent increase in nitrite/nitrate
(NOx) production (Figure 2). Within 5 minutes of the onset of
shear, there was a significant (P<0.02) and dramatic
increase in NOx concentration to a level of
17.8±2.1 nmol/mg protein (compared with 8.7±0.5 nmol/mg protein at
time 0). This burst was followed by a less rapid, sustained
production for the next 55 minutes. Between 60 and 120 minutes,
there was an acceleration of production, albeit at a lower rate
than observed for the first 5 minutes, followed by a final phase (120
to 180 minutes) in which production was less vigorous. At the
end of 3 hours of shear stress, NOx rose to
85.7±19.1 nmol/mg protein, whereas stationary controls (n=8) produced
only 18.4±3.7 nmol/mg protein (4.8±1.3 nmol/mg protein at time 0).
Similar to stationary controls, NOx produced by
monolayers exposed to L-NMMA alone (n=4) increased to only 11.3±3.3
nmol/mg protein (3.4±0.3 nmol/mg protein at time 0) for the same time
period, which did not prove to be significantly different from control
values (P>0.40). This indicates that L-NMMA had no
significant effect on basal production of
NOx. As expected, the shear-induced increase in
NOx could be blocked by the addition of L-NMMA.
At the end of 3 hours, cumulative NOx
concentration in the presence of 20-dyne/cm2
shear stress and L-NMMA (n=4) was 28.42±2.44 nmol/mg protein (6.8±1.4
nmol/mg protein at time 0), which was not significantly different from
control values (P>0.25). It was also not significantly
different from L-NMMA alone (P>0.09).
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As shown in Figure 3, 100 µmol/L
SNAP (n=6) significantly increased endothelial
monolayer Lp by 2.23±0.14-fold
(P<0.01) 3 hours after the addition of this pharmacological
agent. Moreover, 500 µmol/L SNAP elicited an even greater
increase in Lp after 3 hours
(4.8±0.66-fold).
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Figure 4 depicts the direct effect of the
NOS inhibitor L-NMMA in the absence of shear. L-NMMA at
10 µmol/L increased Lp by 1.36±0.19-fold
after 3 hours, whereas L-NMMA at 50 µmol/L increased
Lp by 1.90±0.19-fold after 3 hours. Neither
response was significantly different from control values
(P>0.15). However, for 100 µmol/L L-NMMA,
Lp increased by 2.71±0.33-fold after 3 hours,
and this was significantly greater than control values
(P<0.002).
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The marked increase of Lp in response to shear
stress (Figure 1
) was highly attenuated (P<0.001)
when endothelial cells were treated with 50 or 100
µmol/L L-NMMA (Figure 5). The response
to 20-dyne/cm2 shear stress in the presence of
100 µmol/L L-NMMA was an increase in Lp of
only 2.40±0.32-fold at 3 hours, and in the presence of 50
µmol/L L-NMMA, the response was an increase in
Lp of only 2.30±0.22-fold after 3 hours. At
10 µmol/L, L-NMMA did not significantly alter the shear-induced
increase in Lp, which was 3.87±0.54-fold after 3
hours (P>0.20).
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To determine whether attenuation of the shear response of Lp was in fact due to NOS inhibition and not a side effect of the drug, another set of experiments (n=4) was performed with the use of an inert enantiomer, D-NMMA (100 µmol/L). The baseline Lp in the presence of D-NMMA and 20-dyne/cm2 shear stress was 4.07±0.36x10-7 cm · s-1 · cm H2O-1. D-NMMA, unlike L-NMMA, did not attenuate the shear-induced increase in Lp. At the end of 3 hours of 20-dyne/cm2 shear stress plus D-NMMA, Lp increased by 4.01±0.45-fold, which was not significantly different from the response to 20-dyne/cm2 shear stress alone (P>0.20).
