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Am J Physiol Heart Circ Physiol 287: H100-H106, 2004. First published February 19, 2004; doi:10.1152/ajpheart.00922.2003
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Effects of specific signal transduction inhibitors on increased permeability across rat endothelial monolayers induced by neuropeptide Y or VEGF

Nakako Kurimoto,1 Yong-Shan Nan,4 Zhi-Yong Chen,4 Guo-Gang Feng,2 Toru Komatsu,1 Nobuhisa Kandatsu,1 Junken Ko,1 Norio Kawai,3 and Naohisa Ishikawa2

1Department of Anesthesiology, 2Department of Pharmacology, and 3Department of Anatomy, Aichi Medical University School of Medicine, Nagakute, Aichi, 480-1195; and 4Department of Anesthesiology, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya, 466-8550, Japan

Submitted 26 September 2003 ; accepted in final form 13 February 2004


   ABSTRACT
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 ABSTRACT
METHODS
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Neuropeptide Y (NPY) elevates the permeability of cultured rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O2) possibly by binding to the NPY Y3 receptor. The present study evaluated the effects of NPY compared to vascular endothelial growth factor (VEGF). RAECs were cultured on the upper chamber base of a double-chamber culture system, FITC-labeled albumin was introduced into the chamber, and permeation into the lower chamber was measured. Treatment was with 3 x 10–7 M NPY or 10–7 g/ml VEGF for 2 h along with specific inhibitors. The VEGF receptor-2 tyrosine kinase inhibitor tyrphostin SU-1498 and the protein kinase C inhibitor bis-indolylmaleimide I (GF-109203X) suppressed the VEGF-induced increase in monolayer permeability but not that caused by NPY. Furthermore, although the action of NPY was blocked in a concentration-dependent manner by phospholipase C inhibitor 1-(6-{[(17{beta})-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), it was less sensitive than VEGF. However, the effects of both NPY and VEGF on the permeability of the RAEC monolayer were blocked with equal concentration dependence by STI571 (imatinib mesylate), which is an inhibitor of Abl tyrosine kinase in the nucleus and/or cytoplasm. The myosin light-chain kinase inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) suppressed both NPY- and VEGF-induced increment in permeability by ~70%, whereas the calmodulin-dependent kinase inhibitor DY-9760e could decrease to below the baseline. These results indicate that the NPY Y3-receptor subtype is specifically linked to the effects of STI571 on endothelial cells, and that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with altered intracellular or nuclear signal transduction.

STI571; Abl; hypoxia; Y3 receptor; myosin light-chain kinase; protein kinase C

NEUROPEPTIDE Y (NPY), which is released with norepinephrine from sympathetic nerve terminals when the nerves are overexcited during excessive stress (4), elicits vasoconstriction that may be mediated through the NPY Y1 and Y2 receptors (1). Recently NPY was also recognized as able to mediate increases in vascular permeability probably via the NPY Y3 receptor (12). The DNA sequence for the latter has yet to be elucidated; therefore, identification of the Y3-receptor subtype that participates in physiological functions has focused on the lack of response to the pancreatic peptide YY (PYY) and inhibitory actions of NPY18–36, a Y3 antagonist and also a Y2 agonist (15). Using such criteria, Hirabayashi et al. (12) reported that NPY is a candidate for elevating vascular permeability in the development of neurogenic pulmonary edema via actions on the Y3-receptor subtype. The same characteristics were also demonstrated in rat aortic endothelial cells (RAECs) in monolayer cultures under hypoxic conditions (5% O2) but not with normoxia (18). However, the intracellular mechanisms involved in such actions of NPY have yet to be evaluated.

