Effects of sodium orthovanadate on benzophenanthridine alkaloid formation and distribution in cell suspension cultures of Eschscholtzia californica

Cell suspension cultures of Eschscholtzia californica produce relatively large amounts of benzophenanthridine alkaloids upon elicitation. Sodium orthovanadate is used as an abiotic elicitor to induce alkaloid biosynthesis in cultures of E. californica. The response of the cell culture to this abiotic elicitor is very similar to that observed after elicitation with a biotic elicitor (a carbohydrate fraction from yeast extract). Treatment with orthovanadate leads to alkalinization of the growth medium, a 20-fold induction of the key enzyme tyrosine decarboxylase and increased alkaloid formation (up to 40 mg.L^–1). Cells treated with the yeast elicitor excrete a large portion of alkaloids produced into the growth medium (up to 50 % of total alkaloids) while cells treated with orthovanadate release very small amounts of alkaloids into the medium (less than 10 % of total alkaloids). These results suggest that an active transport system, possibly specific for benzophenanthridine alkaloids, is present in the plasma membrane of E. californica cells. The nature of this putative vanadate-sensitive transporter is not known at present.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Immunolocalization of alkaloids and X-ray microanalysis of elements in lupin seeds

Summary Immunolocalization of alkaloids in lupin seeds (Lupinus spp.) has been performed by cryofixation and conventional methods. Alkaloids were localized in the protein bodies of the cotyledon cells. Some immunogold particles in the walls of these cells were also observed. There were no differences in the sites of localization between the two mentioned methods. X-ray microanalysis of elements showed the presence of P, Mg, S, and K in the protein bodies of cotyledon cells in lupin seeds. The role of K⁺ in alkaloids transport is discussed.     

Influence of chitosan,acetic acid and citric acid on growth and tropane alkaloid production in transformed roots of Brugmansia candida Effect of medium pH and growth phase

The effects of chitosan, acetic acid and citric acid on production and release of hyoscyamine and scopolamine in hairy root cultures of Brugmansia candida were studied. Chitosan and acetic acid were tested at different concentrations and also at different media pH values. At pH 5.5, and at certain concentrations, acetic acid and chitosan increased the content of root scopolamine and hyoscyamine, and promoted the release of both alkaloids. Lowering the pH to 3.5 and 4.5 reduced the accumulation of both alkaloids in the roots, but at a pH of 4.5, their release increased significantly. Acetic and citric acid stimulated the release of scopolamine and hyoscyamine. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regulation of the biosynthesis of ergot alkaloids byPenicillium sizovae

To investigate the regulation of the biosynthesis of ergot alkaloids by end products, the effect of exogenous agroclavine-1 and epoxyagroclavine-1 on their accumulation byP. sizovae was studied. The added alkaloids stimulated considerably their own biosynthesis depending on their concentration and time of introduction. The stimulating effect of both alkaloids and products of their degradation was suggested. Exogenous agroclavine-1 and epoxyagroclavine-1 changed the relation between the quantities of intra-and extracellular alkaloids, thus pointing to their possible influence on the transport processes.     

Transplasmalemma electron transport is changed in simian virus 40 transformed liver cells

Transplasma membrane electron transport activity by fetal rat liver cells (RLA209-15) infected with a temperature-sensitive strain of SV40 has been measured with cells grown at the restrictive temperature (40°C) and permissive temperature (33°C). The transformed cells grown at 33°C had only one-half the rate of external ferricyanide reduction as the nontransformed cells held at 40°C. Both theK _m andV _max for ferricyanide reduction were changed in the transformed state. The change inV _max can be based on a decrease of NADH in the transformed cells. The change in rate with ferricyanide does not depend on change in surface charge. Reduction of external ferricyanide was accompanied by release of protons from the cells. The ratio of protons released to ferricyanide reduced was higher in the transformed cells than in the non-transformed cells. Since the transplasma membrane electron transport has been shown to stimulate cell growth under limiting serum, the changes in the plasma membrane electron transport and proton release in transformed cells may relate to modification of growth control.     

