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The cAMP-PKA cascade is a recognized signaling pathway important in inhibition of inflammatory injury events such as endothelial permeability and leucocyte trafficking, and a critical target of regulation is believed to be inhibition of Rho proteins. Here, we hypothesize that PKA directly phosphorylates GTP dissociation inhibitor (GDI) to negatively regulate Rho activity. Amino acid analysis of GDI[alpha] showed two potential protein kinase A (PKA) phosphorylation motifs, [Ser.sup.174] and [Thr.sup.182]. Using in vitro kinase assay and mass spectrometry, we found that the purified PKA catalytic subunit phosphorylated GDI[alpha]-GST fusion protein and PKA motif-containing GDI[alpha] peptide at [Ser.sup.174], but not [Thr.sup.182]. Transfection of COS-7 cells with mutated full-length GDI[alpha] at [Ser.sup.174] to [Ala.sup.174] (GDI[alpha]Ser.sup.174A]) abrogated the ability of cAMP to phosphorylate GDIct. However, mutation of [Thr.sup.182] to [Ala.sup.182] (GDI[alpha]-[Thr.sup.182]) did not abrogate, and cAMP increased phosphorylation of GDI[alpha] to a similar extent as wild-type GDI[alpha] transfectants. The mutant GDI[alpha]-[Ser.sup.174A], but not GDI[alpha][Thr.sup. 182] was unable to prevent cAMP-mediated inhibition of Rho-dependent serum-response element reporter activity. Furthermore, the mutant GDI[alpha]-[Ser.sup.174A] was unable to prevent the thrombin-induced RhoA activation. Coprecipitation studies indicated that neither mutation of the PKA consensus sites nor phosphorylation alter GDIct binding with RhoA, suggesting that phosphorylation of [Ser.sup.174] regulated preformed GDI[alpha]-RhoA complexes. The findings provide strong support that the selective phosphorylation at [Ser.sup.174] by PKA is a signaling pathway in the negative regulation of RhoA activity and therefore could be a potential protective mechanism for inflammatory injury. adenosine 3',5'-cyclic monophosphate-dependent protein kinase; protein kinase A consensus phosphorylation sites; single-site mutated GDI[alpha]

Abstract: We investigated the activity of [P.sup.21]-activated kinase-1 (Pakl) on myosin light chain phosphorylation and on thrombin-induced barrier dysfunction in human endothelial cells (HMEC). HMEC were infected with recombinant adenoviruses [...]

Abundant evidence indicates that lysophosphatidylcholine (LPC) is proinflammatory and atherogenic. In the vascular endothelium, LPC increases permeability and expression of proinflammatory molecules such as adhesion molecules and cytokines. Yet, mechanisms by which LPC mediates these activities remain unclear and controversial. Recent evidence implicates involvement of a novel subfamily of G protein-coupled receptors (GPR4, G2A, OGR1, and TDAG8) that are sensitive to lysolipids and protons. We previously reported that one of these receptors, GPR4, is selectively expressed by a variety of endothelial cells and therefore hypothesize that the LPC-stimulated endothelial barrier dysfunction is mediated through GPR4. We developed a peptide Ab against GPR4 that detected GPR4 expression in transfected COS 7 cells and endogenous GPR4 expression in endothelial cells by Western blot. Endothelial cells infected with a retrovirus containing small interference RNA (siRNA) to GPR4 resulted in 40-50% decreased GPR4 expression, which corresponded with partial prevention of the LPC-induced 1) decrease in transendothelial resistance, 2) stress fiber formation, and 3) activation of RhoA. Furthermore, coexpression of the siRNA-GPR4 with a siRNA-resistant mutant GPR4 fully restored the LPC-induced resistance decrease. However, extracellular pH of endothelial resistance; small interference ribonucleic acid; actin; RhoA