Vascular permeability may be enhanced by a variety of endogenous substances including bradykinin and histamine. Vascular endothelial growth factor (VEGF) is a potent permeability factor that is associated with vascular endothelial cell growth (8), vascular neogenesis (21), and sprout formation (7). VEGF is also known as a mediator of cancer cell metastasis by facilitating their penetration of the endothelial layer (10). VEGF binds to two receptor subtypes, VEGF receptor-1 [VEGFR-1, also known as Fms-like tyrosine kinase-1 (Flt-1); Refs. 23, 30] and VEGF receptor-2 [VEGFR-2, also known as kinase insert domain-containing receptor/fetal liver kinase-1 (Kdr/Flk-1); Refs. 22, 29]. Recent studies on VEGF-induced permeability revealed that the binding of VEGF to VEGFR-2 initiates autophosphorylation of receptor-coupled tyrosine kinase (31). Consequently, VEGF activates many protein kinases involved in intracellular signal transduction including Rac; phospholipase C{gamma} (PLC{gamma}); protein kinase C (PKC)-{alpha}, -{beta}, and -{zeta}; extracellular regulated kinase (Erk1/2); protein kinase B (Akt); non-receptor-coupled tyrosine kinases (Src and focal adhesion kinase); mitogen-activated protein kinase (MAPK); and phosphatidylinositol 3-kinase (PI3-kinase) (7, 27, 28). Because VEGF is a growth factor for vascular endothelial cells, it may enhance DNA transcription in the nucleus. Recently nucleus protein kinase inhibitors such as STI571 (Gleevec imatinib mesylate; Novartis Pharmaceuticals) have been developed for cancer chemotherapy. STI571 potently inhibits Abl-family tyrosine kinases (c-Abl, v-Abl, and Bcr-Abl) in the nucleus and cytoplasm, which diminishes VEGF-evoked DNA transcription and cell proliferation and moderately or less-potently blocks receptor-coupled tyrosine kinases c-Kit, Flt-1, and Kdr, which are closely linked with the stem cell factor receptor, VEGFR-1, and VEGFR-2, respectively (2, 3). Dudek and Garcia (5) reported that studies of angiogenesis may provide encouraging insights into mechanisms regulating endothelial cell permeability, a key factor for angiogenic processes. However, few studies on the role of transcription factors in increasing vascular permeability have been performed.

Nan et al. (18) reported that NPY enhanced the RAEC monolayer permeability especially under hypoxic conditions (5% O2). Because intracellular cAMP concentrations obtained during hypoxia were much lower than those during normoxia (20% O2), the elevated permeability level obtained during hypoxia seemed to be correlated with a decrease in the intracellular cAMP content. Furthermore, intracellular Ca2+ concentration was increased with PYY to a similar extent as with NPY; therefore, NPY Y3-receptor-mediated permeability is not explained only by Ca2+ concentration changes. The present study was undertaken to evaluate signal transduction pathways for the permeating actions of NPY on RAECs in monolayer cultures compared to VEGF. Effects on increases in monolayer permeability were examined utilizing inhibitors of PKC, PI3-kinase, myosin light chain kinase (MLCK), calmodulin (CaM)-dependent kinase, VEGFR-2 tyrosine kinase, and PLC{gamma}, as well as the Abl protein kinase inhibitor STI571.


   METHODS
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 METHODS
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All procedures were undertaken in accordance with the Guiding Principles in the Care and Use of Animals in the Field of Physiological Sciences of the Physiological Society of Japan (20a) and with the prior approval of the Animal Care Committee of Aichi Medical University.

Isolation and culture of endothelial cells. RAECs were isolated from male Wistar rats (150–200 g, 7–9 wk old) and cultured according to the methods of Suh et al. (26). Rats were anesthetized with ketamine (50 mg/kg body wt im) and pentobarbital sodium (25 mg/kg body wt ip), and their aortas were removed and placed in phosphate-buffered saline (PBS, without Ca2+ or Mg2+). The vessels were cleaned, opened longitudinally, cut into two or three small pieces, and placed with their intimal side down on Matrigel-coated plates in growth medium (GM). The GM contained 10% FCS, 75 µg/ml endothelial cell growth supplement (ECGS), 10 U/ml heparin, 100 U/ml penicillin-streptomycin, 1% L-glutamine, and 100 µM MEM nonessential amino acids in DMEM. After 4–7 days, the pieces were removed and cells were harvested; >90% viability of primary cultured cells was obtained when assessed with Trypan blue. The cells were identified as endothelial cells when they elicited a positive response to anti-von Willebrand factor antibody (Dako) after being fixed with a 7:3 ratio of methanol and acetone.

