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Departments of Surgery and Medical Physiology, Texas A & M University Health Science Center, Temple, Texas 76504
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our previous studies have shown that inflammatory mediators increase microvascular permeability through a phospholipase C-nitric oxide synthase (NOS)-guanylate cyclase cascade. The aim of this study is to delineate in more detail the signaling pathway leading to microvascular hyperpermeability. Endothelial cytosolic calcium and the apparent permeability coefficient of albumin (Pa) were measured in isolated and perfused coronary venules. Histamine stimulated a rapid increase in cytosolic calcium followed by a transient elevation in Pa. The NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) and the guanosine 3',5'-cyclic monophosphate-dependent protein kinase G (PKG) inhibitor KT-5823 abolished the hyperpermeability but did not affect the calcium response to histamine. Similarly, the calcium ionophore ionomycin produced a calcium spike preceding venular hyperpermeability. Blockage of the NOS-PKG cascade inhibited the increase in Pa, whereas the endothelial calcium was still elevated on administration of ionomycin. Furthermore, the relationship between protein kinase C (PKC) and the calcium-NOS-PKG pathway in modulation of venular permeability was investigated. Stimulation of PKC with phorbol 12-myristate 13-acetate (PMA) dramatically increased basal Pa without significantly changing the cytosolic calcium level. The selective PKC inhibitor bisindolylmaleimide abolished the effect of PMA but did not alter the effect of histamines on Pa. In contrast, both L-NMMA and KT-5823 were able to greatly attenuate the increase in Pa caused by PMA. These results suggest that 1) elevation of endothelial cytosolic calcium is an early signaling event preceding nitric oxide (NO) synthesis in the transduction of endothelial hyperpermeability, and 2) activation of PKC may alter the endothelial barrier function partially through the modulation of NO production.
nitric oxide; protein kinase C; protein kinase G; cytosolic calcium; endothelium
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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INFLAMMATORY CYTOKINES increase microvascular permeability by binding to their cognate receptors, leading to activation of a series of second messengers in the endothelial cell (12, 14). Different intracellular signaling processes have been proposed, and considerable evidence suggests the role of endothelial cytosolic calcium and protein kinases in the control of endothelial barrier function (14). The calcium theory was supported by studies demonstrating that calcium is required in the maintenance of endothelial integrity (30) and that calcium ionophores facilitate transendothelial flux of water and proteins (7, 27). Histamine, a typical endogenous mediator that produces leaky sites in postcapillary venules during inflammation, has been shown to promote albumin diffusion by stimulating calcium influx (27). The production of inositol phosphates via phospholipase C (PLC) has been considered as an initial event responsible for calcium mobilization (5, 14), yet the specific site of action of calcium has not been identified. In this regard, calcium may modulate the endothelial barrier property by directly acting on structural proteins or indirectly through other second messengers. Within this context, a potential target of elevated intracellular calcium is constitutive nitric oxide synthase (NOS) in the endothelial cell, because calcium is a potent stimulator of the enzyme (28). Indeed, evidence is accumulating that activation of NOS and subsequent production of nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) plays an important role in upregulation of the transport process by which fluid and macromolecules move across the vascular endothelium (1, 16, 21, 23, 26, 32). However, the stimulating effect of calcium on NOS and the regulatory role of NOS in the barrier function have been studied as separate processes, and evidence is limited regarding the sequential linkage between calcium and NO in the signal transduction of microvascular permeability. Moreover, it is not clear whether the calcium-dependent NO-mediated pathway is involved in macromolecular transflux induced by agonists that are different from histamine, such as phorbol 12-myristate 13-acetate (PMA), which is known to exert a hyperpermeability effect through activation of protein kinase C (PKC; 13-15).
Based on the conventional concepts and our recent findings, we hypothesize that the mechanism of agonist-induced microvascular hyperpermeability involves two different pathways characterized by the activation of NOS-protein kinase G (PKG) and upregulation of PKC, respectively (Fig. 1). On one hand, receptor occupancy by histamine and similar agonists stimulates PLC to produce D-myo-inositol 1,4,5-trisphosphate, which causes an internal release and further influx of calcium, leading to upregulation of NOS and NO production. NO stimulates guanylate cyclase (GC) to produce cGMP, a potent activator of cGMP-dependent PKG. The NO-cGMP-PKG system may participate in the barrier regulatory process through modulation of the structure and function of proteins in the endothelial cytoskeleton, intercellular junction, and cell-matrix contact. On the other hand, activated PLC can catalyze, in parallel with the formation of D-myo-inositol 1,4,5-trisphosphate, the production of diacylglycerol (DAG), which in turn causes upregulation of PKC. PKC may alter endothelial permeability by directly acting on the endothelial structural proteins and/or by indirectly modifying the activity of the common signaling protein NOS. The interaction between the calcium-NOS-PKG and the PKC pathways may play a homogeneous role in modulation of microvascular permeability.
