RESUMEN
INTRODUCTION: Thapsia spp. (Apiaceae) are the major natural source of polyoxygenated guaianolide sesquiterpene lactones known as thapsigargins, which induce apoptosis in mammalian cells via a high affinity inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase. The mechanism of biosynthesis of thapsigargins has not been elucidated, and probable biochemical precursors such as hydrocarbon or oxygenated sesquiterpenes have not been identified in previous phytochemical analyses of essential oils from this genus. OBJECTIVE: To investigate the utility of solid phase micro-extraction (SPME), when compared with classical essential oil distillates, for identifying potential precursors of guaianolide sesquiterpene lactones from Thapsia garganica L. and Thapsia villosa L. type II. METHODOLOGY: A systematic description of the volatile components of roots, flowers, stems and fruits of T. villosa and of root, flower and fruits of T. garganica was constructed via GC-MS analyses of SPME-adsorbed compounds and of essential oils obtained through hydrodistillation of the same tissues. RESULTS: The sesquiterpenoids δ-cadinene, α- and δ-guaiene, elemol and guaiols were found to be major volatile constituents of the roots of T. garganica and T. villosa trapped using SPME. In contrast, these sesquiterpenoids were not detected or were at negligible levels in essential oils, where sesquiterpenoids are potentially converted to azulenes during hydrodistillation. CONCLUSION: The new data reported in this study demonstrates that SPME is a valuable tool for the identification of volatile sesquiterpenes when compared with analysis of essential oils, and we postulate that guaiene is the likely precursor of guaianolide sesquiterpenes from Thapsia.
Asunto(s)
Aceites Volátiles/aislamiento & purificación , Sesquiterpenos de Guayano/biosíntesis , Microextracción en Fase Sólida/métodos , Thapsia/química , Tapsigargina/aislamiento & purificación , Azulenos/metabolismo , Destilación , Flores/química , Frutas/química , Aceites Volátiles/análisis , Aceites Volátiles/química , Aceites de Plantas/análisis , Aceites de Plantas/química , Aceites de Plantas/aislamiento & purificación , Raíces de Plantas/química , Tallos de la Planta/química , Sesquiterpenos de Guayano/metabolismo , Thapsia/metabolismo , Tapsigargina/análisis , Tapsigargina/químicaRESUMEN
In PC Cl3 cells, a continuous, fully differentiated rat thyroid cell line, P2Y(2) purinoceptor activation provoked a transient increase of [Ca(2+)](i), followed by a decreasing sustained phase. The alpha and beta1 protein kinase C (PKC) inhibitor Gö6976 decreased the rate of decrement to the basal [Ca(2+)](i) level and increased the peak of Ca(2+) entry of the P2Y(2)-provoked Ca(2+)transients. These effects of Gö 6976 were not caused by an increased permeability of the plasma membrane, since the Mn(2+) and Ba(2+) uptake were not changed by Gö 6976. Similarly, the Na(+)/Ca(2+) exchanger was not implicated, since the rate of decrement to the basal [Ca(2+)](i) level was equally decreased in physiological and Na(+)-free buffers, in the presence of Gö 6976. On the contrary, the activity of the sarcoplasmic-endoplasmic reticulum Ca(2+)ATPase (SERCA) 2b was profoundly affected by Gö 6976 since the drug was able to completely inhibit the stimulation of the SERCA 2b activity elicited by P2-purinergic agonists. Finally, the PKC activator phorbol myristate acetate had effects opposite to Gö 6976, in that it markedly increased the rate of decrement to the basal [Ca(2+)](i) level after P2Y(2) stimulation and also increased the activity of SERCA 2b. These results suggest that SERCA 2b plays a role in regulating the sustained phase of Ca(2+) transients caused by P2Y(2) stimulation.