Establishment of endothelial cell monolayers for permeability assays. The incubation culture plates comprised two chambers. The base of the upper chamber was a sieve with 3-µm pore size (Chemotaxicell; Kurabo; Osaka, Japan). We used 24-well microplates for the lower chamber. Before use, the upper chamber plate was coated with 50 µl of 50 µg/ml collagen IV and left to dry overnight in a laminar-airflow cabinet. The chambers were then sterilized by rinsing with 70% ethanol and were allowed to dry. RAECs were detached with trypsin-EDTA from culture plates, washed once with fresh GM, and seeded on the upper chamber at a density of 2 x 105 cells per well in 200 µl of GM. They were incubated at 37°C in 5% CO2-95% air for 3–4 days during which time the GM (300 µl) was changed daily.

Incubation of endothelial cells during hypoxia or normoxia. Double chambers with endothelial cell monolayers were then moved to another CO2 incubator so that the cells could be incubated under conditions of hypoxia (5% CO2-5% O2-90% N2) or normoxia (5% CO2-20% O2-75% N2) at 37°C. The incubator was connected to a vinyl bag in which CO2, O2, and N2 gases were mixed at a desired content ratio and pumped at an appropriate velocity. From a gas outlet, PCO2 and PO2 were monitored with inhaled anesthetic gases (isoflurane and sevoflurane) using a respiratory O2-CO2 analyzer (Capnomac; Datex Instrumentarium; Helsinki, Finland). Two hours were allowed to elapse before cells were treated with drugs, and throughout the experiment the incubator door was kept closed except when the incubation medium was changed (which took <3 min).

Measurement of endothelial permeability. Endothelial monolayer permeability was assessed with reference to the filtration velocity of FITC-labeled albumin from the upper to the lower chamber as previously described (14). The incubation medium in the upper chamber was changed to 300 µl of GM that contained a kinase inhibitor or PBS as a control, NPY-VEGF-PBS, and 1% FITC-labeled albumin, while 1 ml of GM that contained 1% BSA was introduced into the lower chamber. After 2 h of incubation, 100-µl aliquots were then aspirated from the lower chamber and diluted with PBS at a 1:5 ratio for measurement of FITC-labeled albumin concentrations. A fluorescent spectrophotometer (Fluoroskan Ascent FL; Labsystems) with an excitation wavelength of 485 nm and an emission wavelength of 538 nm was used for this task. Concentration-dependent responses to NPY and VEGF were first examined with administration of the agents at ranges of 10–8 to 10–6 M for NPY and 3 x 10–9 to 3 x 10–7 g/ml for VEGF.

To evaluate responses of RAECs in monolayer cultures to 3 x 10–7 M NPY and 10–7 g/ml VEGF, RAECs were treated with kinase inhibitors at the following concentrations: 10–5 to 10–10 M each of the MLCK inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9), the CaM-dependent kinase inhibitor DY-9760e, the VEGFR-2-receptor-coupled tyrosine kinase inhibitor tyrphostin SU-1498, the Abl tyrosine kinase inhibitor STI571, the phospholipase C (PLC) inhibitor 1-(6-{[(17{beta})-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), or the PKC inhibitor bis-indolylmaleimide I (GF-109203X); with 10–6 M each of the PI3-kinase inhibitor Wortmannin or the CaM kinase II inhibitor myristoyl-Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu (myristoylated AIP).

Materials. Matrigel-coated plates were purchased from Becton Dickinson (Bedford, MA); DMEM, FCS, trypsin-EDTA, penicillin-streptomycin, L-glutamine, and MEM nonessential amino acid solution were obtained from GIBCO Life Technologies (Eggenstein, Germany); ECGS, Trypan blue, collagen IV, Wortmannin, and FITC-labeled albumin were from Sigma (St. Louis, MO); heparin, BSA, fluo 3-acetoxymethyl ester and Nonidet P-40 were from Wako (Osaka, Japan); Chemotaxicell was from Kurabo (Osaka, Japan); 96-and 24-well cell culture plates and poly-L-lysine were from Becton Dickinson Biosciences (Franklin Lakes, NJ); NPY was from Bachem; VEGF was from R&D Systems; and STI571 and DY-9760e were kindly supplied by Novartis Pharmaceuticals and Daiichi Pharmaceutical (Tokyo), respectively. Tyrphostin SU-1498 and GF-109203X were purchased from LC Laboratories (Boston, MA); U-73122 was from Cayman Chemical; and ML-9 and myristoylated AIP were from Biomol Research Laboratories. Tyrode's solution was composed (in mM) of 137 NaCl, 2.7 KCl, 1 MgCl2, 1.8 CaCl2, 0.2 NaH2PO4, 12 NaHCO3, and 5.5 glucose. ML-9, tyrphostin SU-1498, STI571, and U-73122 were dissolved in glycine + NaCl solution (Sorensen buffer solution; pH was adjusted to 4.5 with 0.1 M HCl) as stock solutions and were diluted with PBS to 0.1% before use. A preliminary study showed that Sorensen buffer solution did not affect the RAEC monolayer permeability.

