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Center for Cardiovascular Sciences, Albany Medical College, Albany, New York 12208-3479
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this study was to determine the signal-transduction pathway that elicits this decrease in permeability. Permeability-decreasing activity of PCM was assessed by the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels. Using a desensitization protocol with cAMP/protein kinase A (PKA)-enhancing agents and pharmacological inhibitors, we determined that the activity of PCM is independent of PKA and PKG. Genistein, an inhibitor of tyrosine kinases, prevented the increase in endothelial electrical resistance. Because lysophosphatidic acid (LPA) has been proposed to be responsible for this activity of PCM and is known to activate the Gi protein, inhibitors of the G protein pertussis toxin and of the associated phosphatidylinositol 3-kinase (PI3K) wortmannin were used. Pertussis toxin and wortmannin caused a 10- to 15-min delay in the characteristic rise in electrical resistance induced by PCM. Inhibition of phosphorylation of extracellular signal-regulated kinase with the mitogen-activated kinase kinase inhibitors PD-98059 and U-0126 did not prevent the activity of PCM. Similar findings with regard to the cAMP protocols and inhibition of Gi and PI3K were obtained for 1-oleoyl-LPA. These results demonstrate that PCM increases endothelial electrical resistance in vitro via a novel, signal transduction pathway independent of cAMP/PKA and cGMP/PKG. Furthermore, PCM rapidly activates a signaling pathway involving tyrosine phosphorylation, the Gi protein, and PI3K.
permeability; signal transduction; Gi protein; phosphatidylinositol 3-kinase; extracellular signal-regulated kinase; tyrosine kinase; lysophosphatidic acid
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PLATELETS SUPPORT THE FUNCTION of the vascular endothelium as a semipermeable barrier to the passage of water and protein. In patients with low platelet counts (thrombocytopenia), petechial and purpuric hemorrhages and edema develop in the skin and mucus membranes (6, 13). In animals, thrombocytopenia has been shown to increase protein permeability in the vasculatures of the lung (18), ear (3), thyroid (8), and heart (19). Repletion with platelet-rich plasma reverses these abnormalities in clinical and experimental settings (3, 8, 18, 19). In vitro experimentation has demonstrated that the addition of platelets or platelet-conditioned medium (PCM) to endothelial cell monolayers decreases protein permeability (2, 10, 30, 31, 34). Although much effort has been focused on identifying the active factors, little is known about the cell-signaling pathway that initiates the permeability-decreasing activity of platelets.
A plethora of literature (4, 14, 20-22, 28, 39) has documented the ability of the cAMP/protein kinase A (PKA) signal-transduction pathway to decrease water and protein transport across the vascular endothelium. The cGMP/protein kinase G (PKG) pathway has also been implicated in tightening the endothelial barrier (12). Therefore, the first objective of the present study was to determine whether platelets decrease endothelial permeability via cAMP/PKA and cGMP/PKG. Two approaches, a desensitization protocol with cAMP-enhancing agents and pharmacological inhibition of PKA or PKG, were taken to determine the importance of the cAMP/PKA- and cGMP/PKG-signaling pathways.
Lysophosphatidic acid (LPA) has been proposed as the active factor responsible for the permeability-decreasing activity of platelets (2). In neurites and fibroblasts, LPA is known to initiate cell signaling via activation of the G proteins, inhibitory (Gi), Gq, and G12/13 (9, 23, 24, 37). Therefore, we also focused on the Gi protein signaling pathway, which can influence the levels of intracellular cAMP, to determine whether tyrosine phosphorylation and specific proteins involved in this pathway, Gi, phosphatidylinositol 3-kinase (PI3K), and the extracellular signal-regulated kinase (ERK), initiate the activity of PCM as well as LPA.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Materials. Bovine endothelial cells derived from pulmonary arteries and pulmonary microvessels were obtained from Vec Technologies, fetal bovine serum was from Summit Biotechnologies, and gentamicin sulfate was from Bioproducts. KT-5720 and H-89 were from Alexis, KT-5823, pertussin toxin, wortmannin, LY-294002, PD-98059, and U-0126 were from Biomol, genistein was from Calbiochem, and 1-oleoyl-LPA was from Avanti Polar Lipids. Electric cell-substrate impedance sensing (ECIS) apparatus and gold-coated electrodes were from Applied Biophysics; SDS-PAGE mini-slab gel was from Bio-Rad; polyclonal antibody specific for phosphorylated ERK (p44/p42, thr 204/tyr 202) was from New England BioLabs; and enhanced chemiluminescence kit was from American International. All other compounds and materials were purchased from Sigma.
