We present a new lung imaging technique based on endoscopic confocal fluorescence microscopy (ECFM), which is a new method that is able to provide cellular and structural assessment of living tissue using a small confocal probe in direct contact with the visceral pleura. To observe distal airspace structure and cellular condition in normal and injured lungs (hyperoxic and bleomycin challenged), we used fluorescent-specific marker contrast and ECFM. Alveolar space ECFM with spectral analyses were performed at 488-nm excitation using FITC-labeled markers or naturally fluorescent dyes. The normal lung was compared with the sick lung, where our in vivo imaging experiments correlated well with results obtained with corresponding ex vivo conventional assays. Four main elements pertaining to the acute lung injury/acute respiratory distress syndrome (ALI/ARDS) pathophysiology and established early key events were specifically studied: alveolar epithelial membrane phenotype, lung cell apoptosis, neutrophil recruitment, and edema. ECFM allowed visualization of (i) fine-tuned ultrastructural lectin (RCA-1) and sialoglycoprotein (RTI40) epithelial cell membrane expression, (ii) YO-PRO-1-related DNA linking of lung cell apoptosis, (iii) PKH2 green fluorescent cell linker-labeled neutrophil tracking in lung microcirculatory network and airspaces, (iv) FITC-dextran plasma contrast and extravasation with edema formation. ECFM provides reliable results to corresponding ex vivo fluorescent methods. ECFM, using the minimally invasive Five-1® optical instrument and specific fluorescent markers, is able to provide real-time potentially useful imaging of live unfixed normal and injured lung tissue with promising developments for improving bedside diagnostic and decision-making therapeutic strategy in patients with ALI.

Angiotensin II (Ang II), through the Ang II type 1 receptor subtype, inhibits basal proliferation of adrenal glomerulosa cells by inducing the disruption of actin stress fiber organization. This effect is observed in cells cultured on plastic or on fibronectin. The aim of the present study was to investigate how Ang II may interfere with extracellular matrix/integrin signaling. In cells treated for 3 d with echistatin (EC) (a snake-venom RGD-containing protein that abolishes fibronectin binding to alpha(5)beta(1) or alpha(v)beta(3) integrins), basal proliferation decreased by 38%, whereas Ang II was unable to abolish basal proliferation. In cells grown on fibronectin, Ang II decreased binding of paxillin to focal adhesions and, similarly to EC, induced a rapid dephosphorylation of paxillin (1 min), followed by an increase after 15 min. Fibronectin enhanced RhoA/B and Rac activation induced by Ang II, an effect abolished by EC. Under basal conditions, paxillin was more readily associated with RhoA/B than with Rac. Stimulation with Ang II induced a transient decrease in RhoA/B-associated paxillin (after 5 min), with a return to basal levels after 10 min, while increasing Rac-associated paxillin. Finally, results reveal that glomerulosa cells are able to synthesize and secrete fibronectin, a process by which cells can stimulate their own proliferative activity when cultured on plastic. Together, these results suggest that Ang II acts at the level of integrin-paxillin complexes to disrupt the well- developed microfilament network, a condition necessary for the inhibition of cell proliferation and initiation of steroidogenesis.

The zona glomerulosa of the adrenal cortex is well-known for its high level of proliferation, compared to the adjacent zona fasciculata, both in in vivo and in vitro conditions. Angiotensin II (Ang II) is a potent growth factor for glomerulosa cells, appearing as a proliferative factor in vivo, under sodium-deficient diet conditions, as well as in vitro, in studies conducted with whole zona glomerulosa. However, in cells maintained in primary culture for 3 days, Ang II rather promotes cellular hypertrophy with a concomitant arrest in basal cell proliferation. The present essay aims at providing experimental arguments supporting such unexpected observations, with particular focus on the modulatory impact of the extracellular environment on Ang II action, namely AT(1) receptor-induced signaling pathways and cell responses.