Asunto(s)
Calcio/metabolismo , Receptores Purinérgicos P2/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Retículo Sarcoplasmático/enzimología , Glándula Tiroides/enzimología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Animales , Bario/metabolismo , Transporte Biológico Activo , Calcio/análisis , Carbazoles/farmacología , Línea Celular , Activación Enzimática , Indoles/farmacología , Maleimidas/farmacología , Manganeso/metabolismo , Microscopía Fluorescente , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Receptores Purinérgicos P2Y2 , Acetato de Tetradecanoilforbol/farmacología , Tapsigargina/análisis , Uridina Trifosfato/metabolismo , Uridina Trifosfato/farmacologíaRESUMEN
Caldesmon is a multifunctional ubiquitous regulator of the actin cytoskeleton, which can affect both actomyosin contractility and actin polymerization. Previous studies showed that caldesmon over-expression in cultured fibroblasts produces effects that resemble those of chemical inhibitors of cellular contractility. Since these inhibitors (H-7, Y-27632, etc.) have been shown to lower intraocular pressure and increase outflow facility from the anterior chamber of the eye, we proposed that caldesmon might be used for gene therapy of glaucoma. In the present study we examined the effects of expression of adenovirus-delivered rat non-muscle caldesmon fused with green fluorescent protein (AdCaldGFP) on the actin cytoskeleton and matrix adhesions in cultured human trabecular meshwork (HTM) cells. In addition, we assessed the effect of caldesmon on the stability of cell-cell junctions in kidney epithelial MDCK cells. Cultured HTM cells demonstrate a well-developed actin cytoskeleton, comprising mainly arrays of parallel actomyosin bundles (stress fibers). Lamellipodial protrusions containing dense actin networks are also observed. Cell-matrix adhesions are dominated by focal adhesions (FAs) associated with the ends of the stress fibers, focal complexes in lamellipodia, and fibrillar adhesions in the central part of the spread cells. Treatment of HTM cells with AdCaldGFP resulted in dose-dependent morphological changes within 24-48 hr post-infection. Cells expressing moderate levels of caldesmon exhibited straight bundles containing actin and myosin II, which were considerably shorter than those in control cells. Short filament bundles in caldesmon over-expressing cells formed arrays consisting of triangular actin structures with small vinculin-positive FAs at their vertices. In addition, the fraction of cells displaying large lamellipodia increased. About 40-50% of the population of caldesmon-expressing cells demonstrated high levels of GFP-caldesmon expression and severe changes in the actin cytoskeleton, manifested by the disappearance of stress fibers and the formation of curved actin- and myosin-containing bundles. These bundles formed together a dynamic network consisting of pulsating loops filling the entire cytoplasm. Addition of thapsigargin, which increases intracellular Ca++ concentration, resulted in a straightening of the curved bundles. Another type of novel actin structures induced by caldesmon over-expression were highly dynamic circular waves that propagated over the affected cells with a velocity about 10 microm min. In cells with disrupted stress fibers, vinculin-containing FAs and tensin-rich fibrillar adhesions had also essentially vanished. However, phosphotyrosine-positive focal complexes were still prominent throughout the lamellipodia of these cells. Over-expression of caldesmon in MDCK cells reduced, in a dose dependent manner, the beta-catenin content at cell-cell adherens junctions and in some cases led to physical disruption of adherens junctions. Thus, caldesmon over-expression induces unique reorganization of the actin cytoskeleton in affected cells, accompanied by disruption of focal and fibrillar cell-matrix adhesions, and destabilization of cell-cell adherens junctions. Inducing such changes in the contractility and actin cytoskeleton of HTM cells in glaucomatous eyes in vivo could produce a therapeutically useful increase in outflow facility.
Asunto(s)
Actinas/análisis , Proteínas de Unión a Calmodulina/farmacología , Citoesqueleto/efectos de los fármacos , Adenoviridae , Adulto , Calcio/metabolismo , Proteínas de Unión a Calmodulina/análisis , Proteínas de Unión a Calmodulina/genética , Adhesión Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Inhibidores Enzimáticos/análisis , Femenino , Expresión Génica/genética , Vectores Genéticos , Proteínas Fluorescentes Verdes/análisis , Humanos , Masculino , Microscopía Fluorescente/métodos , Fibras de Estrés/química , Tapsigargina/análisis , Malla Trabecular/citología , Malla Trabecular/efectos de los fármacos , Vinculina/análisisRESUMEN
Poisoning of livestock by ingestion of Thapsia garganica L, common plant in many countries of North Africa, is described.