RESUMEN
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(R) 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.
Asunto(s)
Lesión Pulmonar Aguda/patología , Microscopía Confocal/métodos , Lesión Pulmonar Aguda/inducido químicamente , Animales , Bleomicina/administración & dosificación , Bleomicina/toxicidad , Endoscopía/métodos , Humanos , Instilación de Medicamentos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/patología , Microscopía Fluorescente/métodos , Neutrófilos/patología , Ratas , Ratas Long-Evans , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
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.
Asunto(s)
Angiotensina II/farmacología , Proliferación Celular/efectos de los fármacos , Fibronectinas/metabolismo , Integrinas/metabolismo , Transducción de Señal/efectos de los fármacos , Zona Glomerular/efectos de los fármacos , Actinas/metabolismo , Animales , Células Cultivadas , Femenino , Fibronectinas/genética , Técnica del Anticuerpo Fluorescente , Adhesiones Focales/metabolismo , Inmunoprecipitación , Integrinas/genética , Paxillin/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Zona Glomerular/citología , Zona Glomerular/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
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.
Asunto(s)
Angiotensina II/metabolismo , Zona Glomerular/citología , Angiotensina II/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Humanos , Integrinas/metabolismo , Receptores de Angiotensina/metabolismo , Transducción de Señal , Zona Glomerular/efectos de los fármacosRESUMEN
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 alpha2, alpha3 and alpha5 were present mainly in the cortex, while alpha1 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 3beta-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.
Asunto(s)
Glándulas Suprarrenales/química , Hormona Adrenocorticotrópica/metabolismo , Proteínas de la Matriz Extracelular/análisis , Integrinas/análisis , Glándulas Suprarrenales/metabolismo , Aldosterona/metabolismo , Animales , Técnicas de Cultivo de Célula , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/análisis , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/análisis , Colágeno Tipo IV/metabolismo , Corticosterona/metabolismo , Activación Enzimática , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibronectinas/análisis , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador , Integrinas/metabolismo , Laminina/análisis , Laminina/metabolismo , Microscopía de Contraste de Fase , Ratas , Ratas Long-Evans , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Calcio/metabolismo , Ácido Linoleico/farmacología , Ácidos Oléicos/farmacología , Zona Glomerular/metabolismo , Aldosterona/metabolismo , Angiotensina II/farmacología , Animales , ATPasas Transportadoras de Calcio/metabolismo , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Ácidos Grasos/metabolismo , Técnicas In Vitro , Inosina Trifosfato/biosíntesis , Ácido Linoleico/metabolismo , Ácidos Oléicos/metabolismo , Oxidación-Reducción , Ratas , Transducción de Señal/efectos de los fármacos , Esteroides/biosíntesis , Zona Glomerular/citología , Zona Glomerular/efectos de los fármacosRESUMEN
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.
Asunto(s)
Neuritas/fisiología , Proteína Quinasa C-alfa/fisiología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Receptor de Angiotensina Tipo 2/fisiología , Angiotensina II/farmacología , Carbazoles/farmacología , División Celular , Línea Celular , Indoles/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Neuritas/ultraestructura , Neuronas/ultraestructura , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Transducción de Señal , Proteínas de Unión al GTP rap1/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Regulación de la Expresión Génica , Proteína Quinasa C/fisiología , Receptor de Melanocortina Tipo 2/biosíntesis , Arrestinas/biosíntesis , Western Blotting , Línea Celular Tumoral , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Epítopos/química , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Indoles/farmacología , Maleimidas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sacarosa/farmacología , Transfección , Arrestina beta 2 , beta-ArrestinasRESUMEN
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-Jun N-terminal kinase inhibitor SP600125 had no effect. Time-course studies revealed that Ang II stimulated phosphorylation of both p42/p44mapk and p38 MAPK but did not activate c-Jun N-terminal kinase. Ang II had no effect on the level of cyclin E expression but increased the expression of the cyclin-dependent kinase, p27Kip1, 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/p44mapk and p38 MAPK pathways, which increase expression of the steroidogenic enzymes, steroidogenic acute regulatory protein and 3beta-hydroxysteroid dehydrogenase and p27Kip1, a protein known to block the cell cycle in G1 phase. Together these results support the key role of Ang II as a stimulus of steroid synthesis rather than a proliferating factor.
Asunto(s)
Angiotensina II/farmacología , Vasoconstrictores/farmacología , Zona Glomerular/citología , Zona Glomerular/efectos de los fármacos , Animales , Proteínas de Ciclo Celular/metabolismo , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Ratas , Ratas Long-Evans , Proteínas Supresoras de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
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).
Asunto(s)
4-Aminopiridina/farmacología , Calcio/metabolismo , Membrana Celular/metabolismo , Células Jurkat/efectos de los fármacos , Proteína Quinasa C/fisiología , 4-Aminopiridina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Inhibidores Enzimáticos/farmacología , Fura-2 , Humanos , Células Jurkat/metabolismo , Toxinas Marinas , Oxazoles/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Proteína Quinasa C/farmacologíaRESUMEN
In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-d-glucamine, 1.8 mm Ca2+, and 100 microM Gd3+ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 microM) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 microM) showed outward rectification, as shown previously for these channels using 100 microM 1-oleoyl-2-acetyl-sn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC50 = 0.8 microM. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels approximately 3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca2+ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Ácidos Hidroxieicosatetraenoicos/farmacología , Animales , Calcio/farmacología , Canales de Calcio/metabolismo , Línea Celular , Femenino , Cobayas , Humanos , Masculino , Meglumina/farmacología , Ratones , Técnicas de Placa-Clamp , Proteínas Recombinantes/metabolismo , Canales Catiónicos TRPC , Canal Catiónico TRPC6RESUMEN
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 (Ca(2+)), 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.