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

Permeabilization of Cinchona ledgeriana cells by dimethylsulphoxide. effects on alkaloid release and long-term membrane integrity

Dimethyl sulphoxide (DMSO) has been used to permeabilize cells of Cinchona ledgeriana in suspension culture and promote the release of intracellular alkaloids. 5–6% v/v is required before any release is seen, and greater than 20% DMSO is required for full release. Even at these high levels of DMSO release is slow, taking in excess of seven hours to reach completion. Conditions which produce significant release of alkaloids have a deleterious effect on cells. Many of the membranes permeabilized did not recover their ability to selectively exclude compounds such as mannitol when the DMSO was removed. It is concluded that DMSO is not a suitable material for inducing alkaloid release in any biotechnological exploitation of alkaloid production by C. ledgeriana.Abbreviations DMSO Dimethyl sulphoxide - 2,4D 2,4-Dichlorophenoxyacetic acid     

Cellular distribution of alkaloids and their translocation via phloem and xylem: the importance of compartment pH

The physico‐chemical background of alkaloid allocation within plants is outlined and discussed exemplarily for pyrrolizidine alkaloids (PAs) and nicotine. The trigger for this discourse is the finding that, for example, PAs, which are taken up from the soil, are translocated in the xylem, whereas – when genuinely present in plants – they are allocated as N‐oxides via phloem. Special emphasis is put on the impact of different pH values in certain compartments, as this entails significant changes in the relative lipophilic character of alkaloids: tertiary alkaloids diffuse readily through biomembranes, while the corresponding protonated alkaloids are retained in acidic compartments, i.e. vacuoles or xylem. Therefore, this phenomenon, well known as the ‘ion trap mechanism’, is also relevant for long‐distance transport of alkaloids. Any efficient allocation of typical tertiary alkaloids within the phloem can thus be excluded. In contrast, due to their strongly increased hydrophilic properties, alkaloid‐N‐oxides or quarternary alkaloids cannot diffuse through biomembranes and, consequently, would be retained in the acidic xylem during translocation. The major aim of this paper is to sharpen the mind for the chemical peculiarities of alkaloids and to consider them adequately in forthcoming investigations on allocation of alkaloids.     

Role of vacuolar transporter proteins in plant secondary metabolism: Catharanthus roseus cell culture

Here the current status of knowledge on some well-characterized transporters located in the vacuolar membrane is reviewed. As different cellular compartments and even different cells may be involved in certain steps of a biosynthetic pathway, the regulation of the flux is not only dependent on structural genes encoding enzymes catabolizing certain steps but also transport has a major regulatory function. The aim of the present review is to give an overview of the present knowledge of transport of secondary metabolites in plants, and to use this information in the context of our knowledge about Catharanthus roseus alkaloid biosynthesis. This should lead to further insight in the possible role of various transporters in the regulation of the biosynthesis of these alkaloids.     

异喹啉类生物碱生物合成、运输、储藏相关细胞生物学研究进展

生物碱是存在于自然界中一类含氮的杂环小分子天然化合物, 约有1.2万多种。其代谢途径往往受到特异酶类的调控, 因此通过对某些特征性关键酶的定位可以确定生物碱的合成、转运和储藏相关的特定细胞或亚细胞结构。该文以异喹啉类生物碱血根碱为主, 对其生物合成、运输和储藏相关的细胞和亚细胞水平上的研究结果进行概述。异喹啉生物碱生物合成主要发生在皮层、筛管、伴胞和乳汁管等组织细胞中, 囊泡、液泡、细胞质基质、微粒体、内质网和叶绿体类囊体等众多细胞器参与生物碱的运输和储存。同时对药用植物中常见的其它几类主要生物碱的相关研究结果进行了讨论。     

Experience influences gustatory responsiveness to pyrrolizidine alkaloids in the polyphagous caterpillar,Estigmene acrea

Electrophysiological recordings from taste sensilla of the caterpillar Estigmene acrea with the pyrrolizidine alkaloid, seneciphylline N-oxide, demonstrated that prior feeding on plants with pyrrolizidine alkaloids caused an increase in responsiveness of the PA-sensitive cells in two sensilla, relative to feeding on plants without such chemicals. Rearing on synthetic diet without pyrrolizidine alkaloids for up to seven generations caused a continuous decline in responsiveness, that could be reversed by experience with powdered Crotalaria pumila in the diet or by pure pyrrolizidine alkaloid, monocrotaline, in the diet. Response to the cardiac glycoside, ouabain, that stimulates one of the two pyrrolizidine alkaloid-sensitive cells, showed a similar decline. Pyrrolizidine alkaloids had no measurable effect on growth and development. Responses in all other taste cells were unaffected. The data are discussed in relation to the possible adaptive significance and the possible mechanisms involved.R.F. Chapman has died since this article was written     