Platelet isolation and preparation of PCM.
Platelets were isolated from fresh nonirradiated platelet packs
purchased from the local blood bank. Isolated platelets were brought to
a concentration of 2 × 109 platelets/ml in modified
(Ca+/Mg2+ free) Tyrode buffer (in M) 0.137 NaCl, 0.003 KCl, 0.012 NaHCO3, 0.006 glucose, and 0.004 NaH2PO4, pH 7.4, incubated in plastic round-bottom tubes for 2 h, and centrifuged at 600 g
for 20 min. The supernatant was designated as PCM and aliquoted and
stored at 4°C for immediate use or at 
80°C for later use.
Endothelial cell culture. Endothelial cells from bovine pulmonary arteries (passages 3-20) and microvessels (passages 3-20) were grown in MCDB-131, 10% fetal bovine serum, and 50 µg/ml of gentamicin sulfate. For the measurement of endothelial electrical resistance using ECIS, cells were seeded onto 1% gelatin-coated gold electrodes (60,000-100,000 cells onto 0.5 cm2 wells). For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), cells were seeded onto 60-mm petri dishes (500,000 cells/dish). Cells were grown to confluence (3-4 days) in a humidified environment maintained at 37°C and 5% CO2. Before commencement of an experiment, cell culture medium was changed from 10% serum to serum-free MCDB-131, and the cells were placed in the ECIS incubator and allowed to equilibrate for 2 h.
Assessment of endothelial permeability.
Continuous measurement of electrical resistance across endothelial cell
monolayers was used to determine changes in endothelial permeability.
Electrical resistance was measured by using the methodology known as
ECIS (7, 28, 30, 35). The basic setup to measure
endothelial electrical resistance is shown in Fig.
1. Endothelial cells were grown to
confluence on small gold electrodes (5 × 10
4
cm2) in culture medium, which functioned as the
electrolyte. The small gold electrode, covered by confluent endothelial
cells, and a larger gold counter electrode (~2 cm2) were
connected to a phase-sensitive, lock-in amplifier. A 1-V, 4,000-Hz
alternating current was supplied through a 1-M
resistor to
approximate a constant current source of 1 µA. Treating the cell-electrode system as a simple series resistance-capacitance circuit, the measured changes in the in-phase voltage and the out-of-phase voltage can be used to calculate these values for resistance and capacitance. Voltage and phase data were stored and
processed with the use of a personal computer. The same computer controlled the output of the amplifier and switched the measurements to
different electrodes in each of two, eight-well arrays during the
course of an experiment. The small size of the cell-seeded electrode is
the critical feature of the system. When electrodes of
103 cm2 or smaller are used, the impedance at
the small electrode dominates the system, allowing this impedance to be
measured and also allowing for assessment of cellular morphology. The
measurement of electrical impedance was obtained in real time every
minute before and for 30-60 min after treatment of the endothelial
cells and reported as the resistive portion of electrical impedance.
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Measurement of the phosphorylation levels of p44/p42 ERK. Endothelial cells were pretreated for 1 h with 10 or 30 µM PD-98059 or with 10 µM U-0126, then treated with PCM. At 10 min, cells were placed on ice, washed with phosphate-buffered saline, and treated with 100 µl of SDS sample buffer with dithiothreitol (62.5 mM Tris · HCl, 2% wt/vol SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% wt/vol bromophenol blue) to lyse the cells. Endothelial cells were scraped from the petri dishes with a rubber policeman, and cell lysates were transferred to a microcentrifuge tube. Samples were boiled for 5 min to denature proteins. Samples (20 µl) were loaded onto each stacker and run on a 10% SDS-PAGE mini-slab gel according to the method of Laemmli (15), and then transferred to a nitrocellulose membrane. Membranes were incubated with a polyclonal antibody to phosphorylated ERK (p44/p42, thr204/tyr202). Enhanced chemiluminescence was used for antibody detection. Phosphorylation levels of p44/p42 ERK were quantified by densitometry and normalized to control conditions.