Asunto(s)
Hormona Adrenocorticotrópica/farmacología , AMP Cíclico/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Canales de Cloruro/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , GMP Cíclico/fisiología , Citoesqueleto/fisiología , Matriz Extracelular/fisiología , Humanos , Integrinas/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Canales de Potasio/fisiologíaRESUMEN
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.
Asunto(s)
Ácidos Hidroxieicosatetraenoicos/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Tráquea/efectos de los fármacos , Tráquea/fisiología , Animales , Bronquios/efectos de los fármacos , Bronquios/fisiología , Calcio/metabolismo , Cationes/metabolismo , Diglicéridos/farmacología , Inhibidores Enzimáticos/farmacología , Cobayas , Indoles/farmacología , Maleimidas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Contracción Muscular/efectos de los fármacos , Técnicas de Placa-Clamp , Proteína Quinasa C/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Colágeno Tipo IV/metabolismo , Fibronectinas/metabolismo , Integrina alfa2beta1/metabolismo , Integrina alfa4beta1/metabolismo , Microdominios de Membrana/metabolismo , Calcio/metabolismo , Fluorescencia , Gangliósido G(M1)/metabolismo , Humanos , Células Jurkat , Microesferas , Factores de TiempoRESUMEN
Previous studies have shown that human fetal adrenal gland from 17- to 20-week-old fetuses expressed pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, which were localized on chromaffin cells. The aim of the present study was to identify PACAP receptor isoforms and to determine whether PACAP can affect intracellular calcium concentration ([Ca(2+)](i)) and catecholamine secretion. Using primary cultures and specific stimulation of chromaffin cells, we demonstrate that PACAP-38 induced an increase in [Ca(2+)](i) that was blocked by PACAP (6-38), was independent of external Ca(2+), and originated from thapsigargin-insensitive internal stores. The PACAP-triggered Ca(2+) increase was not affected by inhibition of PLC beta (preincubation with U-73122) or by pretreatment of cells with Xestospongin C, indicating that the inositol 1,4,5-triphosphate-sensitive stores were not mobilized. However, forskolin (FSK), which raises cytosolic cAMP, induced an increase in Ca(2+) similar to that recorded with PACAP-38. Blockage of PKA by H-89 or (R(p))-cAMPS suppressed both PACAP-38 and FSK calcium responses. The effect of PACAP-38 was also abolished by emptying the caffeine/ryanodine-sensitive Ca(2+) stores. Furthermore, treatment of cells with orthovanadate (100 microm) impaired Ca(2+) reloading of PACAP-sensitive stores indicating that PACAP-38 can mobilize Ca(2+) from secretory vesicles. Moreover, PACAP induced catecholamine secretion by chromaffin cells. It is concluded that PACAP-38, through the PAC(1) receptor, acts as a neurotransmitter in human fetal chromaffin cells inducing catecholamine secretion, through nonclassical, recently described, ryanodine/caffeine-sensitive pools, involving a cAMP- and PKA-dependent phosphorylation mechanism.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Calcio/metabolismo , AMP Cíclico/metabolismo , Neuropéptidos/fisiología , Receptores de la Hormona Hipofisaria/agonistas , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/embriología , Secuencia de Bases , Células Cromafines/citología , Células Cromafines/metabolismo , Citosol/metabolismo , Cartilla de ADN , Humanos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tapsigargina/farmacologíaRESUMEN
In their undifferentiated state, NG108-15 cells express only the angiotensin II (Ang II) type 2 receptor (AT(2)). We have previously shown that Ang II induced neurite outgrowth of NG108-15 cells, a process involving sustained activation of p42/p44(mapk) activity. We have also shown that Ang II stimulates nitric oxide (NO) production. The aim of the present study was to investigate the role of the NO/cyclic GMP (cGMP) cascade in the signal transduction of the AT(2) receptor-stimulated neurite outgrowth. Three-day treatment of cells with dbcGMP induced neurite outgrowth as did Ang II. Preincubation with an inhibitor of cGMP-dependent protein kinase, KT5823, resulted in the formation of short neurites, while in the presence of LY83583 or methylene blue, two inhibitors of guanylyl cyclase, cells resembled control cells with only one or two thin processes. Western blot analyses indicated that nNOS was present in NG108-15 cells. Immunoprecipitation with antiphosphotyrosine antibodies showed that Ang II induced NOS activity and increased cGMP production through a Gi-dependent pathway. However, neither L-NAME, KT5823, nor LY83583 affected the activation of p42/p44(mapk) induced by Ang II, indicating that the pathway NO/guanylyl cyclase/cGMP was not involved in Ang II-induced activation of MAPK. The present results suggest that the neurite outgrowth induced by Ang II results from at least parallel but complementary pathways, one involved in neurite elongation (through the cooperation of MAPK and PKG) and the other involved in sprouting (through cGMP).