Statistical analysis. Differences between means were examined for significance with ANOVA. Statistical significance was evaluated by Scheffé's method (24) at levels of 0.05 and 0.01 with values expressed as means ± SE.


   RESULTS
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 RESULTS
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Concentration-response curves for effects of NPY and VEGF on RAEC monolayer permeability. NPY increased the FITC-labeled albumin concentration in the lower chamber [i.e., the permeability under hypoxic conditions (P < 0.001)] in a concentration-dependent manner and only increased permeability to a small extent during normoxia as shown in Fig. 1A. VEGF concentration-dependently increased the monolayer permeability during normoxia (P < 0.05), and the permeability increased to a much greater extent during hypoxia (P < 0.001) as shown in Fig. 1B. The maximum increase in FITC-labeled albumin concentration obtained during hypoxia was greater than that during normoxia (P < 0.001 in NPY; P < 0.01 in VEGF). The maximum responses to NPY obtained under hypoxic conditions were almost similar to those obtained with VEGF, and the EC50 value for NPY (5 x 10–8 M) was ~77 times greater than for VEGF (3 x 10–8 g/ml) when the molecular mass of VEGF was assumed to be 46 kDa.



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  		Fig. 1. Concentration-response curves for rat aortic endothelial cell (RAEC) monolayer permeability with neuropeptide Y (NPY; A) and vascular endothelial growth factor (VEGF; B). Permeability responses are expressed as FITC-labeled albumin concentrations in the lower-chamber medium measured when 2 h had elapsed after the administration of FITC-labeled albumin into the upper chamber. Data obtained during hypoxia (5% O2; {square} and {circ}) and normoxia (20% O2; {blacksquare} and {bullet}). *P < 0.05; **P < 0.01; ***P < 0.001 vs. absence of NPY or VEGF (no drug); {dagger}P < 0.05; {dagger}{dagger}P < 0.01; {dagger}{dagger}{dagger}P < 0.001 vs. hypoxia; n = 4 animals.

 
Effects of ML-9 and DY-9760e on NPY- and VEGF-induced elevation of RAEC monolayer permeability during hypoxia. ML-9 increased the FITC-labeled albumin concentration in the lower chamber in a concentration-dependent fashion (P < 0.05) and concentration-dependently diminished both NPY- and VEGF-induced increases in permeability (Fig. 2, A and B, respectively). The greatest concentration (10–7 M) of ML-9 lowered the NPY- or VEGF-induced increase in FITC-labeled albumin concentration to a level almost similar to that obtained with the same concentration of ML-9 alone.



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  		Fig. 2. Effects of myosin light chain kinase (MLCK) inhibitor 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine HCl (ML-9) on NPY- and VEGF-induced increases (A and B, respectively) in RAEC monolayer permeability. Permeability responses are expressed as FITC-labeled albumin concentrations in the lower-chamber medium and were measured when 2 h had elapsed after administration of FITC-labeled albumin into the upper chamber. Concentration of NPY used was 3 x 10–7 M; that of VEGF was 10–7 g/ml. Shown are NPY- or VEGF-induced permeability responses in the presence of each concentration of ML-9 ({square} and {circ}) and responses to ML-9 alone ({blacksquare} and {bullet}). **P < 0.01; ***P < 0.001 vs. ML-9 alone; {dagger}P < 0.05 vs. no drug; n = 4 animals.

