María Perassolo

Induction of Rat Intestinal P-glycoprotein by Spironolactone and Its Effect on Absorption of Orally Administered Digoxin

The effect of the diuretic spironolactone (SL) on expression and function of intestinal P-glycoprotein (P-gp), as well as its impact on intestinal absorption of digoxin, was explored. Rats were treated with daily doses of 200 μmol/kg b.wt. of SL intraperitoneally for 3 consecutive days. The small intestine was divided into four equal segments of ∼25 cm, with segment I being the most proximal. Brush-border membranes were isolated and used in analysis of P-gp expression by Western blot analysis. P-gp content increased in the SL group by 526, 292, 210, and 622% over controls for segments I, II, III, and IV, respectively. Up-regulation of apical P-gp was confirmed by immunofluorescence microscopy. P-gp transport activity was explored in intestinal sacs prepared from segment IV using two different model substrates. Serosal to mucosal transport (efflux) of rhodamine 123 was 140% higher, and mucosal to serosal transport (absorption) of digoxin was 40% lower in the SL group, both indicating increased P-gp function. In vivo experiments showed that intestinal absorption of a single dose of digoxin administered p.o. was attenuated by SL pretreatment. Thus, concentration of digoxin in portal and peripheral blood was lower in SL versus control groups, as well as its accumulation in kidney and liver. Urinary excretion of digoxin was significantly decreased in the SL group, probably reflecting decreased systemic availability of digoxin for subsequent urinary elimination. We conclude that SL induces P-gp expression with potential impact on intestinal absorption of substrates with therapeutic application.

Role of reactive oxygen species and proline cycle in anthraquinone accumulation in Rubia tinctorum cell suspension cultures subjected to methyl jasmonate elicitation

Elicitors are compounds or factors capable of triggering a defense response in plants. This kind of response involves signal transduction pathways, second messengers and events such as Reactive Oxygen Species (ROS) generation, proline accumulation and secondary metabolite production. Anthraquinone (AQs) biosynthesis in Rubia tinctorum L. involves different metabolic routes, including shikimate and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. It has been proposed that the proline cycle could be coupled with the pentose phosphate pathway (PPP), since the NADP+ generated by this cycle could act as a cofactor of the first enzymes of the PPP. The end-product of this pathway is erithrose-4-phosphate, which becomes the substrate of the shikimate pathway. The aim of this work was to study the effect of methyl jasmonate (MeJ), a well-known endogenous elicitor, on the PPP, the proline cycle and AQs production in R. tinctorum cell suspension cultures, and to elucidate the role of ROS in MeJ elicitation. Treatment with MeJ resulted in AQs as well as proline accumulation, which was mimicked by the treatment with a H₂O₂-generating system. Both MeJ-induced effects were abolished in the presence of diphenyliodonium (DPI), a NADPH oxidase inhibitor (main source of ROS). Treatment with the elicitor failed to induce PPP; therefore, this route did not turn out to be limiting the carbon flux to the shikimate pathway.

Increasing anthraquinone production by overexpression of 1-deoxy-D-xylulose-5-phosphate synthase in transgenic cell suspension cultures of Morinda citrifolia

A Morinda citrifolia cell line was obtained by overexpresion of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) from Catharanthus roseus, a key enzyme of the metabolic pathway of anthraquinones (AQs). This cell line increased AQs production by about 24% compared to the control cell line. This transgenic cell line which carries dxs cDNA isolated from Catharanthus roseus, was achieved by direct transformation of cell suspension cultures of M. citrifolia using a hypervirulent Agrobacterium tumefaciens strain. The effects of the overexpression of the dxs gene also resulted in increased levels of dxs mRNA transcripts and DXS activity compared to the control cell line. In addition, total phenolics and phenylalanine ammonia-lyase activity were evaluated and were significantly higher in the transgenic line than in controls.

Biosynthesis of Sesquiterpene Lactones in Plants and Metabolic Engineering for Their Biotechnological Production

In the present chapter, we review some aspects of the biosynthesis of sesquiterpene lactones and its regulation in different medicinal and aromatic plants used in the pharmaceutical industry. In this sense, we describe the mevalonate and the 2-C-methyl-D-erythritol 4-phosphate pathways, which generate the corresponding isoprenoid precursors (isopentenyl diphosphate and dimethylallyl diphosphate), as well as the late pathways that lead to sesquiterpene lactone biosynthesis. This chapter also analyses the role of the transcription factors involved in the regulation of sesquiterpene lactone biosynthesis and the different biotechnological approaches that have been developed for sesquiterpene lactone production. In vitro plant cell cultures (comprising micropropagation and plant cell suspension, shoot and root cultures) have emerged as a production platform for many plant secondary metabolites, since they allow their production under controlled conditions and shorter production cycles. The characterisation and isolation of genes involved in the regulation of sesquiterpene lactone biosynthetic pathways have allowed the design of metabolic engineering strategies to increase the production of these metabolites. Moreover, we discuss different strategies to increase sesquiterpene lactone production through genetic engineering. We also focus on the metabolic engineering of the artemisinin biosynthetic pathway in Artemisia annua. This metabolic pathway has become a model system not only for the biotechnological production of sesquiterpene lactones but also for the improvement of other plant secondary metabolic pathways. Finally, we analyse the successful expression of the complete artemisinin biosynthetic pathway in Escherichia coli and Saccharomyces cerevisiae, which has led to the efficient accumulation of artemisinic acid in these microorganisms.