The expression of main extracellular matrix (ECM) and their integrins were studied in the adult rat adrenal gland. Collagen I, IV (CI, CIV), laminin (LN) and fibronectin (FN) expression was observed surrounding each glomerulosa cell and as long fibrils between the cords of fasciculata cells. In the medulla, FN was present around chromaffin cells or bordering blood vessels. Integrin alpha 2, alpha 3 and alpha 5 were present mainly in the cortex, while alpha 1 was present in the medulla. In culture, all ECM favoured proliferation of both glomerulosa and fasciculata cells, while protein synthesis was lower on FN and LN in glomerulosa cells. CIV promoted ACTH-induced proliferation whereas FN favoured ACTH-induced protein synthesis in glomerulosa cells. Except for LN, ECM increased expression of 3 beta-hydroxysteroid dehydrogenase and enhanced basal aldosterone, although corticosterone secretion was only enhanced by CI and CIV In fasciculata cells, the potency of ACTH-induced cAMP production was lower on ECM, compared with plastic. Moreover, ACTH, but not ECM activated mitogenic-activated protein kinase p38 and stress-activated protein kinases. Glomerulosa and fasciculata cells grown on CI and CIV had a polygonal morphology, while cells grown on LN appeared as clusters of small rounded cells. On FN, the glomerulosa cells exhibited polygonal morphology while fasciculata cells appeared as clusters of small rounded cells. Together, these results indicate that ECM modulates basal and ACTH-induced cell functions, with FN, CI and CIV specifically favouring steroid secretion, as opposed to LN which inhibits secretion while promoting proliferation.

The aim of this study was to investigate the short-term regulation of the ACTH receptor human (h) melanocortin receptor 2 (MC2R) by transfection of a c-Myc-tagged hMC2R in the M3 cell line and assess its membrane expression by indirect immunofluorescence. Stimulation with ACTH induced production of cAMP with EC(50) values ranging from 7.6-11.9 nM in transient and stable transfectants, respectively. Pretreatment with ACTH induced a dose-dependent loss of cAMP production, from 1 pm up to 10 nM. Desensitization was also time dependent, with 70% loss of maximal responsiveness occurring after 15-min pretreatment with 10 nM ACTH, followed by a plateau up to 60 min. The decrease in hMC2R responsiveness was abrogated by individual treatment with protein kinase A (PKA) or protein kinase C inhibitors, H-89 and GF109203X. However, when added simultaneously, receptor responsiveness was raised over the maximal hMC2R activity observed in control cells. ACTH-induced loss of cAMP production was accompanied by receptor sequestration into intracellular vesicles (maximum after 30-min exposure). Cotransfection of M3 cells with the c-Myc-tagged hMC2R and beta-arrestin-2-green fluorescence protein along with sucrose treatment revealed that beta-arrestin-2-green fluorescence protein and c-Myc-hMC2R were redistributed in similar intracellular vesicles through a clathrin-dependent, but caveolae-independent, process. Sucrose pretreatment blocked receptor desensitization, indicating that hMC2R desensitization and internalization are interrelated. Moreover, preincubation with H-89 abrogated hMC2R internalization, whereas GF109203X had no effect. In conclusion, the present results indicate that PKA and protein kinase C act synergistically to induce hMC2R desensitization, but only PKA is essential for receptor internalization, highlighting the complex nature of the short-term regulatory pattern of this receptor.