To further confirm the role of NOS in the shear stress response of Lp, we examined the effect of another NOS inhibitor, L-NAME (100 µmol/L), in the presence of 20-dyne/cm2 shear stress. Similar to 100 µmol/L L-NMMA, 100 µmol/L L-NAME (baseline Lp 4.71±0.19x10-7 cm · s-1 · cm H2O-1, n=5) highly attenuated the shear-induced increase in Lp. At the end of the third hour, normalized Lp was elevated only 2.25±0.12-fold, which was significantly lower than Lp in response to 20-dyne/cm2 shear stress alone (P<0.001).
To examine the role of the GC/cGMP/PKG pathway, endothelial cell monolayers were exposed to 20-dyne/cm2 shear stress in the presence of a GC inhibitor, LY-83,583 (1 µmol/L, Ki 2 µmol/L). The inhibitor did not significantly alter the shear-induced increase in Lp. At the end of 3 hours, 20-dyne/cm2 shear stress in the presence of LY-83,583 (baseline Lp 2.94±0.33x10-7 cm · s-1 · cm H2O-1, n=4) induced an increase in endothelial Lp of 5.27±0.47-fold, which was not significantly different from the shear-induced increase in the absence of the inhibitor (P>0.15). Consistent with the LY-83,583 data, addition of a cell-permeant stable analogue of cGMP, 8-bromo-cGMP, had no significant effect (P>0.50) in altering baseline Lp (2.96±0.63x10-7 cm · s-1 · cm H2O-1, n=6). At the end of 3 hours of exposure, 8-bromo-cGMP (1 mmol/L) increased Lp by 1.58±0.39-fold, whereas stationary control increased Lp by 1.46±0.17-fold in the same time period. Similarly, the PKG inhibitor KT5823 (1 µmol/L, Ki 234 nmol/L) had no significant effect in altering the shear-induced increase in Lp. At the end of 3 hours, 20-dyne/cm2 shear stress in the presence of KT5823 (baseline Lp 2.58±0.68x10-7 cm · s-1 · cm H2O-1, n=5) led to an increase in Lp of 4.11±0.44-fold, which was not significantly different from the response in the absence of the inhibitor (P>0.25). These data indicate that the GC/cGMP/PKG pathway does not play a significant role downstream from NO in BAECs.
Another pathway that is known to be affected (inhibited) by NO in BAECs
is glycolysis, through the enzyme GAPDH.21 It is clear in
Figure 6 that use of the putative
glycolysis inhibitor IAA led to a dose-dependent increase
in endothelial Lp. At the lowest
dose (10 µmol/L), IAA did not increase Lp
above control levels after 3 hours of exposure (P>0.10).
IAA (30 µmol/L) was required to elicit a significant increase in
Lp at the end of 3 hours (3.31±0.11-fold,
P<0.01), which was comparable to that induced by shear
stress. This IAA-induced increase in Lp could be
reversed by addition of the cell-permeant stable analogue of cAMP,
DBcAMP (1 mmol/L). Within 30 minutes of the addition of DBcAMP,
Lp returned to original baseline levels. A
similar return of Lp to baseline within 30
minutes of the addition of DBcAMP (1 mmol/L) was reported by Sill
et al6 after stimulating an increase in BAEC
Lp with shear stress. As much as an 18-fold
increase in Lp after 3 hours was induced by
100 µmol/L IAA (P<0.001, data not shown).
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Having established that IAA causes an increase in
Lp, we next examined whether the effects of shear
stress and IAA were interactive. To test this hypothesis, we added IAA
at a concentration that did not affect baseline
Lp (10 µmol/L) along with
20-dyne/cm2 shear stress and then measured
Lp for 3 hours (Figure 7). Compared with
20-dyne/cm2 shear stress alone,
20-dyne/cm2 shear stress in the presence of IAA
elicited a greatly potentiated response (8.25±0.88-fold increase in
Lp after 3 hours, P<0.001).
Furthermore, this enhanced response could be reversed nearly to
baseline within 30 minutes of adding DBcAMP (1 mmol/L).