Experimental drugs. PCM was administered to endothelial cell monolayers as a 1:4 dilution of the stock concentration (2 × 109 platelets/ml) of PCM. LPA was dissolved in a Ca2+-free phosphate-buffered saline solution to yield a stock concentration of 500 µM and diluted further in serum-free MCDB-131. Isoproterenol and 8-bromo-cAMP were dissolved in MCDB-131. Pertussis toxin was reconstituted in sterile water and diluted in MCDB-131; the vehicle control was sterile water with 10 mM NaP and 50 mM NaCl. Forskolin, KT-5720, H-89, KT-5823, genistein, wortmannin, LY-294002, and U-0126 were dissolved initially in 100% dimethyl sulfoxide (DMSO) to yield a stock concentration and diluted further in serum-free MCDB-131. PD-98509 was also dissolved initially in 100% DMSO, but the primary dilution was made in MCDB-131 containing 5% fetal bovine serum.
Statistics. Each study that measured endothelial electrical resistance consisted of at least five different experiments. Western blots were analyzed quantitatively from at least three separate experiments. Data were analyzed using a two-way analysis of variance with repeated measures (38). Differences between treatment groups from the control group were analyzed with the Newman-Keuls post hoc test. Statistical significance was set at P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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PCM functions via a cAMP/PKA-independent signaling pathway.
A desensitization protocol was used first to determine whether PCM
decreases endothelial permeability via cAMP/PKA. Isoproterenol (1 µM), forskolin (10 µM), and 8-bromo-cAMP (1 mM) were incubated individually with endothelial cell monolayers as two separate challenges that were 45 min apart. Cells responded to the first challenge of each of these agents with an increase in electrical resistance (Fig. 2). After the second
challenge with each agent, electrical resistance did not increase,
indicative of desensitization. In contrast, desensitization was not
observed when PCM was administered as the second challenge (Fig. 2).
Similar responses were obtained with 2 and 5 µM isoproterenol,
respectively (data not shown). When the challenges were reversed, PCM
administered as the first treatment desensitized the increase in
endothelial electrical resistance induced by the second challenge of
isoproterenol, 8-bromo-cAMP, and PCM (Fig.
3).
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PCM does not function via PKG.
To determine whether PKG was responsible for the increased electrical
resistance induced by PCM, the endothelial cell monolayers were
pretreated for 1 h with 5 or 10 µM KT-5823, an inhibitor of PKG,
and were then administered PCM or 1 mM 8-bromo-cGMP, a cell-permeable
analogue of cGMP. KT-5823 blocked the increase in endothelial
electrical resistance induced by 8-bromo-cGMP (Fig. 5A) but did not inhibit the
increased electrical resistance induced by PCM (Fig. 5B).
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Inhibitor of tyrosine kinase activity prevents PCM activity.
There are a number of steps in cell signaling that require tyrosine
phosphorylation. Pretreatment with the tyrosine kinase inhibitor
genistein for 1 h at 50 and 100 µM prevented the increase in
endothelial electrical resistance induced by PCM (Fig.
6).
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Inhibitors of Gi and PI3K delay PCM activity.
The recently proposed active platelet factor LPA (2) has
been shown to activate the Gi pathway, which is pertussis
toxin sensitive (24, 37) and involves PI3K (9, 11,
23, 32). To determine whether the PCM-induced increase in
endothelial electrical resistance is initiated by a
pertussis-toxin-sensitive Gi protein, endothelial cell
monolayers were pretreated for 3 h with 50 and 100 ng/ml of
pertussis toxin, and were then treated with PCM. Pertussis toxin
blocked the characteristic rapid rise in endothelial electrical
resistance induced by PCM (Fig.
7A). Electrical resistance eventually increased to a similar level as that induced by PCM but the
increase was delayed for 10 to 15 min. Similar results were
obtained by using two mechanistically different inhibitors of
PI3K, wortmannin, and LY-294002. At doses that have been reported to be
specific for PI3K, wortmannin (15 and 30 nM; Fig. 7B) and LY-294002 (0.5 and 1 µM; data not shown) also caused a delay of 10 to
15 min in the initial, rapid rise in endothelial electrical resistance
induced by PCM.
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Inhibition of ERK phosphorylation does not prevent PCM activity.
