Ghassan Bkaily

Function of the endothelinB receptor in cardiovascular physiology and pathophysiology

One of the two receptors by which the potent vasoactive effects of endothelin (ET)-1 are mediated is the ET(B) receptor (ET(BR)), which is found in several tissues, but, more importantly from a cardiovascular point of view, on the endothelial cell. The endothelial cell also has the unique capability of releasing ET-1, as well as other factors, such as the endothelial-derived relaxing factors and prostacyclin, which counteract the myotropic effects of the peptide. The secretory and contractile responses to ET-1 rely on G-protein-coupled ET(BR)s, as well as ET(A)-G-protein-coupled receptor-like proteins. The mitogenic properties of ET-1 via ET(A) receptors (ET(AR)s) coupled to mitogen-activated protein kinases and tyrosine kinases on the vascular smooth muscle may occur in conjunction with the anti-apoptotic characteristics of the endothelial ET(BR)s. Interestingly, most of the relevant antagonists and agonists for both ET(AR)s and ET(BR)s have been developed by the pharmaceutical industry. This highlights the therapeutical potential of compounds that act on ET receptors. In normal as well as in physiopathological conditions, the ET(BR) plays an important role in the control of vascular tone, and must be taken into account when using ET receptor antagonists for the treatment of cardiovascular diseases. For the management of congestive heart failure, renal failure and primary pulmonary hypertension, the most recent literature supports the use of selective ET(AR) antagonists rather than mixed antagonists of ET(AR)s and ET(BR)s. Nonetheless, validation of this view will have to await the first clinical trials comparing the actions of ET(A) to mixed ET(A)/ET(B) receptor antagonists.

Modulation of Pro-inflammatory Gene Expression by Nuclear Lysophosphatidic Acid Receptor Type-1

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.

Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP3 Receptors

We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE2 in tissues and presence of perinuclear PGE2 receptors (EP). We presently studied mechanisms by which PGE2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE2 and selective EP3 receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP3 immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP3 receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear KCa channels were detected, and their functionality corroborated by NS1619-induced Ca2+ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca2+ chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as KCa channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-&kgr;B inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca2+ transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE2 through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope KCa channels, protein kinases, and NF-&kgr;B; the roles for nuclear EP3 receptors seem different from those on plasma membrane.

The use of confocal microscopy in the investigation of cell structure and function in the heart, vascular endothelium and smooth muscle cells

In recent years, fluorescence microscopy imaging has become an important tool for studying cell structure and function. This non invasive technique permits characterization, localisation and qualitative quantification of free ions, messengers, pH, voltage and a pleiad of other molecules constituting living cells. In this paper, we present results using various commercially available fluorescent probes as well as some developed in our laboratory and discuss the advantages and limitations of these probes in confocal microscopy studies of the cardiovascular system.

Proinflammatory Gene Induction by Platelet-Activating Factor Mediated Via Its Cognate Nuclear Receptor

It has been postulated that intracellular binding sites for platelet-activating factor (PAF) contribute to proinflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCECs) produced PAF-molecular species in response to H2O2. Using FACS analysis, we demonstrated the expression of PAF receptors on cell and nuclear surfaces of PCECs. Confocal microscopy studies performed on PCECs, Chinese hamster ovary cells stably overexpressing PAF receptors, and isolated nuclei from PCECs also showed a robust nuclear distribution of PAF receptors. Presence of PAF receptors at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with methylcarbamate-PAF evoked a decrease in cAMP production and a pertussis toxin-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a pertussis toxin-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei methylcarbamate-PAF evoked the expression of proinflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2) and was associated with augmented extracellular signal-regulated kinase 1/2 phosphorylation and NF-κB binding to the DNA consensus sequence. COX-2 expression was prevented by mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 and NF-κB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of the proinflammatory gene COX-2.

Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1

Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor l-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-κB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.