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid that can be generated by pathological activities. We investigated the hypothesis that LPC signals increase in endothelial permeability. Stimulation of human dermal microvascular endothelial cells and bovine pulmonary microvascular endothelial cells with LPC (10-50 [micro]M) induced decreases (within minutes) in transendothelial electrical resistance and increase of endothelial permeability. LPC activated (within 5 min) membrane--associated PKC phosphotransferase activity in the absence of translocation. Affinity-binding analysis indicated that LPC induced increases (also by 5 min) of GTP-bound RhoA, but not Rac 1 or Cdc42. By 60 min, both signaling pathways decreased toward baseline. Inhibition of RhoA with C3 transferase inhibited ~50% of LPC-induced resistance decrease. Pretreatment with PKC inhibitor Go6983 (concentrations selective for classic PKC), PMA-induced depletion of PKC[alpha], and transfection of antisense PKC[alpha] oligonucleotide each prevented 40-50% of the LPC-induced resistance decrease. Furthermore, these three PKC inhibition strategies inhibited 60-80% of the LPC-induced GTP-bound RhoA. These results show that LPC directly impairs the endothelial barrier function that was dependent, at least in part, on cross talk of PKC[alpha] and RhoA signals. The evidence indicates that elevated LPC levels can contribute to the activation of a proinflammatory endothelial phenotype. protein kinase C; signal transduction

The atherogenic serum lysophosphatidycholine (LPC) is known to mediate vascular endothelial responses ranging from upregulation of adhesion molecules and growth factors to secretion of chemokines and superoxide anion. We investigated whether endothelial cells express receptors for LPC, which may account for their actions. Human brain microvascular (HBMEC) and dermal microvascular endothelial cells (HMEC) were prepared for RT-PCR analysis for possible expression of the G protein-coupled receptors, GPR4 and G2A, which are believed to be specific LPC receptors. Results, indicated that HBMEC expressed low basal GPR4 MRNA, but stimulation with tumor necrosis factor-[alpha] (TNF-[alpha]) (100 U/ml) or [H.sub.2][O.sub.2] (50 [micro]mol/l) for 2 h or overnight upregulated expression severalfold. In contrast, HMEC expressed high basal GPR4 mRNA, which was not further increased by either TNF-[alpha] or [H.sub.2][O.sub.2] stimulation. Another LPC receptor, G2A, was not detected in either endothelial cell type. Competition binding studies were made to evaluate specific binding of [[sup.3]H]LPC to the intact endothelial cell monolayer. Basal specific [[sup.3]H]LPC binding in HBMEC was approximately eight times lower than in HMEC; however, TNF-[alpha] or [H.sub.2][O.sub.2] stimulation increased [[sup.3]H]LPC binding on HMBEC but not HMEC. The results indicated that GPR4 expression was consistent with specific [[sup.3]H]LPC binding. Overall, we report that endothelial cells selectively expressed GPR4, a specific LPC receptor. Furthermore, GPR4 expression by HBMEC, but not HMEC, was increased by inflammatory stresses. We conclude that endogenous GPR4 in endothelial cells may be a potential G protein-coupled receptor by which LPC signals proinflammatory activities. G protein-coupled receptors 4 and 2A; oxidants; tumor necrosis factor-[alpha]; specific binding

Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 [micro]M forskolin plus 1 [micro]M IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of [sup.32]P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of ~23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction. Rho guanosine 5'-triphosphate; protein kinase A inhibitor; guanine nucleotide dissociation inhibitor; endothelial resistance

Shear stress stimulates nitric oxide (NO) production by phosphorylating endothelial NO synthase (eNOS) at [Ser.sup.1179] in a phosphoinositide-3-kinase (PI3K)- and protein kinase A (PKA)-dependent manner. The eNOS has additional potential phosphorylation sites, including [Ser.sup.116], [Thr.sup.497], and [Ser.sup.635]. Here, we studied these potential phosphorylation sites in response to shear, vascular endothelial growth factor (VEGF), and 8-bromocAMP (8-BRcAMP) in bovine aortic endothelial cells (BAEC). All three stimuli induced phosphorylation of eNOS at [Ser.sup.635], which was consistently slower than that at [Ser.sup.1179]. [Thr.sup.497] was rapidly dephosphorylated by 8-BRcAMP but not by shear and VEGF. None of the stimuli phosphorylated [Ser.sup.116]. Whereas shear-stimulated [Ser.sup.635] phosphorylation was not affected by phosphoinositide-3-kinase inhibitors wortmannin and LY-294002, it was blocked by either treating the cells with a PKA inhibitor H89 or infecting them with a recombinant adenovirus-expressing PKA inhibitor. These results suggest that shear stress stimulates eNOS by two different mechanisms: 1) PKA- and PI3K-dependent and 2) PKA-dependent but PI3K-independent pathways. Phosphorylation of [Ser.sup.635] may play an important role in chronic regulation of eNOS in response to mechanical and humoral stimuli. endothelial cells; vascular endothelial growth factor; cAMP