To determine the involvement of ERK, a primary kinase activated by the
Gi signaling pathway, the upstream kinase mitogen-activated protein kinase kinase (MEK) was inhibited with PD-98059 (10 and 30 µM) or U-0126 (10 µM). Pretreatment for 1 h with 10 or 30 µM PD-98059 (Fig. 8A) or 10 µM
U-0126 (Fig. 8B) had no effect on PCM activity, although
there was a 3-min delay in the onset of PCM activity in the presence of
U-0126. However, these doses of PD-98059 and U-0126 significantly
inhibited the increased phosphorylation of ERK in PCM-treated cells
(Fig. 8, C and D). The lack of a correlation between the effects of MEK inhibition on endothelial electrical resistance and ERK phosphorylation demonstrated that PCM does not
decrease endothelial permeability via activation of ERK.
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LPA also functions independently of cAMP and is affected by
inhibitors of Gi and PI3K.
Because LPA may be the active factor in PCM and is known to activate
the Gi protein, we repeated the experiments involving cAMP,
Gi, and PI3K using 1-oleoyl-LPA (10 µM). The
desensitization protocol with the cAMP-enhancing agents and the PKA
inhibitor, KT-5720, produced similar responses with LPA as occurred
with PCM (Fig. 9). Whereas the
two challenges of 8-bromo-cAMP resulted in a desensitization response,
LPA was not inhibited by the prior challenge of 2 mM 8-bromo-cAMP.
Similarly, 2 µM KT-5720 prevented the 8-bromo-cAMP response but
did not prevent the rapid increase in endothelial electrical resistance
induced by LPA. KT-5720 did attenuate the maximum increase in
electrical resistance after LPA treatment; however, this attenuation
may have been influenced by the two pretreatments, DMSO and KT-5720
with DMSO. Electrical resistance decreased initially due to the DMSO
vehicle, then rebounded back to a higher level in the KT-5720 group
than in the vehicle DMSO group, which would affect the calculation of
the normalized experimental-to-baseline values.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The objective of the present study was to determine the signal-transduction pathway that PCM activates to decrease vascular endothelial permeability. Permeability was assessed by the real-time measurement of electrical resistance across endothelial cell monolayers. Desensitization with three cAMP/PKA-enhancing agents and inhibitors of PKA and PKG did not prevent the increase in endothelial electrical resistance induced by PCM. Inhibition of ERK activity also had no effect on the activity of PCM. Instead, an inhibitor of tyrosine kinases prevented PCM activity, and inhibitors of the Gi protein and PI3K delayed by ~10 min the onset of activity of PCM. These findings indicate that PCM rapidly increases electrical resistance across endothelial cell monolayers via a novel signal-transduction pathway that involves the activity of tyrosine kinases, the Gi protein, and PI3K. Similar findings were observed when LPA was substituted for PCM, except that the inhibitor of the Gi protein, pertussis toxin, did not cause a delay in activity but instead prevented the activity of LPA.
Permeability across endothelial cells grown as a monolayer was assessed with a methodology known as ECIS for electric cell-substrate impedance sensing. This methodology was developed to study the dynamic behavior of cells in culture (7, 35). ECIS measures in real-time the changes in impedance of an endothelial cell monolayer to a weak, noninvasive, alternating current signal. We have demonstrated that measurements of increases and decreases in electrical resistance correlate with decreases and increases, respectively, in 125I-labeled albumin clearance and in microscopic gaps across endothelial cell monolayers (28, 30). The increase in endothelial electrical resistance induced by PCM in this study also correlates with the PCM induced decrease in permeability of sodium fluorescein (mol wt 342) across endothelial cell monolayers derived from bovine aorta (2, 10).
Thrombocytopenia increases vascular permeability, resulting in petechial and purpuric hemorrhages in the skin and tissue edema (3, 8, 18, 19). Repletion with platelet-rich plasma improves these conditions (3, 8, 18, 19). This permeability-decreasing activity of platelets appears to reside in a releasable, soluble factor because PCM consistently increases electrical resistance and decreases albumin permeability across endothelial cell monolayers derived from bovine aorta, pulmonary arteries, and pulmonary microvessels (2, 10, 30, 31, 34). Many soluble factors have been proposed, studied, and rejected as the active factor released from platelets. These factors include serotonin, norepinephrine, cyclooxygenase products, adenosine, adenine nucleotides, and a protein (30, 31, 34). The most recently proposed active platelet factor is a phospholipid LPA (2).
Although much effort has been generated to identify this soluble
platelet factor, there is a paucity of information concerning the
cellular mechanisms by which the active platelet factor functions to
decrease endothelial permeability. Two signal-transduction pathways
have been studied in depth with regards to endothelial permeability.