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We have previously tested, in part, the above hypothesis by examining the sequence of events from PLC activation to NO production and PKG upregulation during stimulation by various agonists and growth factors (32, 34, 36). This study was undertaken to delineate the signaling process in greater detail with respect to the linkage among calcium, PKC, and the NOS-PKG cascade. Specifically, the aims of this study were 1) to define the contribution and location of the calcium signal in the transduction of histamine-induced microvascular hyperpermeability and 2) to examine the effect of PKC on coronary venular permeability and its relationship with the NO pathway.
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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General Preparation
Pigs weighing 9-13 kg were anesthetized with pentobarbital sodium (25 mg/kg iv) and heparinized (250 U/kg iv). After a tracheotomy and intubation the animal was ventilated. A left thoracotomy was performed, and the heart was electrically fibrillated, excised, and placed in 4°C physiological saline. The coronary sinus was cannulated, and 5 ml of india ink-gelatin-physiological salt solution were infused to clearly define venular microvessels. This solution was prepared by adding 0.2 ml of india ink (Koh-I-Noor, Bloomsbury, NJ) and 0.35 g of porcine skin gelatin to 10 ml of warm physiological salt solution and was filtered through P8 filter paper (Fisher Scientific, Pittsburgh, PA). A recent study (10) has demonstrated that the perfusion of the ink-gelatin mixture increases the basal permeability of coronary arterioles from 5.7 ± 2.3 × 10
7 cm/s to 8.7 ± 3.3 × 107 cm/s,
raising the possibility that the venules exposed to the ink-gelatin
solution during isolation were in a state of increased basal
permeability. In this regard, the hyperpermeability
effects of the agonists reported in this study might be underestimated. However, a large body of evidence supports the argument that the barrier function of the isolated venules is not seriously damaged by
the ink-gelatin solution. Information regarding the validation and
limitation of the technique has been provided in detail in our previous
publications (33).
Solutions and Perfusates
An albumin-physiological salt solution (APSS) was used as a bathing solution while the microvessels were being dissected. It contained the following (in mM): 145.0 NaCl, 4.7 KCl, 2.0 CaCl2, 1.17 MgSO4, 1.2 NaH2PO4, 5.0 glucose, 2.0 pyruvate, 0.02 EDTA, and 3.0 3-(N-morpholino)propanesulfonic acid buffer. After 1% bovine serum albumin was added, the solution was buffered to a pH of 7.40 at 4°C and then filtered through a Millex-PF 0.8-µm filter unit (Millipore, Bedford, MA). The APSS used to perfuse the vessels had the same composition as mentioned previously but was buffered to a pH of 7.40 at 37°C. For the cytosolic calcium measurement, the fura 2-acetoxymethyl ester (AM) solution was prepared fresh daily at a concentration of 1 µM in APSS containing 0.1% dimethyl sulfoxide (DMSO). Before use the solution was agitated and filtered through an 0.8-µm pore filter.Isolated and Perfused Microvessel Preparation
The methods for isolating and cannulating coronary venules have been described in detail in our previous study (33). Briefly, a suitable venule (length 0.8-1.2 mm, diameter 20-60 µm) was dissected from surrounding myocardium in the dissecting chamber containing APSS at 4°C with the aid of a Zeiss stereo dissecting microscope. The vessel was transferred to a cannulating chamber, which was mounted on a Zeiss axiovert microscope. The isolated vessel was cannulated with a micropipette on each end and secured with 11-0 suture (Alcon, Fort Worth, TX). A third smaller pipette was inserted into the inflow pipette. The vessel was perfused with either APSS through the outer inflow pipette or APSS containing fluorescein isothiocyanate-albumin through the inner inflow pipette. Each cannulating micropipette was connected to a reservoir so that the vessel was perfused at a relatively constant intraluminal pressure and flow rate. The bath solution in the chamber was maintained at 37°C and pH 7.4 throughout the experiments. The image of the vessel was projected onto a Hamamatsu charge-coupled device intensified camera and was displayed on a high-resolution monochromatic video monitor and recorded onto a VHS video recorder. Diameter of the vessel was measured on-line with a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX).Measurement of Venular Permeability
The permeability of the vessel was measured with a fluorescence ratio technique (9). With an optical window of a video photometer positioned over the venules and adjacent space on the monitor, the fluorescent intensity from the window was measured and digitized on-line by a Power Macintosh computer. In each measurement the isolated venule was first perfused with APSS through the outer inflow pipette to establish a baseline intensity. The venular lumen was then rapidly filled with fluorochromes by switching the perfusion to the inner inflow pipette. This produced an initial step increase, followed by a gradual increase, in the intensity of fluorescence. There was a step decrease of intensity when the fluorescently labeled molecules were washed out of the vessel lumen by switching the perfusion back to the outer inflow pipette. The apparent solute permeability coefficient of albumin (Pa) was calculated using the equation Pa = (1/
If)
(dIf/dt)o
(r/2), where, If is the initial step increase
in fluorescent intensity,
(dIf/dt)o is the initial rate of gradual increase in intensity as solutes diffuse
out of the vessel into the extravascular space, and
r is the venular radius.