Comparison of the influence of different elicitors on hyoscyamine and scopolamine content in hairy root cultures of <Emphasis Type="Italic">Brugmansia candida</Emphasis>

Summary Hairy roots of Brugmansia candida were exposed to different elicitors, such as pectinase, B. candida root homogenate, Hormonema ssp. homogenate, and the acetate control buffer. Pectinase increased intracellular hyoscyamine (200–300%) and the release of both alkaloids up to 1500% (scopolamine) and 1100% (hyoscyamine). However, the increment observed in both alkaloids in roots with the acetate control buffer was superior than with pectinase, obtaining increases of 700% in hyoscyamine and 200% in scopolamine. The B. candida root homogenate enhanced the accumulation (50–600%) and specific production of both alkaloids (ca. 150%). Hormonema ssp. homogenates induced different responses according to the original medium in which the fungus was cultured. The effect of each elicitor is discussed.     

Tropane alkaloid production by Agrobacterium rhizogenes transformed hairy root cultures of Atropa baetica Willk. (Solanaceae)

Atropa baetica hairy root cultures were induced after infecting stem segments with Agrobacterium rhizogenes strain ATCC 15834. Accumulation of the tropane alkaloids atropine and scopolamine by hairy roots cultured in half- and full-strength Murashige and Skoog (MS) medium was high, although this was not growth associated. These alkaloids were also released into both liquid media. Higher tropane alkaloids present both in hairy roots and liquid medium occurred in half MS medium, showing a clear relationship between slow growth of cultures and higher product accumulation. The pH of both nutrient media varied as culture progressed, and seemed to be associated with the release of scopolamine. GC-MS analyses showed the presence of a new compound, namely tigloylpseudotropine; moreover, 3α-isobutyryloxytropane, formerly found only in plant leaf tissue, was also identified in the hairy roots. Received: 18 August 1997 / Revision received: 30 November 1997 / Accepted: 20 January 1998     

A highly selective alkaloid uptake system in vacuoles of higher plants

Vacuoles were isolated from different plant cell cultures and the transport mechanism for alkaloid uptake at the tonoplast membrane, as well as the compartmentation of enzymes and products inside the cells were investigated. While serpentine, the major alkaloid of Catharanthus roseus cells, is definitely located inside the vacuole, two key enzymes of the indole-alkaloid pathway, strictosidine synthase and a specific glucosidase, are located in the cytosol. Transport of alkaloids across the tonoplast into the vacuolar space has been characterized as an active, engergy-requiring mechanism, which is sensitive to the temperature and pH of the surrounding medium, stimulated by K⁺ and Mg²⁺, and inhibited by N,N-dicyclohexylcarbodiimid and Cu²⁺. The alkaloids accumulate inside the vacuoles against a concentration gradient, and the uptake system is specific for alkaloids indigenous to the plant from which the vacuoles have been isolated.Abbreviation DCCD N,N-dicyclohexylcarbodiimid Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Induction of apoptosis in a human erythroleukemic cell line K562 by Tylophora alkaloids involves release of cytochrome c and activation of caspase 3

Tylophora alkaloids are plant products known for their antiasthamatic and antiproliferative activities. The underlying cellular changes resulting from inhibition of proliferation were investigated. Tylophora alkaloids induced apoptosis in K562 cells with characteristic apoptotic features like nuclear condensation, apoptotic body formation, flipping of membrane phosphatidylserine, activation of caspase 3 and release of mitochondrial cytochrome c. These studies suggest that the Tylophora alkaloids, in addition to their antiproliferative effects also induce apoptosis in erythroleukemic cells. These observations imply that Tylophora alkaloids could be useful molecules for their antiproliferative activity and for induction of apoptosis in tumor cells.     