 
DY-9760e, a CaM-dependent kinase inhibitor, concentration-dependently decreased the FITC-labeled albumin concentration in the lower chamber (P < 0.05) and concentration-dependently diminished both NPY- and VEGF-induced increases in permeability (Fig. 3, A and B, respectively). The greatest concentration (10–6 M) of DY-9760e lowered the NPY- or VEGF-induced increase in FITC-labeled albumin concentration to a level almost similar to that obtained with the same concentration of DY-9760e alone.



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  		Fig. 3. Effects of calmodulin (CaM)-dependent protein kinase inhibitor DY-9760e on NPY- and VEGF-induced increases (A and B, respectively) in RAEC monolayer permeability. Permeability responses are expressed as FITC-labeled albumin concentrations in the lower-chamber medium, which was measured when 2 h had elapsed after administration of FITC-labeled albumin into the upper chamber. NPY concentration was 3 x 10–7 M; that of VEGF was 10–7 g/ml. Shown are NPY- or VEGF-induced permeability responses in the presence of each concentration of DY-9760e ({square} and {circ}) and responses to DY-9760e alone ({blacksquare} and {bullet}). *P < 0.05; **P < 0.01; ***P < 0.001 vs. DY-9760e alone; {dagger}P < 0.05 vs. no drug; n = 4 animals.

 
Effects of tyrphostin SU-1498 on NPY- and VEGF-induced elevations of RAEC monolayer permeability in hypoxia. Tyrphostin SU-1498, a VEGFR-2 tyrosine kinase inhibitor, clearly inhibited permeability responses to VEGF in a concentration-dependent fashion (P < 0.001 compared to NPY) but exerted no inhibitory action in the case of NPY (Fig. 4). The highest concentration of tyrphostin SU-1498 completely inhibited the VEGF-induced permeability response to the baseline level obtained without any drug. Tyrphostin SU-1498 alone hardly affected the RAEC monolayer permeability.



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  		Fig. 4. Effects of tyrphostin SU-1498 on NPY- and VEGF-induced increases in RAEC monolayer permeability. Permeability response is expressed as the percentage of the maximum response to NPY ({square}) or VEGF ({bullet}) obtained at high concentrations (3 x 10–7 M and 10–7 g/ml, respectively) after subtracting basal permeability values without NPY and VEGF. *P < 0.05; ***P < 0.001 vs. NPY; n = 4 animals.

 
Effects of U-73122 and GF-109203X on NPY- and VEGF-induced elevations of RAEC monolayer permeability under hypoxic conditions. As shown in Fig. 5A, the PLC inhibitor U-73122 inhibited both VEGF- and NPY-induced increases in monolayer permeability in a concentration-dependent fashion. However, U-73122 inhibited the NPY-induced responses less potently than those caused by VEGF (P < 0.05). The highest concentration of U-73122 inhibited the permeability responses of the RAEC monolayer to either NPY or VEGF to slightly below the zero line. Alone, U-73122 decreased the baseline of permeability in the absence of NPY and VEGF (but not significantly).



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  		Fig. 5. Effects of 1-(6-{[(17{beta})-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122; A) and bis-indolylmaleimide I (GF-109203X; B) on NPY- and VEGF-induced increases in RAEC monolayer permeability. Permeability responses are expressed as a percentage of the maximum response to NPY ({square}) or VEGF ({bullet}) obtained at high concentration (3 x 10–7 M and 10–7 g/ml, respectively) after subtraction of basal permeability levels without NPY and VEGF. *P < 0.05; **P < 0.05; ***P < 0.05 vs. NPY; n = 4 animals.

 
The PKC inhibitor GF-109203X inhibited the VEGF-induced increase in monolayer permeability in a dose-dependent manner. The increase in permeability caused by NPY, in contrast, was hardly affected (P < 0.001 compared to VEGF; Fig. 5B). The highest concentration of GF-109203X completely inhibited the permeability responses to VEGF to the zero level. GF-109203X alone, i.e., in the absence of NPY and VEGF, hardly affected the RAEC monolayer permeability.