Measurement of Endothelial Cytosolic Calcium in Isolated Venules
The changes in the endothelial cytosolic calcium were measured with a fluorescence ratio technique with the aid of a microscope photometry system (Photon Technology), which consisted of a PowerFilter high-speed dual-wavelength illuminator, a high-grade quartz fiber optic bundle, a D-104B single channel microscope photometer, and a 710 photon-counting photomultiplier tube (PMT). The cell-permeable form of a fluorescent dye, fura 2-acetoxymethyl ester (fura 2-AM), was used as the calcium indicator. To load the endothelium with the indicator, the porcine venule was cannulated as previously described and perfused for 20 min at a pressure gradient of 20 cmH2O through the inner inflow pipette with APSS containing 1 µM fura 2-AM and 0.1% DMSO. After loading was completed, the dye remaining in the extracellular space was washed off by switching the perfusion to the outer inflow pipette containing APSS for 20 min. The loading and washing procedures were performed in the dark at room temperature. The cannulated vessel was then aligned on the optical axis of a Zeiss axiovert microscope, which was connected to the PMT equipped with the photometry system. Emission fluorescence at 510 nm during excitation at 340 and 380 nm was detected by the PMT photon counting system through the measuring window positioned over the venule and recorded with FeliX, computer software associated with the photometer system. In each experiment, the excitation wavelength alternated between 340 and 380 nm at a rate of 650/s, and the fluorescence intensities at both wavelengths were recorded for 10 min at a 5-s interval. Background correction was automatically controlled by the program through subtraction of the background values from the measured values in real time. Because the wall of isolated venules mainly consisted of endothelial cells, the contribution of other cells to the fluorescence signals was small and therefore ignored. The ratio of fluorescent intensity at 340 vs. 380 nm was calculated with the computer program and presented as an index of the intracellular calcium level. To obtain the absolute concentration of calcium as a function of the 340/380 nm fluorescence ratio, the standard calibration experiment was conducted as previously described (7, 20). However, because the conclusion drawn from the study does not critically depend on the absolute value of intracellular calcium concentrations, the data are primarily represented as the fluorescence ratio.Chemicals and Drugs
The selective PKC inhibitor bisindolylmaleimide (BIM), the specific PKG inhibitor KT-5823, and the calcium ionophore ionomycin were ordered from Calbiochem (San Diego, CA). PMA, NG-monomethyl-L-arginine (L-NMMA), NG-monomethyl-D-arginine (D-NMMA), and 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) were also from Calbiochem. The chemicals used to make the perfusate, including fluorescein isothiocyanate-albumin, were purchased from Sigma Chemical (St. Louis, MO). Histamine and sodium nitroprusside (SNP) were also purchased from Sigma. Bovine serum albumin was obtained from United States Biochemical (Cleveland, OH). The calcium indicator fura 2-AM was from Molecular Probes (Eugene, OR). Ionomycin, KT-5823, and BIM were diluted with 0.1% DMSO in physiological salt solution.Experimental Protocol
In each experiment the cannulated venule was perfused at a constant perfusion pressure gradient of 20 cmH2O. According to our previous investigations (35, 36), this approach produced an approximate intraluminal pressure of 10 cmH2O and a flow velocity of 7 mm/s. The preparation was equilibrated for 45-60 min after cannulation, and the measurements were conducted under a temperature of 36-37°C and a pH of 7.35-7.45. In each vessel, limited (4-6) interventions were applied. The preparations were washed three times and allowed to equilibrate for 10-15 min between interventions. In some vessels the permeability and the calcium response were monitored over 4 h to ensure that the function of the venular endothelium was not significantly altered with time. For each experimental condition Pa was measured two to three times at specific time points, and the average value was reported.Effects of histamine, ionomycin, and PMA on cytosolic calcium.