Vascular endothelial cells express isoforms of protein kinase A inhibitor. Am J Physiol Cell Physiol 282: C59-C66, 2002. First published September 5, 2001; 10.1152/ ajpcell.00256.2001.--The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI[alpha], human PKI[gamma] and mouse PKI[beta] sequences detected PKI[alpha] and PKI[gamma] mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI[alpha] and PKI[gamma] were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI[alpha] mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI[gamma] mRNA. Western blot analysis indicated that PKI[gamma] protein was detected in all three cell types, whereas PKI[alpha] was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI[alpha] and PKI[gamma], which may be physiologically important in endothelial barrier function. cAMP; protein kinase inhibitor; endothelial resistance; barrier function

Immunofluorescence studies and cytosolic calcium determination were undertaken to determine the effects of protein kinase C (PKC) activation on the synthesis and activity of the proteolytically activated thrombin receptor 1 (PAR-1). Results indicate that PKC-catalyzed phosphorylation of PAR-1 leads to receptor inactivation. Further, PKC contributes to the loss of PAR-1 on the cell surface and to the heterologous desensitization of the receptor as well as to the inhibition of cellular signal transduction through calcium.

The thrombin receptor domains responsible for receptor activation in endothelial cells were studied using site-specific antibodies. Results of experiments on polyclonal antibodies directed against peptide sequences corresponding to postulated binding sites of thrombin suggested a critical role for the thrombin receptor's NH2-terminal extension and loop 2 sites. These binding sites influenced receptor activation in endothelial cells, which in turn lead to increased intracellular calcium and transendothelial permeability to albumin.

Hydrogen peroxide-induced increase in Ca2+ determines the increased permeability of endothelium. Hydrogen peroxide increases the Ca2+ intake within 10 seconds of loading the bovine pulmonary microvessel endothelial cells (BMVEC) with Ca2+ sensitive membrane permeant fluorescent dye fura 2-AM. Pretreatment of BMVEC monolayers with Ca2+ chelator, BAPTA-AM (1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl) decreases hydrogen peroxide-induced increase in endothelial permeability. This increase is independent of Ca2+ influx.

The integrin binding to extracellular matrix proteins controls a crucial determinant of the endothelial permeability and alpha-thrombin and Gly-Arg-Gly-Asp-Ser-Pro increases endothelial cell monolayer permeability by various mechanisms. Using cultured bovine pulmonary microvascular endothelial cell monolayers and the calculation of hydraulic conductivity, the contribution of integrin receptors to the regulation of endothelial permeability was studied.

Rise in the vascular endothelial permeability in response to histamine and alpha-thrombin inflammatory mediators results in the activation of two regulatory pathways, protein kinase C (PKC) and myosin light-chain kinase (MLCK), by second messengers to damage the endothelial barrier function. The PKC pathway regulates actin-based systems and the phosphorylation of linking proteins to cause actin redistribution, high paracellular transport and cell rounding, while the MLCK pathway results in centripetal cell retraction. Cadherins linked to actin-based systems modulate the development of actin-related tight junctions.

TRAP-14, the synthetic thrombin receptor peptide, and alpha thrombin lead to pulmonary vasoconstriction of guinea pig lungs. TRAP-14 and alpha thrombin lead to increase in Ca ion concentrations in the smooth muscle cells of pulmonary artery and inverse desensitization of vasoconstriction, suggesting an enzymatic cleavage of thrombin's receptor, leading to pulmonary vasoconstriction. The vascular smooth muscle cells become the site of action.