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These data suggested that inhibition of glycolysis might be the pathway downstream from NO that leads to an increase in Lp. To further test this hypothesis, experiments were conducted with a more specific inhibitor of glycolysis, DOG (10 mmol/L),23 24 or an inhibitor of mitochondrial ATP synthesis, oligomycin (1 µmol/L),25 in glucose-free medium. First, compared with normal experimental medium (MEM–1% BSA) in which endothelial Lp increased 1.46±0.17-fold at the end of 3 hours, Lp in a glucose-free medium (DMEM–glucose-free 1% BSA) increased 1.47±0.13-fold (P>0.50). Second, unlike IAA, neither DOG nor oligomycin significantly altered the 20-dyne/cm2 shear stress–induced increase in Lp: at the end of 3 hours, BAEC Lp increased 5.21±0.27-fold (P>0.25) and 4.84±0.45-fold (P>0.40) for DOG and oligomycin, respectively. Finally, another pathway by which NO can inhibit ATP synthesis is via OONO-. The addition of scavengers of reactive oxygen species, deferoxamine (1 mmol/L)26 27 or ascorbate (10 mmol/L),28 failed to significantly alter the 20-dyne/cm2 shear stress–induced increase in Lp. At the end of 3 hours, Lp increased 4.80±0.81-fold (P>0.30) and 5.49±0.66-fold (P>0.25) for deferoxamine and ascorbate, respectively.
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The present study addresses the role of NO in mediating the shear-induced increase in Lp of BAEC monolayers. We observed that an exogenous NO donor, SNAP, increased endothelial Lp in a dose-dependent manner and that the response of Lp to 500 µmol/L SNAP was comparable in magnitude to that elicited by 20-dyne/cm2 shear stress, although the dynamics of the responses were different (Figures 1
and 3). The differences in dynamics of the Lp
response to SNAP and shear stress were probably the result of differing
rates of NO release associated with the 2 mechanisms. We also
demonstrated that shear stress of 20 dyne/cm2
caused a multiphasic NO release that was greatly attenuated by the NOS
inhibitor L-NMMA (Figure 2). These results are
similar to those in human umbilical vein endothelial
cells reported by Kuchan and Frangos.8
Another important finding was that NOS inhibitors blocked
the shear-induced increase in Lp. When Figures 4 and 5 are compared, it is apparent that after 3 hours,
the increase in Lp induced by
20-dyne/cm2 shear stress in the presence of
100 µmol/L L-NMMA is not significantly different from the
increase in Lp in response to 100 µmol/L
L-NMMA alone (P>0.50). Therefore, the attenuation observed
in response to 100 µmol/L L-NMMA is a total inhibition of the
shear-induced response. Even at 50 µmol/L L-NMMA, there is total
inhibition of the shear-induced response (P>0.20). These
data support the central role of NO in mediating shear-induced
increases in endothelial monolayer
Lp. It should also be noted that Ranjan et
al29 showed that in BAECs, shear stress alters
constitutive NOS within 3 hours but has no effect on inducible NOS
levels. This suggests that constitutive NOS has been inhibited in our
experiments.
Our results are consistent with the observations of Meyer and Huxley,12 who superfused the NO donor sodium nitroprusside into frog mesenteric venular capillaries and recorded 2.6-fold elevation in Lp. Subsequently, Rumbaut et al,13 using a similar experimental protocol, demonstrated that microvascular Lp decreases when vessels are superfused with the NOS inhibitor L-NMMA. In related experiments dealing with solute transport, Yuan et al5 measured the permeability coefficient of albumin in isolated porcine coronary venules at various intraluminal perfusion velocities and observed a 48% increase in permeability when velocity increased from 7 to 13 mm/s. The flow-induced permeability change was completely abolished by addition of L-NMMA. Investigators who examined the effect of NOS inhibition on agonist-meditated endothelial transport properties also observed significant attenuation of agonist-induced increases in permeability. Hughes et al14 showed that L-NMMA and L-NAME inhibited substance P–induced edema formation in the dorsal skin of male Wistar rats. Mayhan15 also demonstrated in the hamster cheek pouch that L-NMMA significantly decreased the formation of histamine-induced leaky sites. Similarly, Noel et al16 reported that inhibition of NOS with L-NMMA attenuated the platelet activating factor–induced and histamine-induced increase in permeability of FITC-dextran in the hamster cheek pouch.