The changes in the endothelial cytosolic calcium were examined in
isolated coronary venules by measuring the fluorescence intensities
exerted by the calcium indicator at 340 and 380 nm, representing the
levels of free and bound calcium. The basal level of fluorescence
intensities was first measured in the absence of agonists. Then
histamine (104 M),
ionomycin (106 M), or PMA
(106 M) was added to the
suffusion bath, and the measurement was continued over a 10-min period.
In separate experiments vessels were pretreated for 20 min with either
L-NMMA
(104 M), a specific
inhibitor of NOS, or KT-5823
(106 M), a selective
inhibitor of PKG. The changes in the intracellular calcium on
administration of the same doses of the agonists (histamine, ionomycin,
and PMA) were measured in the presence of the inhibitor. The
agonist-induced calcium changes were compared between control and
L-NMMA- or KT-5823-treated
venules. In some vessels, the calcium response to
L-NMMA or KT-5823 alone was
monitored to ensure that the inhibitor did not alter the basal level of
intracellular calcium.
Effects of NOS-PKG inhibitors on hyperpermeability responses to
histamine and ionomycin.
This experiment was performed to further test the hypothesis that the
NOS-PKG cascade is located downstream of the pathway secondary to the
elevation of intracellular calcium. Although the above experiment
answered the question of whether blockage of NOS or PKG affects
histamine-induced changes in intracellular calcium, this study examines
the effect of the blockers on venular permeability stimulated by the
agonist. The apparent
Pa was measured in isolated coronary venules during administration of histamine (104 M) before and after
treatment with the NOS inhibitor
L-NMMA
(104 M) or the PKG
inhibitor KT-5823 (106 M).
Control studies with D-NMMA
(104 M), the biologically
inactive form of L-NMMA, were
performed to ensure the specific inhibitory effect of
L-NMMA on NOS. To support the
sequential relationship between calcium and NOS-PKG, the effects of the
calcium ionophore ionomycin
(106 M) on
Pa were examined
in venules before and after inhibition of NO production or PKG
activation. Furthermore, the time courses of histamine- or
ionomycin-induced changes in intracellular calcium and permeability
were compared between inhibitor-treated and -untreated vessels. In each
experiment Pa was
measured two to three times at 5-7 min after administration of
histamine or ionomycin, and the values were averaged to represent the
permeability response to the respective agonists.
Relationship between PKC and NOS cascade.
Although it is well known that activation of PKC dramatically increases
microvascular permeability, interactions of PKC with other signaling
molecules have not been well studied. This experiment was performed to
evaluate the downstream mechanism of PKC activation with a focus on its
relationship with the NOS-GC-PKG cascade. On one hand, the possible
involvement of PKC in the NO pathway was tested by measuring the
permeability response to the NO system activators during inhibition of
PKC. On the other hand, the specific inhibitors of NOS and PKG were
applied to examine whether blockage of the NO pathway has an effect on
PKC-stimulated albumin transflux. In the first experiment, BIM was
chosen to block PKC activity because it is a highly selective and
cell-permeable inhibitor of the enzyme (31) and because it inhibits
identically the cytosolic and membrane-derived PKC (4). The
dose-response effects of BIM
(107-105
M) were examined in isolated venules under the basal
conditions and in the presence of the PKC activator PMA
(106 M). Based on the
dose-response experiment and previous literature (4, 31), the dose of
106 M was selected to block
PKC activity in the venule study. Vessels were incubated with BIM
(106 M) for 20 min, and
Pa was measured
before and after application of the NOS activator histamine
(104 M), the GC activator
SNP (105 M), or the PKG
activator 8-BrcGMP (104 M).
The effects of BIM on the basal
Pa and
PMA-induced changes in
Pa were used as
controls. In the second experiment, the effect of PKC activation during
inhibition of the NO cascade was examined. Venules were treated for 20 min with the PKG inhibitor KT-5823 (106 M), the NOS inhibitor
L-NMMA
(104 M), or the inactive
NOS inhibitor D-NMMA
(104 M), and
Pa was measured
during activation of PKC with PMA
(106 M). The changes in
Pa in response to
the PKC activator were compared before and after inhibition of NOS or
PKG. In each experiment, Pa was measured
at 5-7 min after administration of the respective agonists.