The cAMP/PKA-signaling pathway is well known to prevent and reverse an
increase in vascular permeability induced by a variety of mediators,
diseases, and syndromes (4, 14, 20-22, 28, 39). The
effect of the cGMP/PKG-signaling pathway on endothelial permeability,
however, is controversial and may be cell, tissue, and/or organ
specific (12). When we considered the long list of
publications involving the above two signaling pathways, we initially
asked the question whether one of these two pathways is responsible for
the permeability-decreasing activity of PCM. Two approaches were taken:
the first used a desensitization protocol with three different
cAMP/PKA-enhancing agents and the second used inhibitors of PKA and
PKG. The first challenge with the use of isoproterenol (a
2-adrenergic receptor agonist), forskolin (a
cell-permeable activator of adenylate cyclase), or 8-bromo-cAMP (a
cell-permeable analogue of cAMP) increased endothelial electrical resistance with an activity profile similar to that of PCM. This first
challenge prevented (desensitized) the change in electrical resistance
when the second challenge (45 min apart) with the same agent was
administered. In contrast, these three cAMP/PKA-enhancing agents did
not prevent the activity of PCM when PCM was administered as the second
challenge. The inhibitors of PKA (KT-5720 and H-89) and of PKG
(KT-5823) blocked their respective kinase activators, isoproterenol,
forskolin, 8-bromo-cAMP, and 8-bromo-cGMP but had no effect on the
activity of PCM. Furthermore, 8-bromo-cGMP consistently initiated a
slower increase in endothelial electrical resistance compared with PCM.
We conclude from these studies that PCM increases endothelial
electrical resistance via a cellular mechanism independent of PKA and PKG.
Interestingly, when the challenges were reversed in the desensitization protocol, PCM significantly attenuated the increased endothelial electrical resistance induced by isoproterenol and 8-bromo-cAMP. This desensitization of the isoproterenol response could result from activation of the Gi protein by PCM and downregulation of the cAMP pathway. The mechanism for desensitization of the 8-bromo-cAMP response is less clear. It is possible that the active factors in PCM and cAMP/PKA activate parallel pathways that converge on common final targets that mediate the increase in endothelial electrical resistance, our assessment of endothelial barrier function. According to the data in Figs. 2 and 3, activation of the signal transduction pathway that is independent of cAMP would have a greater effect on modulating endothelial barrier function.
We next determined whether PCM might influence endothelial permeability via the Gi-ERK-signaling pathway. The recently proposed active platelet factor LPA (2) is known to stimulate signal-transduction pathways initiated by Gi, Gq, and G12/13 protein-coupled receptors (9, 23, 24). We focused initially on the Gi-p21ras-ERK pathway. The Gi-ERK-signaling cascade was blocked using inhibitors of upstream proteins, Gi, PI3K, and ERK kinase (or MEK), and an inhibitor of tyrosine kinases.
Genistein, an inhibitor of the activity of tyrosine kinases, prevented PCM activity. There are a number of steps in the Gi-ERK signaling pathway that require tyrosine phosphorylation. A tyrosine kinase has been proposed as an early mediator in the suspected pathway (9, 23); MEK, the upstream kinase of ERK, is a dual specific kinase that causes phosphorylation of ERK at tyrosines and threonines; assembly of the protein scaffold involved in Ras activation is dependent on tyrosine phosphorylation; and internalization of the G-protein-coupled receptor complex, which may be important in activation of downstream kinases, is dependent on the tyrosine phosphorylation of dynamin (1).
Inhibitors of the Gi protein (pertussis toxin) and of PI3K (wortmannin and LY-294002) resulted in a 10-15 min delay in the characteristic rapid rise in endothelial electrical resistance induced by PCM. These findings indicate that tyrosine phosphorylation and both the Gi protein and PI3K are involved in the PCM-signaling cascade. Because the inhibitors of Gi and PI3K only delayed the increase in electrical resistance, other G proteins and signal-transduction pathways could be involved in the cellular response and/or the active platelet factor may internalize into the cell in some way and bypass upstream signaling proteins. One possible explanation is that the active platelet factor initiates a fast signal-transduction pathway involving Gi and PI3K and a slower receptor pathway that bypasses Gi and PI3K. On the other hand, the active platelet factor may initiate a slower signal- transduction event after internalization. The proposed active platelet factor LPA appears to function via receptor activation as it is not active when microinjected into cells (36). Another candidate for the active platelet factor is sphingosine 1-phosphate, a phospholipid, which also mimics the activity of PCM and LPA (J. T. Roberts, P. A. Vincent, C. A. Morton, and F. L. Minnear, unpublished observations). Sphingosine 1-phosphate activates similar G protein-coupled receptors as does LPA and has been reported to be active after internalization (36), although this latter effect is controversial (17).