Effects of STI571 on NPY- and VEGF-induced elevations of RAEC monolayer permeability in hypoxia. The Abl tyrosine kinase inhibitor STI571 concentration-dependently inhibited both NPY- and VEGF-induced elevations of RAEC monolayer permeability as shown in Fig. 6. The dose-response curves of STI571 obtained against NPY- and VEGF-induced increases in permeability were very similar; IC50 values were almost 2 x 10–8 M. The highest concentration of STI571 completely inhibited the permeability responses of RAECs to either NPY or VEGF to the baseline level. STI571 alone (in the absence of NPY and VEGF) hardly affected the RAEC monolayer permeability.



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  		Fig. 6. Effects of STI571 (Gleevec imatinib mesylate) on NPY- and VEGF-induced increases in RAEC monolayer permeability. Permeability responses are expressed as a percentage of the maximum response to NPY ({square}) or VEGF ({bullet}) obtained at high concentration (3 x 10–7 M and 10–7 g/ml, respectively) after subtraction of basal permeability levels without NPY and VEGF; n = 4 animals.

 
Effects of Wortmannin and myristoylated AIP on NPY- and VEGF-induced elevations of RAEC monolayer permeability during hypoxia. The PI3-kinase inhibitor Wortmannin (10–6 M) showed no effects on either NPY- or VEGF-induced increases in RAEC monolayer permeability (Table 1). Wortmannin alone hardly affected the RAEC monolayer permeability in the absence of NPY and VEGF. The CaM kinase II inhibitor myristoylated AIP (10–6 M) strongly inhibited both NPY- and VEGF-induced permeability to a level below the baseline value obtained without any agents. Myristoylated AIP alone decreased the FITC-labeled albumin concentration in the absence of NPY and VEGF from 748 ± 138 to 253 ± 102 mg/l.


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  		Table 1. Effects of protein kinase C inhibitors on neuropeptide Y-induced and vascular endothelial growth factor-induced RAEC monolayer permeability

 

   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
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Dudek and Garcia (5) recently reviewed the intracellular signal transduction mechanism for vascular endothelial permeability with a special reference to two different forces: contractile and tethering. Briefly, a disturbance between these forces such as greater intensity for the contractile compared with the tethering force may induce an imbalance between barrier dysfunction and integrity and lead to increased enhancement of vascular permeability. Endothelial cell contraction may be caused by the actin-myosin interaction being regulated in an MLCK-dependent manner and maintained by Rho kinase, which inhibits myosin light-chain phosphatase. Besides such an MLCK-dependent mechanism, an increase in endothelial permeability induced by PKC activation may occur in an MLCK-independent manner and result in actin polymerization through phosphorylation of caldesmon, an actin-, myosin-, and CaM-binding protein that is present in actomyosin cross-bridges. Caldesmon-mediated regulation of actomyosin ATPase may be modified by the actin cross-linking proteins filamin and gelsolin; the former participates in barrier regulation via CaM kinase II activation. Thus endothelial cell permeability may be evoked through stress-fiber formation, which is associated with an MLCK-dependent pathway and an MLCK-independent path such as actin polymerization. On the other hand, the tethering forces may be derived from cell-to-cell and cell-to-matrix contacts, mediated by molecules such as occludin, cadherin, and platelet endothelial cell adhesion molecule-1, which are linked to the endothelial cytoskeleton. The regulatory mechanism for the tethering forces, however, is still to be evaluated and is possibly associated with activation of tyrosine and/or serine-threonine kinases, and so on, involved in cell proliferation and migration.

The results obtained in the present study showed that ML-9 and DY-9760e inhibited permeability responses of RAECs to both NPY and VEGF, which indicates the possible participation of MLCK- and CaM-dependent kinases in NPY- and VEGF-induced permeability. These kinases may be activated via an increase in intracellular Ca2+ released by inositol 1,4,5-trisphosphate, which is a product of PLC (11), but previously Nan et al. (18) showed a loss of difference in increasing intracellular Ca2+ concentration between NPY and PYY, the latter of which elicited no action on permeability. Such a controversial result suggests that an MLCK-mediated actin-myosin interaction may modulate the stress-fiber formation in association with the MLCK-independent pathway. But whether NPY causes such an MLCK-independent actin polymerization is still unclear. Furthermore, high concentrations of DY-9760e significantly lowered the baseline permeability as obtained with no drugs (see Fig. 5). Similarly, the CaM kinase II inhibitor myristoylated AIP (10–6) strongly lowered the FITC-labeled albumin concentration in the presence of NPY or VEGF to below the baseline level. These results indicate that a CaM-dependent protein kinase, possibly CaM kinase II, may play a role in maintaining the basal permeability level.