Data Analysis
For each experimental condition Pa was measured two to three times, and the values were averaged and presented. To compare the changes in Pa before and after administration of the agonists or inhibitors, the absolute values of Pa were also normalized to the control values obtained before the treatment and were reported as percentages of the controls. In the experiments on calcium, the level of cytosolic calcium was presented as the ratio of fluorescence at 340/380 nm. All the data were reported as means ± SE. Analysis of variance was applied to test the significance of the changes in venular permeability in response to the agonists and inhibitors. Fisher's protected least-significant-difference analysis was used to evaluate the significance of intergroup differences. A value of P < 0.05 was considered significant for the comparisons. For all experiments n is given as the number of vessels studied, with each vessel representing a separate animal.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Data Cogent to Calcium-NOS-PKG Pathway
The changes in endothelial cytosolic calcium were measured in isolated and perfused coronary venules in the presence of various agonists. The ratio of fluorescence intensities acquired at the dual wavelengths represented the intracellular level of free calcium vs. bound calcium. The mean basal fluorescence ratio was 0.67 ± 0.03 (n = 6). Figure 2 shows representative calcium traces from single experiments. A typical pattern of calcium responses to histamine in six vessels was a biphasic increase in the fluorescence ratio with a rapid peak followed by a several-minute plateau (Fig. 2A). The onset and the time course of changes in calcium were closely related to those in Pa. Treatment of the vessels with either L-NMMA (Fig. 2B) or KT-5823 (Fig. 2C) did not have a significant effect on the basal level of fluorescence ratios (0.68 ± 0.03 with L-NMMA and 0.66 ± 0.02 with KT-5823). In the majority of vessels the inhibitors did not significantly change the magnitude and the time course of the calcium response to histamine. However, both L-NMMA and KT-5823 abolished the increase in Pa caused by histamine. Figure 3 summarizes the effects of L-NMMA, D-NMMA, and KT-5823 on histamine-induced changes in coronary venular permeability. Under control conditions histamine increased Pa by 189.21 ± 20.63% from a basal value of 2.70 ± 0.32 × 106 cm/s to 5.09 ± 0.82 × 106 cm/s
(n = 5, diameter 55.60 ± 2.66 µm). This hyperpermeability effect was not altered by
D-NMMA
(n = 4, Pa values were
3.70 ± 0.34 × 106
cm/s before and 7.40 ± 0.88 × 106 cm/s after histamine)
but was diminished during inhibition of NOS with
L-NMMA
(Pa = 3.12 ± 0.14 × 106
cm/s at control and 2.10 ± 0.52 × 106 cm/s after histamine in
the presence of
L-NMMA,
n = 4), as well as during blockage of PKG
with KT-5823, where
Pa slightly, but not significantly, increased from 2.98 ± 0.38 × 106 cm/s to 3.76 ± 0.36 × 106 cm/s in
response to histamine in the presence of KT-5823
(n = 4). Similarly, the calcium
ionophore ionomycin induced a calcium spike preceding the increase in
Pa
(n = 6; Fig.
4A).
Inhibition of NOS with L-NMMA
(Fig. 4C) and PKG with KT-5823 (Fig.
4B) did not affect the elevation in
intracellular calcium but prevented the permeability response to
ionomycin. As shown in Fig. 5, ionomycin increased Pa by
193.21 ± 12.08% from a basal value of 2.36 ± 0.26 × 106 cm/s to 4.49 ± 0.26 × 106 cm/s
(n = 4, diameter 53.25 ± 2.93 µm). The effect was inhibited in the presence of
L-NMMA
(Pa was from 3.06 ± 0.23 × 106 cm/s
at control to 3.97 ± 0.27 × 106 cm/s after ionomycin,
n = 5, diameter 53.00 ± 1.92 µm)
or KT-5823 (Pa
was from 2.50 ± 0.23 × 106 cm/s at control to 2.80 ± 0.13 × 106 cm/s
after ionomycin, n = 4, diameter 51.75 ± 1.75 µm). It should be noted that neither histamine nor
ionomycin significantly altered the venular diameter. Therefore, the
Pa values
reflected the changes in the barrier function of the endothelium.
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Data Cogent to PKC Pathway
Similar to the results obtained from our previous studies (36), activation of PKC by treating the vessels with the specific activator PMA caused a dramatic increase in coronary venular permeability. Importantly, there were no changes in the level of endothelial cytosolic calcium on administration of PMA (n = 3) (Fig. 6). Furthermore, the PKC inhibitor BIM did not significantly alter the basal Pa but abolished the hyperpermeability effect of PMA in a dose-dependent fashion (Fig. 7). Under control conditions PMA caused a dramatic increase in Pa by 290.94 ± 78.31% (n = 7, diameter 63.86 ± 3.14 µm). This effect was greatly attenuated in the presence of 107 M BIM
(Pa increased by
156.08 ± 18.66%, n = 4, diameter
59.75 ± 2.32 µm) and was abolished at higher concentrations of
the inhibitor (139.97 ± 20.82% at
106 M BIM,
n = 4, diameter 43.25 ± 3.08 µm,
and 133.15 ± 4.91% at 105 M BIM,
n = 4, diameter 58.00 ± 1.08 µm). In contrast, BIM did not significantly alter the
hyperpermeability effects of the agents known to activate the NO
pathway (Fig. 8). Under control conditions the increases
in Pa on
administration of histamine, SNP, and 8-BrcGMP were 189.21 ± 20.63% (n = 5, diameter 55.60 ± 2.66 µm), 267.59 ± 63.96% (n = 4, diameter 66.50 ± 11.32 µm), and 176.86 ± 23.48% (n = 7, diameter 54.86 ± 0.68 µm), respectively. After treatment with BIM, these agonists were
still able to increase the
Pa value by
184.30 ± 16.89% (n = 9), 223.93 ± 32.49% (n = 3), and 187.44 ± 5.49% (n = 5), respectively.