A decline in endothelial hydraulic conductivity (Lp), a measure of liquid permeability across endothelial and matrix barrier, is affected by albumin and lectin Ricinus communis agglutinin (RCA) in bovine pulmonary microvascular endothelial cell monolayers. This effect of albumin and RCA is consistent with the response associated with the binding of albumin and RCA to extracellular matrix (ECM). The permeability-reducing effect of these molecules is hindered by the administration of protamine sulfate, a polycation, thus suggesting the mechanism whereby albumin and RCA interact with ECM and render the endothelial layer to function as a barricade to liquid infiltration.

An assay of the kinetics of the binding of fibroblasts to endothelin (ET), a 21-amino acid peptide possessing vasoconstricting features, reveals the outline of the molecluar mechanism involved in the cross-linking of endothelial cells and fibroblasts, relating it to the molecular mechanisms of the smooth muscle and dermal fibroblasts. This raises speculations about the possible function of the peptide ET in site-directed activity in the orbital connective tissue. The reactivity of orbital fibroblasts to ET-1 of 10(-9) moles is accompanied by a steep transient rise in free intracellular Ca2+ concentration.

Serine/threonine (Ser/Thr) protein phosphatases (PPs) are implicated in the recovery from endothelial barrier dysfunction caused by inflammatory mediators. We hypothesized that Ser/Thr PPs may regulate protein kinase C (PKC), a critical signaling molecule in barrier dysfunction, in the promotion of barrier recovery. Western analysis indicated that bovine pulmonary microvascular endothelial cells (BPMECs) expressed the three major Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506 (a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A or okadaic acid potentiated the thrombin-induced increase in PKC phosphotransferase activity. FK506 also potentiated thrombin-induced PKC-[Alpha] but not PKC-[Beta] phosphorylation. FK506 but not calyculin A or okadaic acid inhibited recovery from the thrombin-induced decrease in transendothelial resistance. Neither FK506 nor okadaic acid altered the thrombin-induced resistance decrease, whereas calyculin A potentiated the decrease. Downregulation of PKC with phorbol 12-myristate 13-acetate rescued the FK506-mediated inhibition of recovery, which was consistent with the finding that the thrombin-induced phosphorylation of PKC-[Alpha] was reduced during the recovery phase. These results indicated that PP2B may play a physiologically important role in returning endothelial barrier dysfunction to normal through the regulation of PKC. protein kinase C-[Alpha]; transendothelial resistance; FK506; endothelial permeability

Lum, Hazel, and Kenneth A. Roebuck. Oxidant stress and endothelial cell dysfunction. Am J Physiol Cell Physiol 280: C719-C741, 2001.--Reactive oxygen species (ROS) are generated at sites of inflammation and injury, and at low levels, ROS can function as signaling molecules participating as signaling intermediates in regulation of fundamental cell activities such as cell growth and cell adaptation responses, whereas at higher concentrations, ROS can cause cellular injury and death. The vascular endothelium, which regulates the passage of macromolecules and circulating cells from blood to tissues, is a major target of oxidant stress, playing a critical role in the pathophysiology of several vascular diseases and disorders. Specifically, oxidant stress increases vascular endothelial permeability and promotes leukocyte adhesion, which are coupled with alterations in endothelial signal transduction and redox-regulated transcription factors such as activator protein-1 and nuclear factor-[Kappa]B. This review discusses recent findings on the cellular and molecular mechanisms by which ROS signal events leading to impairment of endothelial barrier function and promotion of leukocyte adhesion. Particular emphasis is placed on the regulation of cell-cell and cell-surface adhesion molecules, the actin cytoskeleton, key protein kinases, and signal transduction events. endothelial permeability; leukocyte transmigration; actin filament; cadherin; occludin; intercellular adhesion molecule-1; selectins; redox-sensitive signal transduction; nuclear factor-[Kappa]B

Lum, Hazel, H. Ari Jaffe, Irena T. Schulz, Anwar Masood, Amlan RayChaudhury, and Richard D. Green. Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction. Am. J. Physiol. 277 (Cell Physiol. 46): C580-C588, 1999.--We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An [E1.sup.-], [E3.sup.-], replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 [micro]M thrombin increased transendothelial albumin clearance rate (0.012 [+ or -] 0.003 and 0.035 [+ or -] 0.005 [micro]l/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKIresulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction ofluciferase activity. The results show that Ad-mediated overexpression PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function. endothelial permeability; adenovirus; cAMP-dependent protein kinase