Increased RAEC monolayer permeability caused by NPY differed from that induced by VEGF at least with regard to sensitivity to the VEGFR-2-coupled tyrosine kinase inhibitor tyrphostin SU-1498, GF-109203X, and U-73122. The PKC inhibitor GF-109203X inhibited the VEGF-evoked permeability elevation in a concentration-dependent manner but did not exert any inhibitory action in the NPY case. The IC50 value for U-73122 (a PLC inhibitor) in inhibiting the permeability changes caused by NPY was much greater than that for VEGF. Because PKC activity may depend on the amount of diacylglycerol present, which is another product of PLC (11), the difference between NPY and VEGF in sensitivity to the PLC inhibitor indicates that different subtypes of PLC and PKC may be involved in NPY- and VEGF-induced increases in RAEC monolayer permeability. Reportedly, PKC activation induced by phorbol myristate acetate, tumor necrosis factor, or pertussis toxin enhances bovine pulmonary endothelial cell permeability but not human umbilical vein endothelial cell permeability, which likely reflects differences in PKC isotype-specific expression (5, 25). In the former, the permeability responses were observed without significant increases in myosin light-chain phosphorylation. In such a context, a PKC-mediated, MLCK-independent pathway may be involved in the VEGF-induced increase in RAEC monolayer permeability but not in the NPY-induced change.

Furthermore, in the present study, STI571 clearly inhibited both VEGF- and NPY-induced elevations of RAEC monolayer permeability in a concentration-dependent fashion and with similar IC50 values. According to the manufacturer's data for STI571 (obtained by in vitro estimation; Ref. 2), the IC50 values reported for the inhibitory actions on Abl-family protein kinases such as c-Abl, v-Abl, and Bcr-Abl were 0.025–0.038 µM, which are almost identical to those obtained for the inhibitory action of STI571 in the present study. These values are much smaller (1:1,000) than those for VEGFR-2 tyrosine kinase Kdr and VEGFR-1 tyrosine kinase Flt-1, which were 10.7 and 19.6 µM, respectively. Multiple structural and functional domains have been found in the Abl molecules, especially in the DNA- and actin-binding domains (20). Thus Abl activation exerts a regulatory function in cell proliferation, survival, and apoptotic response depending on cell type or specific extracellular stimuli. Because Abl-family tyrosine kinases are transcription factors that exist in the nucleus, STI571 is used as a therapeutic drug for chronic myelogenous leukemia, lung cancers, and gastrointestinal stromal tumor (2, 3, 13). Abl molecules locate in the cytoplasm as well and act as a non-receptor-coupled protein tyrosine kinase and interact with actin to participate in cell migration (32). Woodring et al. (33) suggested that reciprocal regulation between F-actin and the c-Abl tyrosine kinase may provide a self-limiting mechanism in the control of cytoskeleton dynamics: purified c-Abl tyrosine kinase may be inhibited by F-actin, and in turn, occurrence of F-actin microspikes may depend upon Abl activity. Platelet-derived growth factor (PDGF) and epithelium growth factor (EGF) may activate Abl in fibroblasts and evoke plasma membrane ruffling (lamellipodial exploration) and filopodial exploration through actin polymerization (32), possibly independent of MLCK. Whether such a role for Abl is closely related to the NPY-induced increase in permeability across RAEC monolayers, i.e., through another MLCK-independent pathway, is still to be evaluated.

Although Kdr is known as a tyrosine kinase whose activation evokes vascular permeability with VEGF (16, 31, 34), the IC50 values for STI571 differed between the VEGF-induced increase in permeability and Kdr activities. In this context, the possibility that VEGF-induced permeability may be mediated through Abl activation in nucleus and/or cytoplasm deserves consideration. It was recently reported that NPY stimulates cell proliferation in several tissues via binding to NPY Y1- or Y2-receptor subtypes (35). It remains unclear whether proliferation of RAECs may also be induced by NPY mediated by the NPY Y3-receptor subtype, and whether such a proliferation may be responsible for the increase in permeability (12).