Expression of endogenous nuclear bradykinin B2 receptors mediating signaling in immediate early gene activation

Bradykinin (BK) represents a pro‐inflammatory mediator that partakes in many inflammatory diseases. The mechanism of action of BK is thought to be primarily mediated by specific cell surface membrane B2 receptors (B2Rs). Some evidence has suggested, however, the existence of an intracellular/nuclear B2R population. Whether these receptors are functional and contribute to BK signaling remains to be determined. In this study, by mean of Western blotting, 3D‐confocal microscopy, receptor autoradiography and radioligand binding analysis, we showed that plasma membrane and highly purified nuclei from isolated rat hepatocytes contain specific B2R that bind BK. The results depicting B2R nuclear expression in isolated nuclear organelles were reproduced in situ on hepatic sections by immunogold labeling and transmission electron microscopy. Functional tests on single nuclei, by means of confocal microscopy and the calcium‐sensitive probe fluo‐4AM, showed that BK induces concentration‐dependent transitory mobilization of nucleoplasmic calcium; these responses were blocked by B2R antagonist HOE 140, not by the B1R antagonist R954 and, were also found in wild‐type C57/Bl6 mice, but not in B2R‐KO mice. In isolated nuclei, BK elicited activation/phosphorylation of Akt, acetylation of histone H3 and ensuing pro‐inflammatory iNOS gene induction as determined by Western blot and RT‐PCR. ChIP assay confirmed binding of acetylated‐histone H3 complexes, but not B2R, to promoter region of iNOS gene suggesting that B2R‐mediated gene expression is bridged with accessory downstream effectors. This study discloses a previously undescribed mechanism in BK‐induced transcriptional events, via intracrine B2R‐mediated signaling, occurring in rat autologous hepatic cells. J. Cell. Physiol. 216: 234–244, 2008.

Regulation of the calcium slow channel by cyclic GMP dependent protein kinase in chick heart cells

In order to assess the interaction between the cAMP-dependent and the cGMP-dependent phosphorylation pathways on the slow Ca2+ current (ICa(L)), whole-cell voltage-clamp experiments were conducted on embryonic chick heart cells. Addition of 8Br-cGMP to the bath solution reduced the basal (unstimulated) ICa(L). Intracellular application of the catalytic subunit of PK-A (PK-A(cat); 1.5 μM) via the patch pipette rapidly potentiated ICa(L) by 215±16% (n=4); subsequent addition of 1 mM 8Br-cGMP to the bath reduced the amplitude of ICa(L) towards the initial control values (123±29%). Intracellular application of PK-G (25 nM pre-activated by 10−7 M cGMP), rapidly inhibited the basal ICa(L) by 64±6% (n=8). Heat-denatured PK-G was ineffective. Subsequent additions of relatively high concentrations of 8Br-cAMP (1 mM) or isoproterenol (ISO, 1–10 μM) did not significantly remove the PK-G blockade of ICa(L). The results of the present study suggest that: (a) 8Br-cGMP can inhibit the basal or stimulated (by PK-A(cat)) ICa(L) in embryonic chick myocardial cells. (b) PK-G applied intracellularly inhibits the basal ICa(L).

PAF activation of a voltage-gated R-type Ca2+ channel in human and canine aortic endothelial cells.

By the use of fura‐2 and digital imaging techniques, [K]o depolarization or PAF (10−9 m) were shown to induce a sustained increase of [Ca]i in human or canine single aortic vascular endothelial cells (VEC) that was insensitive to nifedipine but sensitive to (−)‐PN200–110 or to lowering of [Ca]o. The PAF‐induced effect on [Ca]i was blocked by the PAF receptor antagonist, WEB2170. Our results suggest that [K]o depolarization and PAF increase [Ca]i via the activation of R‐type Ca2+ channels.

Presence of functional endothelin-1 receptors in nuclear membranes of human aortic vascular smooth muscle cells.

Our previous work showed that the nucleus plays a role in excitation-contraction coupling and that the channels and receptors could be present at the nuclear membrane. In the study reported here, the objective was to test the hypothesis that endothelin-1 (ET-1) receptors are functional at the level of the nuclear membranes and that their stimulation importantly regulates free nucleoplasmic Ca2+ level. Using a Fluo-3 Ca2+ measurement technique in human vascular smooth muscle cells (HVSMC), superfusion with increasing concentrations of extracellular ET-1 induced a dose-dependent sustained increase of free cytosolic ([Ca]c), nuclear ([Ca]n) Ca2+ and contraction with an EC50 near 3 x 10(-10) M. Like the extracellular ET-1, the cytosolic application of ET-1 using the perforated sarcolemma membrane technique, induced a dose-dependent increase of nuclear free calcium of HVSMC with an EC50 of 2 x 10(-11) M. These results strongly suggest that ET-1 receptors are functional at the level of the nuclear membranes. Furthermore, the sensitivity of ET-1 receptors at the nuclear membrane level seems to be higher than that of the receptors at the sarcolemma membrane. Finally, our results suggest that cytosolic ET-1 may play a role in preventing HVSMC nuclear calcium overload, thus protecting the cells from apoptosis.