Furthermore, BIM did not change the time course and the transience of
the effect of histamine on venular permeability. Consistent with our
previous investigation, neither the agonists nor the inhibitors caused
changes in the vessel diameter. The data indicate that inhibition
of PKC blocked the effect of PMA but did not affect the
hyperpermeability response mediated by the NOS-GC-PKG cascade.
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In contrast, inhibition of the NOS-PKG cascade greatly attenuated the
hyperpermeability effect of PMA (Fig. 9). In
L-NMMA-treated vessels, PMA
caused an increase in
Pa from a basal
value of 4.28 ± 0.44 × 106 cm/s to 7.26 ± 1.08 × 106 cm/s
(n = 8), which was in an extent less
than the control response. Similarly, PMA caused a lower but still high
Pa in venules
treated with KT-5823 (2.66 ± 0.24 × 106 cm/s at control and
4.39 ± 0.63 × 106
cm/s after PMA in the presence of KT-5823,
n = 6).
D-NMMA did not exert any
inhibitory effect on PMA-induced hyperpermeability (n = 3). Taken together, these data
suggest that the calcium-NOS-GC-PKG cascade is an independent but
common pathway leading to agonist-induced protein transflux across the
coronary venular endothelium, whereas PKC activation mediates the
hyperpermeability reaction partially through the NO
pathway.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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This study focused on the interaction of different signaling molecules during modulation of macromolecular permeability in coronary venules. The measurements of cytosolic calcium transience and albumin permeability in the endothelium of isolated venules provided some interesting results. First, histamine and ionomycin caused a rapid elevation in endothelial cytosolic calcium preceding the increase in endothelial permeability to albumin, whereas PMA induced a hyperpermeability response unaccompanied by intracellular calcium changes. These data indicate a heterogeneity of calcium dependence among different hyperpermeability mediators. Second, the NOS and PKG blockers inhibited the increases in venular permeability but failed to block the calcium spikes during stimulation of histamine and ionomycin. The dissociation of the permeability effect from the calcium response suggests that elevation in endothelial cytosolic calcium is an early signaling event that occurs upstream from NO synthesis. Finally, the relationship between PKC and NOS was examined. Although the PKC inhibitor did not affect the hyperpermeability effects of histamine, SNP, and cGMP, which are known to stimulate the NO pathway, inhibition of NO synthesis or PKG activity was able to attenuate the permeability response to PMA, a PKC activator. Therefore, we suggest that there are at least two independent signaling pathways that contribute to the pathophysiological regulation of coronary venular permeability. One mechanism involves a rapid elevation in endothelial cytosolic calcium followed by NO synthesis and PKG activation. The other is characterized by activation of PKC. However, interactions occur between the two pathways, in that PKC may exert its action partially by modulating the synthesis of NO.
Endothelial Cytosolic Calcium and NO Production in Modulation of Venular Permeability
The control of cytosolic calcium is critical in the physiological and pathophysiological regulation of endothelial function. Calcium is required for the maintenance of endothelial integrity, and alterations in calcium produce dynamic changes in endothelial structure and barrier properties (30). Elevation of endothelial cytosolic calcium has been considered as a primary trigger of transendothelial flux of macromolecules on stimulation by some inflammatory mediators such as histamine (27). Supporting the importance of calcium in acute inflammatory reactions is an in vivo observation that histamine-caused leakage in postcapillary venules of the hamster cheek pouch is dependent of extracellular calcium (17). A possible mechanism for the increase in intracellular calcium is provided by the finding that histamine stimulates inositol phosphate production via a G protein associated with PLC (5). In this instance, PLC hydrolysis of phosphotidylinositol bisphosphate and subsequent calcium mobilization comprise the initial intracellular signaling processes after agonist binding (14). However, the later events that occur downstream from calcium elevation remain to be clarified. The proposed sites of action of calcium include actin-myosin contraction, endothelial cell-cell adhesion, and cell-matrix interaction (6, 14). Although all these processes are very important to the barrier property, they are generally considered as the determinants of endothelial integrity that ultimately control the structure or morphology of the endothelial monolayer (6, 14). In other words, there may be some intermediate molecules that reside within the cell to transduce the signals from calcium to structural proteins in the cytoskeleton and intercellular junction. Within this context, a potential second messenger of calcium is NOS. In view of the potent stimulating effect of calcium on endothelial constitutive NOS (28) and the key role of NOS in mediation of agonist-induced microvascular hyperpermeability (16, 21, 23, 26, 34), it is possible that the conventionally emphasized central effect of calcium relies on the activity of NOS and is transduced by NO. This hypothesis has not been directly tested due to technical difficulties in measuring cytosolic calcium and macromolecular permeability in the intact microvessel.