The aim of the present study was to investigate whether protein kinase C (PKC) isoforms may be among the putative candidates implicated in the primary effects of the Ang II type 2 (AT2) receptor. Western blot analyses revealed the presence of PKC alpha,epsilon, iota, and zeta in NG108-15 cells. After a 3-d treatment with 3 nm Gö6976, a specific inhibitor of classical PKC isoforms, cells were characterized by the presence of one elongated process similar to that observed after treatment with Ang II or with CGP42112, a selective AT2 receptor agonist. Similar findings were observed in cells expressing a dominant-negative mutant of PKC alpha (K368A). Inhibition of PKC alpha in NG108-15 cells also decreased cell number and proliferation. In conditions of acute stimulation, Ang II induced a time-dependent and transient inhibition of PKC alpha activity, as well as a decrease in PKC alpha levels associated with the membrane. Treatment of cells with Gö6976 was also found to inhibit p21(ras) (between 1-10 min) but stimulated Rap1 activity (1-5 min) in a time-course similar to that of Ang II. Incubation of NG108-15 cells with Gö6976 (3 nm) inhibited basal p42/p44(mapk) phosphorylation, but failed to interfere with its activation by the AT(2) receptor, indicating that inhibition of PKC alpha is not directly involved in the Rap1-MEK-p42/p44(mapk) cascade. Taken together, these results indicate that PKC alpha is a primary target of the AT2 receptor. Inhibition of PKC alpha leads to a decrease in both p21(ras) activity and cell proliferation, which may facilitate AT2 receptor signaling through p42/p44(mapk), thereby leading to neurite outgrowth.

EKODE, an epoxy-keto derivative of linoleic acid, was previously shown to stimulate aldosterone secretion in rat adrenal glomerulosa cells. In the present study, we investigated the effect of exogenous EKODE on cytosolic [Ca(2+)] increase and aimed to elucidate the mechanism involved in this process. Through the use of the fluorescent Ca(2+)-sensitive dye Fluo-4, EKODE was shown to rapidly increase intracellular [Ca(2+)] ([Ca(2+)](i)) along a bell-shaped dose-response relationship with a maximum peak at 5 microM. Experiments performed in the presence or absence of Ca(2+) revealed that this increase in [Ca(2+)](i) originated exclusively from intracellular pools. EKODE-induced [Ca(2+)](i) increase was blunted by prior application of angiotensin II, Xestospongin C, and cyclopiazonic acid, indicating that inositol trisphosphate (InsP(3))-sensitive Ca(2+) stores can be mobilized by EKODE despite the absence of InsP(3) production. Accordingly, EKODE response was not sensitive to the phospholipase C inhibitor U-73122. EKODE mobilized a Ca(2+) store included in the thapsigargin (TG)-sensitive stores, although the interaction between EKODE and TG appears complex, since EKODE added at the plateau response of TG induced a rapid drop in [Ca(2+)](i). 9-oxo-octadecadienoic acid, another oxidized derivative of linoleic acid, also increases [Ca(2+)](i), with a dose-response curve similar to EKODE. However, arachidonic and linoleic acids at 10 microM failed to increase [Ca(2+)](i) but did reduce the amplitude of the response to EKODE. It is concluded that EKODE mobilizes Ca(2+) from an InsP(3)-sensitive store and that this [Ca(2+)](i) increase is responsible for aldosterone secretion by glomerulosa cells. Similar bell-shaped dose-response curves for aldosterone and [Ca(2+)](i) increases reinforce this hypothesis.

The role and signaling properties of the low-affinity neurotensin receptor (NTS2) are still controversial. In particular, it is unclear whether neurotensin acts as an agonist, inverse agonist, or antagonist at this site. In view of the growing evidence for a role of NTS2 in antinociception, the elucidation of the pharmacological and coupling properties of this receptor is particularly critical. In the present study, we demonstrate that in Chinese hamster ovary (CHO) cells expressing the rat NTS2 receptor, neurotensin (NT), levocabastine, neuromedin N, and the high-affinity NT receptor antagonist SR48692 [2-{[1-(-7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbonyl]amino}adamantane-2-carboxylic acid] all bind to and activate the NTS2 receptor. This activation is followed by ligand-induced internalization of receptor-ligand complexes, as evidenced by confocal microscopy using a fluorescent NT analog. All compounds tested produced a rapid and sustained activation of extracellular signal-regulated kinases 1/2 (ERK1/2) but were without specific effect on Ca2+ mobilization. The agonist-induced activation of ERK1/2 was completely abolished by preincubation of the cells with the endocytosis inhibitors phenylarsine oxide and monodansylcadaverine as well as overexpression of a dominant-negative mutant of dynamin 1 (DynK44A), indicating that receptor internalization was required for ERK1/2 activation. NTS2-induced activation of ERK1/2 was not species-specific, because the same agonistic effects of NT and analogs were observed in CHO cells transfected with the human NTS2 receptor. In conclusion, this study demonstrates that NTS2 is a bona fide NT receptor and that activation of this receptor by NT or NT analogs results in an internalization-dependent activation of the ERK1/2 signaling cascade.