Wortmannin, an inhibitor of PI3-kinase, elicited no inhibitory actions on either NPY- or VEGF-induced increases in permeability in the present study, which suggests that PI3-kinase does not play a role in increasing permeability. It is therefore interesting that Takahashi et al. (27) demonstrated that VEGF-induced activation of Raf-MEK-MAP kinase and DNA synthesis are mainly mediated by a PKC-dependent pathway rather than by Ras-dependent or PI3-kinase-dependent pathways.

Thus far, all NPY-receptor subtypes except the Y3-receptor subtype are GTP-binding-protein-coupled receptors (15). Because STI571 showed no inhibitory action on NPY-induced vascular smooth muscle contraction caused through NPY Y1- and Y2-receptor subtypes (1, 6), signal transduction through Y1- and Y2-receptor subtypes seemed different from that through the Y3-receptor subtype, which may be specifically linked to Abl in endothelial cells. Lack of any inhibitory action of tyrphostin SU-1498 on NPY-induced monolayer permeability suggests that NPY may directly elicit permeability through NPY Y3-receptor binding but not indirectly by releasing VEGF as reported by Nan et al. (18). As additional evidence for support of direct action of NPY, differences in the actions between NPY and VEGF were obtained in the inhibitory responses of RAEC monolayers to PLC or PKC inhibitors during hypoxia. In experiments performed under hypoxic conditions, VEGF may be released from endothelial cells (7) and thereby increase the permeability, but it generally takes a long time for these effects to appear. It was reported that the amount of VEGF produced in human umbilical vein endothelial cells begins to increase from 24 h after incubation with hypoxia (5% O2) and then levels off at 48 h (17). Because the present study was carried out within 2–5 h after hypoxia was induced, it is not likely that VEGF contributed to the NPY-induced elevation of permeability (18). Nevertheless, the other unknown pathway through VEGF receptors or facilitated release cannot be ruled out.

Furthermore, the permeability responses obtained for both VEGF and NPY during hypoxia were significantly greater than those during normoxia. Although the mechanisms involved have yet to be elucidated, hypoxia may enhance the effects of both VEGF and NPY on RAEC monolayer permeability, probably by interacting with a signal transduction pathway that causes a decrease in cAMP (9, 18). Noll et al. (19) described NPY as reducing macromolecule permeability across coronary endothelial monolayers through modulation of cAMP-dependent signal transduction. Respecting such a difference in the permeability results obtained with NPY, Nan et al. (18) pointed out differences in O2 tension, vascular beds, and NPY-receptor subtypes.

In conclusion, NPY elicits permeability action on RAEC monolayers under hypoxic conditions to a greater extent than during normoxia with much less potency and almost similar efficacy when compared to VEGF. It is likely that a PKC-mediated pathway may be involved in the VEGF-induced increase of permeability, but this pathway likely has little effect on NPY-induced increase. It is noteworthy that STI571, an Abl tyrosine kinase inhibitor that acts in the nucleus and/or cytoplasm, equally inhibited the effects of both NPY and VEGF on permeability, which suggests a close relationship with signal transduction pathways for their actions. NPY- or VEGF-induced increases in RAEC monolayer permeability are still to be evaluated with special respect for participation of Abl tyrosine kinase and interaction with tethering or contractile forces. Taken together, we propose that NPY, a sympathetic coneurotransmitter, may increase vascular permeability in association with intracellular and/or nucleus signal transduction via the NPY Y3-receptor subtype.


   GRANTS
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 ABSTRACT
 METHODS
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 DISCUSSION
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This study was supported in part by Grant 12670097 from the Japanese Ministry of Education, Science, Sports, Culture, and Technology, Japan.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Ishikawa, Dept. of Pharmacology, Aichi Medical Univ., School of Medicine, Nagakute, Aichi Gun, Aichi Prefecture 480-1195, Japan (E-mail: nao{at}aichi-med-u.ac.jp).

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.


   REFERENCES
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 RESULTS
 DISCUSSION
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