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Our study reports for the first time the measurement of agonist-elicited changes in endothelial calcium and permeability of isolated and perfused intact coronary venules. The method has the advantage of the precise measurement of endothelial cytosolic calcium and albumin permeability in the absence of neutrophils and with little influence from hemodynamic and parenchymal factors (33). Under such conditions, histamine induced a rapid and biphasic rise in intracellular calcium followed by an increase in albumin permeability in the endothelium of coronary venules. The onset and time course of changes in calcium and Pa were closely related. Similarly, the calcium spike on administration of ionomycin preceded the elevation in Pa in the presence of the ionophore. The pattern of calcium responses to histamine and ionomycin is consistent with that observed in cultured endothelial cells (27) and perfused frog mesenteric capillaries (7). More importantly, we found that histamine- and ionomycin-induced increases in venular permeability were abolished, whereas the calcium responses were not significantly altered by inhibition of NOS or PKG. This indicates that elevation in endothelial cytosolic calcium occurs before NOS activation. Therefore, NO, rather than calcium, is a common mediator toward the downstream of the pathway leading to microvascular hyperpermeability. Although calcium is an important signaling molecule it may not be essential in mediation of permeability responses to certain agonists. Indeed, the present study suggests that PMA-induced increases in coronary venular permeability are not calcium dependent.
A limitation of this study is that the association of calcium and NOS was not directly tested, and it is therefore unconfirmed that the presence of calcium is required in the signaling process triggered by histamine. In this regard, a measurement of the permeability response to the agonist in the absence of intracellular and extracellular calcium would directly address the question. However, depletion of calcium could largely alter the basal permeability of the endothelium by disrupting the cell-cell and cell-matrix attachment, rendering results meaningless. On the other hand, we were unable to measure the activity and products of NOS in isolated and perfused microvessels due to technical difficulties. Therefore, we tested the calcium-NOS-PKG cascade by using pharmacological approaches. Our results support the sequential association between calcium and NOS. A recent study on perfused frog mesenteric microvessels (7) has also demonstrated that calcium-dependent release of NO is a necessary step to increase microvascular hydraulic conductivity in response to ionomycin and ATP.
One of the major aims of this study was to evaluate the relationship of calcium and NO in the signal transduction of coronary venular hyperpermeability. Specifically, we were interested in testing the hypothesis that elevation in endothelial calcium occurs upstream from NO production, rather than the route of calcium mobilization or the absolute value of intracellular calcium. Therefore, we report the trend of calcium responses in the form of fluorescence ratio at 340/380 nm. Regarding the concentration and source of calcium, previous studies (7, 27, 35) have shown that histamine, ATP, or ionomycin stimulates an increase in endothelial cytosolic calcium from a resting level of 50-100 to 500-1,000 nM, and that the biphasic response of calcium is due to an initial release from intracellular pools followed by an influx of extracellular calcium.