Angiotensin II (Ang II) is one of the most important stimuli of rat adrenal glomerulosa cells. The aim of the present study was to investigate whether Ang II can stimulate cell proliferation and/or hypertrophy and investigate pathways and intracellular targets. A 3-d treatment with Ang II (5-100 nM), through the Ang II type 1 receptor subtype, abolished cell proliferation observed in control cells but increased protein synthesis. Preincubation with PD98059 (a MAPK kinase inhibitor) abolished basal proliferation and had no effect on basal protein synthesis but did reverse the effect of Ang II on protein synthesis. The p38 MAPK inhibitor SB203580 reversed the inhibitory effect on cell proliferation and abolished the increase in protein synthesis, whereas the c-JunN-terminal kinase inhibitor SP600125 had no effect. Time-course studies revealed that Ang II stimulated phosphorylation of both p42/p44(mapk) and p38 MAPK but did not activate c-JunN-terminal kinase. Ang II had no effect on the level of cyclin E expression but increased the expression of the cyclin-dependent kinase, p27(Kip1), an effect abolished in cells preincubated with SB203580 and PD98059. In conclusion, in cultured rat glomerulosa cells, a 3-d treatment with Ang II increases protein synthesis, with a concomitant decrease in proliferation. These effects are mediated by both the p42/p44(mapk) and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase and p27(Kip1), a protein known to block the cell cycle in G(1) phase. Together these results support the key role of Ang II as a stimulus of steroid synthesis rather than a proliferating factor.

Since its discovery, the AT2 receptor of angiotensin II has been one of the most controversial G protein-coupled receptors. The AT2 receptor is widely distributed in the fetus, but in most tissues, its expression is dramatically diminished few hours after birth. These observations have led to the hypothesis that this receptor may play an important role during fetal development. During the last decade, many studies have been conducted to elucidate the role of the AT2 receptor in many different tissues and cell lines. Apart from a well-described action in cell apoptosis, one of the major roles attributed to the AT2 receptor of angiotensin II is its involvement in cellular differentiation. The AT2 receptor is involved in differentiation of many tissues. For example, in cells from neuronal origin, activation of the AT2 receptor was shown to induce neurite out-growth and elongation, to modulate neuronal excitability, and to promote cellular migration. In steroidogenic tissues, the AT2 receptor is associated with the development of the human fetal adrenal gland, where it induces apoptosis and probably cell migration, as well as of granulosa ovarian cells, where it promoted ovulation and oocyte maturation. The AT2 receptor is also transiently expressed in smooth muscle cells where it may play a role in vasculogenesis, and in adipocytes where it induces production of prostacyclin (PGI2) involved in preadipo-cyte differentiation. This chapter describes in detail how the AT2 receptor of angiotensin II acts on cellular differentiation, with a particular emphasis on neuronal differentiation. Indeed, signaling mechanisms involved in the AT2 effects are various and can be classified as atypical. For instance, AT2 receptor activation promotes mitogen-activated protein kinase (MAPK) cascade through a p21ras-independent pathway, and stimulates, in a parallel way, nNOS/cGMP/PKG signaling; both are essential to promote neurite outgrowth.