The studies cited above indicate that NO increases
endothelial transport rates, but there are studies that
exhibit an opposite action of NO. For example, Oliver30
demonstrated that the bradykinin-induced increase in sucrose
permeability was enhanced when endothelial cells were
exposed to NOS inhibitors. Kubes10 and Kurose
et al11 also showed an enhancement of albumin
permeability when vessels were exposed to L-NAME in feline small
intestinal microvessels and in mesenteric venules of male
Sprague-Dawley rats, respectively. Even in our own system, opposing
results were observed. L-NMMA (100 µmol/L), which was expected
to have an action opposite that of SNAP, actually elicited an increase
in Lp (Figure 4), albeit of lesser
magnitude than that induced by SNAP (Figure 3). When shear
stress was present to stimulate higher levels of NO, the addition
of L-NMMA (Figure 5) attenuated Lp. Thus,
the effects of NO on endothelial transport properties
are somewhat controversial. They appear to depend on the specific
species and vascular origin of the endothelium under
consideration and possibly the level of NO concentration within the
cells.
Although we have demonstrated that NO mediates shear-induced increases in endothelial Lp, the mechanism downstream from NO is of great interest. It has been well established that NO stimulates soluble GC, which elevates the level of cGMP.31 Meyer and Huxley12 have shown that frog mesenteric capillary Lp is elevated by a cGMP-dependent mechanism. Studies performed by MacMillan-Crow et al32 indicate that BAECs possess a cGMP-dependent protein kinase that is partially associated with the cytoskeleton. Because it is believed by many that endothelial cells undergo cytoskeletal rearrangement to alter their transport properties, these lines of evidence suggest a potential pathway for shear-induced increase in Lp; ie, shear induces elevation of NO/cGMP/PKG, leading to rearrangement of the cytoskeleton and alteration of endothelial Lp. Another possibility was suggested by van Hinsbergh,18 who pointed out that in vitro studies that used human endothelial cells show that cGMP affects endothelial permeability directly (by activating PKG) and indirectly (by activating cAMP-degrading phosphodiesterases). Previous studies have shown that the intracellular level of cAMP is critical in maintaining endothelial barrier properties.33 34 By using both a PKG inhibitor and a GC inhibitor, we were able to test for the relevance of these 2 downstream mechanisms in the overall shear stress–Lp response.
Our data show that inhibition of PKG with KT5823 does not attenuate the
shear-induced increase in Lp after 3 hours,
indicating that the cGMP/PKG pathway is not involved. Furthermore,
direct exposure to a stable analogue of cGMP, 8-bromo-cGMP (at a
relatively high concentration), had no effect on baseline
Lp, and inhibition of GC with LY-83,583 did not
alter the shear stress–Lp response, confirming
that the cGMP/phosphodiesterase pathway is also inoperative. These
findings are consistent with a recent study by Gooch et
al,35 who reported that BAECs (received from our
laboratory) actually release very low basal levels of cGMP. Moreover,
stimulation of these cells with shear stress of 22
dyne/cm2 or with spermine–NO complex
(30 µmol/L) had no effect in altering the production of
cGMP. In contrast, the endothelial cells derived from
human umbilical veins released substantially higher (
20-fold) basal
levels of cGMP, and this level could be increased 20-fold with the
addition of 5 µmol/L spermine–NO complex and 2.5-fold with
application of 22-dyne/cm2 shear
stress.36 Taken together, our studies as well as the
findings of Gooch et al provide strong evidence that the NO/GC/cGMP
pathway does not play a significant role in the shear
stress–Lp response in BAECs. An alternate
mechanism downstream from NO must mediate this response.