Evidence for Multidrug Resistance-1 P-Glycoprotein-dependent Regulation of Cellular ATP Permeability

The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)¹ family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl⁻ current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd³⁺ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways. Received: 20 November 2000/Revised: 25 May 2001     

In‐depth proteome mining of cultured Catharanthus roseus cells identifies candidate proteins involved in the synthesis and transport of secondary metabolites

Madagascar periwinkle (Catharanthus roseus) is the major source of terpenoid indole alkaloids, such as vinblastine or vincristine, used as natural drugs against various cancers. In this study, we have extensively analyzed the proteome of cultured C. roseus cells. Comparison of the proteomes of two independent cell lines with different terpenoid indole alkaloid metabolism by 2D‐DIGE revealed 358 proteins that differed quantitatively by at least a twofold average ratio. Of these, 172 were identified by MS; most corresponded to housekeeping proteins. Less abundant proteins were identified by LC separation of tryptic peptides of proteins from one of the lines. We identified 1663 proteins, most of which are housekeeping proteins or involved in primary metabolism. However, 63 enzymes potentially involved in secondary metabolism were also identified, of which 22 are involved in terpenoid indole alkaloid biosynthesis and 16 are predicted transporters putatively involved in secondary metabolite transport. About 30% of the proteins identified have an unclear or unknown function, indicating important gaps in knowledge of plant metabolism. This study is an important step toward elucidating the proteome of C. roseus, which is critical for a better understanding of how this plant synthesizes terpenoid indole alkaloids.     

Propentofylline and Other Adenosine Transport Inhibitors Increase the Efflux of Adenosine Following Electrical or Metabolic Stimulation of Rat Hippocampal Slices

Abstract: Propentofylline is a novel neuroprotective agent that has been shown to act as an adenosine transport inhibitor as well as an adenosine receptor antagonist. In the present series of experiments we have compared the effects of propentofylline with those of known adenosine transport inhibitors and receptor antagonists on the formation of adenosine in rat hippocampal slices. The ATP stores were labeled by incubating the slices with [³H]-adenine. The total ³H overflow and the overflow of endogenous and ³H-labeled adenosine, inosine, and hypoxanthine were measured. Adenosine release, secondary to ATP breakdown, was induced both by hypoxia/hypoglycemia and by electrical field stimulation. Propentofylline (20–500 µM) increased the release of endogenous and radiolabeled adenosine, without increasing the total release of purines. Thus, the drug altered the pattern of released purines, i.e., increasing adenosine and decreasing inosine and hypoxanthine. This pattern, which was observed when purine release was induced both by electrical field stimulation and by hypoxia/hypoglycemia, was shared by the nucleoside transport inhibitor dipyridamole (1 µM) and by mioflazine (1 µM) and nitrobenzylthioinosine (1 µM). By contrast, other xanthines, including theophylline (100 µM) and 8-cyclopentyltheophylline (10 µM), enprofylline (100 µM), or torbafylline (300 µM), if anything, increased the total release of purines without alterations of the pattern of release. These results indicate that nucleoside transport inhibitors can decrease the release of purines from cells and at the same time increase the concentration of extracellular adenosine, possibly by preventing its uptake and subsequent metabolism. This change in purine metabolism may be beneficial with regard to cell damage after ischemia. The results also indicate that propentofylline behaves in such a potentially beneficial manner.     

The role of pH gradients across the plasmalemma of Catharanthus roseus and its involvement in the release of alkaloids

During growth, Catharanthus roseus cells exhibit an acidification of the culture medium that may be controlled by Ca²⁺. With a view to enhance the productivity of alkaloids by plant cells, the effect of extracellular pH modifications on the excretion processes has been investigated. Ca²⁺ dependent proton pumping leads to the release of various lipophilic amine-like compounds (benzylamine, methylamine, nicotine) initially accumulated by the cells, but also facilitates the excretion of endogenous ajmalicine. Once released in the medium, these compounds are however taken up again by the cells, probably as the charged form. For the alkaloid contained in C. roseus some evidence suggests that the diffusible form comes from the cytosolic compartment and not from the storage vacuoles. This appears to be a major production limitation to the use of pH gradients in order to favour alkaloid excretion.