Numerous studies have demonstrated that the cellular environment could alter cell functions. The aim of the present study was to determine the extracellular matrix components and their integrin receptors within the rat adrenal gland and to evaluate the role of these components in rat adrenal glomerulosa cells behavior. RT-PCR and Western blot experiments showed that integrins a5 and av as well as their fibronectin receptor partners b1 and b3, were present in rat glomerulosa cells in culture. Furthermore, we report that rat glomerulosa cells in culture plated on poly-l-lysine coverslips are able to produce and secrete ECM components in their environment. Laminin (LN), fibronectin (FN), and collagen I (Col I) are found to be secreted and still in interaction with the glomerulosa cells. Finally, immunohistochemistry experiments done on paraffin section of rat adrenal glands, using specific antibodies, showed that FN, Col I, and LN present differential expression throughout the adrenal glands. Given that we showed that Arg-Gly-Asp (RGD) peptide and a fibronectin matrix could stimulate aldosterone secretion, these results suggested that the binding of integrins to extracellular matrix components could be important for adult rat adrenal glands homeostasis.

Integrins are responsible for adhesion and activation of several intracellular cascades. The present study was aimed at determining whether the interaction between fibronectin and integrins could generate pathways involved in physiological functions of rat adrenal glomerulosa cells. Immunofluorescence studies and adhesion assays showed that fibronectin was the best matrix in promoting the formation of focal adhesion. Binding of glomerulosa cells to fibronectin, but not to collagen I or poly-L-lysine, involved the integrin-binding sequence Arg-Gly-Asp (RGD). Activation of glomerulosa cells with Arg-Gly-Asp-Ser (RGDS) induced an increase in [Ca(2+)](i), whereas fibronectin triggered a release of Ca(2+) from InsP(3)-sensitive Ca(2+) stores. Aldosterone secretion induced by ACTH, angiotensin II, and RGDS and proliferation were improved on fibronectin, compared with poly-L-lysine. The RGDS peptide induced a transient increase in the activity of the p42/p44(mapk), independent of phosphatidylinositol-3 kinase and protein kinase C. Integrins alpha(5) and alpha(V) as well as their fibronectin receptor partners beta(1) and beta(3), were identified. These results suggest that in rat adrenal glomerulosa cells, binding of the alpha(5)beta(1), alpha(v)beta(1), or alpha(v)beta(3) integrins to fibronectin is involved in the generation of two important signaling events, increase in intracellular calcium, and activation of the p42/p44(mapk) cascade, leading to cell proliferation and aldosterone secretion.

4-aminopyridine (4AP) is a general blocker of voltage-dependent K+ channels. This pyridine derivative has also been shown to inhibit T cell proliferation, to modulate immune responses and to alleviate some of the symptoms associated with neurological disorders such as multiple sclerosis, myasthenia gravis and Alzheimer's disease. 4AP triggers a Ca2+ response in lymphocytes, astrocytes, neurons and muscle cells but little is known about the regulation of the 4AP response in these cells. We report that 4AP induced a non-capacitative transplasma membrane influx of Ca2+ in Jurkat T lymphocytes. The influx of Ca2+ was not affected by activation or inhibition of protein kinase A (PKA). In contrast, activation of protein kinase C (PKC) by phorbol myristyl acetate (PMA), mezerein or 1-oleoyl-2-acetyl-sn-glycerol (OAG) inhibited the influx of Ca2+ triggered by 4AP. The inhibitory effect of PKC could be prevented by prior exposure of the cells to the PKC inhibitor GF 109203X. Under these conditions, mezerein and OAG no longer inhibited the 4AP-dependent Ca2+ response. Inhibition of serine and threonine protein phosphatases PP1 and PP2A by treating the cells with calyculin A (CalA) reduced the Ca2+ response to 4AP. Okadaic acid (OA) had no effect, suggesting an involvement of PP1. A combination of CalA and OAG (or PMA) abolished the influx of Ca2+ induced by 4AP, adding further evidence to the importance of protein phosphorylation in the modulation of the 4AP response. Our data suggest that the transplasma membrane influx of Ca2+ triggered by 4AP in Jurkat T cells can be modulated by the opposite actions of PKC and protein serine and threonine phosphatase(s).