Interactions Between NO and PKC in Modulation of Venular Permeability
The endothelial cell responds to a wide array of messages carried by hormones, neurotransmitters, growth factors, cytokines, and other cells. The efficient operation of the endothelial barrier requires coordination and integration between different signaling pathways that are triggered by various mediators. Our current findings suggest that at least two different signaling events occur in the endothelium of coronary venules that are responsible for agonist-induced changes in the barrier function. We have previously demonstrated that histamine increases venular permeability via a PLC-NOS-GC cascade (34). Recently, we have extended the hypothesis to PKG, a second messenger of cGMP (32, 36). The current study further includes intracellular calcium as an early intermediate located upstream from NO production. We suggest that PLC-calcium-NOS-GC-PKG comprises a signaling cascade in the pathophysiological regulation of the endothelial permeability. It is likely that this pathway plays a dominating role in mediation of histamine-elicited transvenular flux of albumin, because the response could be abolished or greatly attenuated only by inhibitors of the related enzymes but not affected during inhibition of cyclooxygenase (34) or PKC.In addition to the calcium-initiated response, other mechanisms may be involved in the regulatory process associated with certain agonists that are different from histamine. For example, PKC has been known as an important second messenger in the regulation of microvascular barrier function during stimulation by phorbol esters, DAG, thrombin, bradykinin, and platelet-activating factor (6, 11, 13-15, 22). A significant finding of the current study is that activation of PKC with the phorbol ester PMA increased venular permeability via a mechanism independent of calcium. This is in agreement with other investigations in which PKC activators caused albumin transflux across the endothelial monolayer unaccompanied by alterations in the level of endothelial cytosolic calcium (3). The dissociation of changes in the endothelial permeability and the intracellular calcium content suggests that the PKC activators, unlike histamine-type agonists, act as barrier modulators through a pathway that is not triggered by calcium mobilization. However, how PKC alters the barrier function is still a controversial issue. A novel study in the hamster cheek pouch has demonstrated that the increase in microvascular permeability during stimulation of PKC requires the production of NO (25), indicating that NOS may be a target protein of PKC. Considering the effect of PKC on the activity of NOS, although some studies showed that pharmacological inhibition as well as depletion of PKC increased the expression of NOS mRNA and enhanced the release of NO in cultured endothelial cells derived from large vessels (24), opposite data were reported in which PKC activation promoted NO production in various cells, including cardiac myocytes and vascular smooth muscle cells (8, 19, 29). The disparity between the two results may be explained by the difference in experimental preparations and conditions. As in the postcapillary venular endothelium where leaky sites are formed on inflammatory stimulation (18), there is no direct evidence showing the relationship between PKC activation and NO production. Unfortunately, our current technique did not allow us to measure the enzyme activity and products in isolated and perfused venules. Nevertheless, studies with pharmacological approaches have revealed that the hyperpermeability effect of PKC-activating agents could be blocked by NOS inhibitors (13, 16, 22, 25, 26), supporting the concept that PKC displays its signaling effect by modulating the activity of NOS in the endothelium. Our observation that inhibition of NOS attenuated PMA-augmented permeability is favorable to the regulatory relationship between PKC and NO. In fact, we emphasize that PKC-mediated changes in venular permeability involve, in part, the production of NO. Because inhibition of NOS did not totally abolish the effect of PMA, mechanisms that are independent of NO should be considered.
The molecular mechanism responsible for the upregulation of PKC has not been specified. A potential reason for the lack of understanding is that the enzyme consists of at least eight isoforms that may respond to different signals and exert different actions on the downstream proteins (14). Generally, it is accepted that PLC hydrolysis of phosphotidylinositol bisphosphate produces DAG, leading to PKC activation (2). In this study, we used a phorbol ester that is known to directly stimulate PKC bypassing PLC, rendering difficulties in verification of the link between PLC and PKC in the signaling pathway leading to microvascular permeability. On the other hand, we found that PLC was an early signal before NO production in the mediation of histamine's effect on venular permeability (34), which was not affected by the PKC inhibitors. This is interesting because if PLC activation causes PKC upregulation, then blockage of PKC should at least attenuate the histamine-elicited, PLC-mediated response. An explanation for the insignificant effect of the PKC inhibitor is that PKC is not necessarily activated after PLC activation by histamine. Alternatively, different isoforms of PKC are involved in the signaling process, of which the one turned on by PLC may not have a significant effect on the endothelial permeability.
In summary, we provide evidence for a sequential linkage between the elevation in endothelial cytosolic calcium and the activation of NOS during mediation of agonist-elicited venular hyperpermeability. The study further supports the regulatory importance of the PLC-calcium-NOS-GC-PKG cascade and the PKC pathway in the process of microvascular exchange. During inflammation, activated PKC may modulate the endothelial barrier function directly and/or indirectly through stimulating the NOS activity. Through this interaction, the two pathways may act in concert to regulate microvascular permeability.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Harris J. Granger for the valuable comments on the theoretical aspect of the study. We also appreciate Dacia Cottle for technical assistance.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-52221 and American Heart Association Grant-in-Aid 95009170.
Address for reprint requests: Y. S. Yuan, Dept. of Surgery and Medical Physiology, Texas A & M Univ. Health Science Center, 1901 South First St., Bldg. 4, Temple, TX 76504.
Received 11 March 1997; accepted in final form 30 July 1997.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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) and apparent permeability coefficient to albumin
(Pa; 
) in
isolated and perfused coronary venules on stimulation with histamine
under control conditions (A), during
inhibition of NOS with
NG-monomethyl-L-arginine
(L-NMMA;
B), and during inhibition of PKG
with KT-5823 (C).
Significant vs.
control response to histamine in absence of
L-NMMA and KT-5823.