Previously, we have shown that the shear-induced increase in BAEC
Lp can be highly attenuated by elevating the
level of cAMP through exogenous (DBcAMP) and endogenous
(3-isobutyl-1-methylxanthine) means.6 The effects of cAMP
analogues in preventing permeability increases in response to a variety
of chemical agonists have been reported previously.33 34 A
novel pathway linking NO and cAMP was suggested by recent studies
showing that NO leads to reversible inhibition of a key glycolytic
enzyme, GAPDH, via S-nitrosylation of the active site
cysteine residue in intact bovine pulmonary artery
endothelial cells20 and
BAECs.21 Because endothelial cells rely
primarily on glycolysis for the production of
ATP,37 inhibition of glycolysis leads to a reduction in
the intracellular concentration of ATP and, thus, cAMP. To test for the
role of glycolytic activity in the shear
stress–Lp response, we used a putative
inhibitor of glycolysis, IAA. Our results (Figure 6)
show that addition of IAA leads to a dose-dependent increase in
endothelial Lp. Moreover, we
observed that in the presence of 10 µmol/L IAA (a concentration
at which the baseline Lp was not altered), the
effect of 20-dyne/cm2 shear stress on
endothelial Lp was greatly
potentiated (Figure 7) and that this potentiated response could
be reversed by the addition of DBcAMP. Thus, even though IAA by itself
did not significantly alter endothelial
Lp, it had a synergistic effect in enhancing the
shear-induced increase. This strongly suggests that shear stress and
IAA mediate their effects on Lp through the same
pathway, potentially via inhibition of glycolysis.
It is important to note, however, that IAA is a potent acetylating agent that can nonselectively inhibit susceptible sulfhydryl groups within a cell.38 To further explore the metabolic pathway, more selective inhibitors were incorporated. DOG (a selective inhibitor of glycolysis) and oligomycin (an inhibitor of the citric acid cycle) failed to alter the shear stress–Lp response, indicating that the metabolic pathway may not mediate the physiological response.
Peroxynitrite represents another intermediate species through which NO can inhibit ATP synthesis. NO forms OONO- by reacting with a superoxide anion radical (O2· -),39 and OONO- has been shown to inhibit ATP synthesis by inhibiting aconitase, the rate-limiting enzyme in the citric acid cycle.40 However, consistent with the results obtained with DOG and oligomycin, scavengers of reactive oxygen species (deferoxamine or ascorbate) also failed to alter the shear-induced increase in Lp. Taken together, these data confirm that the metabolic pathway does not play a role in mediating the shear stress—NO–Lp response.
Our findings are consistent with those of Bolin et al,38 who demonstrated that the IAA-induced increase in vascular permeability of isolated rabbit lung was independent of ATP synthesis. Similarly, Wilson et al24 showed that low glucose and DOG, as well as DOG plus antimycin-A (a metabolic inhibitor), can greatly inhibit ATP but not affect albumin or dextran permeability in pulmonary arterial endothelial cells and that H2O2 will also inhibit ATP but does increase albumin permeability.
In conclusion, the present study has shown that NO is a key signaling molecule mediating the shear stress–Lp response. In addition, we have demonstrated that neither the GC/cGMP/PKG pathway nor the metabolic pathway plays a prominent role in mediating the shear stress–Lp response. We do, however, show that endothelial Lp is sensitive to inhibition by IAA. Moreover, shear stress and IAA may mediate their effects on Lp through the same pathway. The exact nature of this downstream pathway remains to be explored but may involve a direct inhibition of adenylate cyclase, as has been suggested recently.41
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Acknowledgments |
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This study was supported by National Institutes of Health (NIH) training grant T32-GM-08619-01, NIH grant HL-57093, NIH grant HL-35549, and NASA grant NAG3-1871. The authors would like to thank Dr Keith Gooch for his invaluable assistance and comments and Dr Esther Brooks-Asplund for her expertise in statistical analysis.
Received June 21, 1998; accepted August 12, 1999.
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