Because inhibitors of the Gi protein and PI3K significantly modified the permeability-decreasing activity of PCM and are known to modify the activity of ERK, the next logical step was to determine whether ERK was involved. MEK, the upstream kinase of ERK, was inhibited with PD-98059 at 10 and 30 µM or with 10 µM U-0126. These doses of PD-98059 and U-0126 had no affect on PCM activity. However, these doses of PD-98059 and U-0126 lowered the basal phosphorylation of ERK and prevented the PCM-induced increase in ERK phosphorylation. The lack of a correlation between the effects of PD-98059 and U-0126 on electrical resistance and ERK phosphorylation indicates that PCM does not function via ERK to increase endothelial electrical resistance.
Taken together, these results indicate that PCM rapidly increases endothelial electrical resistance via a novel, signal transduction pathway involving tyrosine kinases, the Gi protein, and PI3K. However, PCM can also initiate a signaling cascade downstream or parallel to the Gi protein or P13 kinase to cause a slower increase in endothelial electrical resistance. This novel signaling pathway does not involve PKA, PKG, or ERK.
Substitution of 1-oleoyl-LPA (10 µM) for PCM in the above protocols involving cAMP and inhibitors of the Gi protein and PI3K yielded similar results. The only exception was that pertussis toxin, the inhibitor of Gi, prevented the activity of LPA and delayed the activity of PCM. Interestingly, sphingosine 1-phosphate was also affected by the above experimental protocols in the same way as LPA (unpublished observations).
The cellular mechanism for this endothelial barrier activity of PCM has
not been studied. cAMP-enhancing agents also tighten the endothelial
barrier and the cellular mechanism is not clear, although some of the
cellular targets are known. It has been proposed that cAMP-enhancing
agents affect cellular targets that initiate cell spreading and alter
the adherens junction proteins, cell-specific cadherins and catenins,
that mechanically hold cells together. This is supported by studies
showing that an increase in intracellular cAMP causes the inactivation
of myosin light-chain kinase (5, 33), a decrease in
cellular isometric tension (25), remodeling of actin
filaments (16), and cell spreading. A decrease in
isometric tension and cell spreading by cAMP could counteract the
ability of agents such as thrombin, which increase cellular isometric tension and cause cell contraction, to loosen the endothelial barrier.
However, Moy et al. (25) directly measured cellular isometric tension as well as myosin light-chain phosphorylation and
concluded that cAMP-enhancing agents protect the thrombin-induced decrease in endothelial electrical resistance independently of the
development of isometric cellular tension. cAMP-enhancing agents may
also stabilize the adherens junction. An increase in intracellular cAMP
has been shown to stabilize within 10 min the transvascular flux of
125I and the peripheral localization of epithelial
(E)-cadherin and ZO-1 in the presence of low calcium in epithelial
cells (26, 27) and to increase by 2 h the peripheral
localization of E-cadherin in brain microvascular endothelial cells
(29). Sphingosine 1-phosphate, a lysophospholipid that is
synthesized in platelets (40) and that mimics the
PCM-induced increase in endothelial electrical resistance (unpublished
observations), has been reported to increase within 1 h the
localization of vasoendothelial (VE)-cadherin and 
-, -, and
-catenins to cell-cell contacts of human umbilical vein endothelial
cells (17). Microinjection of Clostridium
botulinum C3 exoenzyme to inhibit Rho or microinjection of
dominant-negative Rac reduced the peripheral localization of
VE-cadherin and -catenin by sphingosine 1-phosphate in these cells.
Therefore, PCM as well as cAMP-enhancing agents may function to tighten
the endothelial barrier at the level of the actin cytoskeleton and/or
the adherens junction.
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ACKNOWLEDGEMENTS |
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The authors thank Wendy Hobb for editorial assistance.
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FOOTNOTES |
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This study was supported by American Heart Association Grant AHA-97-127A.
Address for reprint requests and other correspondence: F. L. Minnear, Center for Cardiovascular Sciences, MC-8, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208-3479 (E-mail: minneaf{at}mail.amc.edu).
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.
Received 5 December 2000; accepted in final form 25 July 2001.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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