The angiotensin II (Ang II) type 2 receptor (AT(2)) is a member of the seven-transmembrane domain, G-protein coupled receptor family. This receptor is ubiquitously distributed in the fetus but, in most tIssues, its expression dramatically falls in the first few hours after birth. Based on this observation, the hypothesis that this receptor could be involved in fetal development was raised and, over the past ten Years, many studies have tried to identify a role for the AT(2) receptor using many different tIssues and cell lines. To date, one of the major roles associated with the Ang II AT(2) receptor concerns its ability to induce neuronal differentiation. Indeed, in cells of neuronal origin, activation of the AT(2) receptor was shown to induce neurite outgrowth and elongation, modulate neuronal excitability, promote cellular migration and, in particular conditions, induce neuronal cell death. Regarding its signaling mechanisms, the AT(2) receptor still represents one of the most controversial G-protein coupled receptors since it does not stimulate the production of any of the classical second messengers. This review summarizes knowledge of the functions and the signaling mechanisms involved in the actions of the AT(2) receptor in neurons and cells of neuronal origin. Based on its altered expression in neurological disorders, a role for the AT(2) receptor in control of neuronal plasticity is proposed.

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.

Collagen type IV (CnIV) and fibronectin (Fn) were used as ligands to study the distribution of alpha(2)beta(1) and alpha(4)beta(1) integrins in low-density, detergent-resistant microdomains (DRM) of Jurkat lymphocytes. CnIV-coated microspheres induced (optical trapping) the redistribution of GM(1)-associated fluorescence from the cell periphery to the area of contact. This was not observed in cells treated with beta-methyl cyclodextrin (MCD). Fn- or bovine serum albumin-coated microspheres did not modify the peripheral distribution of fluorescence. These observations were confirmed by confocal microscopy. Western blot analysis of cells exposed to surfaces coated with CnIV revealed that the alpha(2)-subunit was initially present at low levels in DRM, became strongly associated after 40 min, and returned to basal levels after 75 min. Fn induced a slight recruitment of the beta(1)-integrin alpha(4)-subunit in DRM after 5 and 10 min, followed by a return to basal levels. Neither CnIV nor Fn triggered significant changes in the distribution of the beta(1)-subunit in DRM. Fn- and CnIV-coated microspheres or surfaces coated with these ligands triggered a MCD-sensitive mobilization of Ca(2)(+). MCD did not alter the state of the Ca(2)(+) reserves. The differential distributions of the alpha(2)beta(1) and alpha(4)beta(1) integrins in DRM may provide one additional step in the regulation of outside-in signaling involving these integrins.

ACTH is the major regulator of adrenal cortex function, having acute and chronic effects on steroid synthesis and secretion. The precise molecular mechanisms by which ACTH stimulates steroid synthesis and secretion, as well as cell hypertrophy, survival, and migration are still poorly understood. Several studies have shown that ACTH action is mediated not only by cyclic adenosine monophosphate (cAMP), but also by calcium (Ca2+), both interacting closely through positive feedback loops to enhance steroid secretion. However, in spite of the evidence that ACTH could stimulate other signaling pathways, such as inositol phosphates and diacylglycerol or mitogenic‐activated protein kinase pathway (MAPK), none is as potent as cAMP. Recent data indicate that duration and potency of the cAMP production could be modulated by several isoforms of adenylyl cyclases and phosphodiesterases. In addition, calcium is probably not a first second messenger per se; rather, there are several arguments indicating that its increase occurs following cAMP production. Finally, in addition to steroid secretion, ACTH, through cAMP, is a survival factor, protecting cells against apoptosis. All of the effects of ACTH are dependent on cytoskeleton integrity. In summary, after 30 years of intensive research in this field, cAMP remains the first obligatory second messenger of ACTH action. However, recent work emphasizes that cell environment (matrix and cytoskeleton) probably interacts with cAMP to coordinate functions other than steroid secretion. Microsc. Res. Tech. 61:275–287, 2003. © 2003 Wiley‐Liss, Inc.