Pyrrolizidine alkaloids as oviposition stimulants for the cinnabar moth,Tyria jacobaeae

In choice experiments with artificial leaves, we tested related pyrrolizidine alkaloids (PAs) for their stimulatory effects on the oviposition of the cinnabar moth, a specialist on the PA-containing plant Senecio jacobaea. The PAs from S. jacobaea that we tested stimulated oviposition. Monocrotaline also stimulated oviposition although this PA is not found in plants of the genus Senecio. The moths preferred ovipositing on filter paper with a PA mixture extracted from S. jacobaea to ovipositing on filter paper with single PAs. Senkirkine, heliotrine, and retrorsine did not stimulate oviposition. The nonactive retrorsine differs only in one OH group to the active senecionine, indicating that small structural differences alter the stimulatory activity of PAs. However, a PA mixture extracted from a nonhost plant, Senecio inaequidens, that consisted of 81% of the nonactive retrorsine did stimulate oviposition. Oviposition preferences between Senecio species seem to be determined by chemical compounds other than PAs.     

Toxicity of Pyrrolizidine Alkaloids to Spodoptera exigua Using Insect Cell Lines and Injection Bioassays

Pyrrolizidine alkaloids (PAs) are feeding deterrents and toxic compounds to generalist herbivores. Among the PAs of Jacobaea vulgaris Gaertn, jacobine and erucifoline are the most effective against insect herbivores as indicated by correlative studies. Because little is known about the effect of jacobine and erucifoline as individual PAs, we isolated these compounds from their respective Jacobaea chemotypes. These PAs and other commercially available senecionine-like PAs, including senecionine, seneciphylline, retrorsine, and senkirkine, were tested as free base and N-oxide forms at a range of 0–70 ppm. Feeding bioassays using live insects are closer to the natural pattern but require relatively large amounts of test compounds. We, therefore, compared the toxicity of PAs using both Spodoptera exigua cell line and larval injection bioassays. Both bioassays led to similar results in the order of PA toxicity, indicating that the cell lines are a valuable tool for a first toxicity screen. Testing individual PAs, jacobine and erucifoline were the most toxic PAs, suggesting their major role in plant defense against generalist herbivores. Senkirkine and seneciphylline were less toxic than jacobine and erucifoline but more toxic than retrorsine. Senecionine was not toxic at the tested concentrations. For all toxic PAs, the free base form was more toxic than the N-oxide form. Our results demonstrate that structural variation of PAs influences their effectiveness in plant defense.     

Pyrrolizidine alkaloids from Senecio jacobaea affect fungal growth

We investigated the growth-reducing effects of pyrrolizidine alkaloids (PAs) from Senecio jacobaea on nine plant-associated fungi (five strains of Fusarium oxysporum, two of F. sambucinum, and two of Trichoderma sp). Fungal growth was monitored on water agar media containing different concentrations of monocrotaline, retrorsine, or a purified extract of PAs from S. jacobaea. The growth rate of six strains was inhibited by PAs at the highest test concentration (3.33 mM), with the magnitude of the inhibition (7–35%) being dependent upon the specific fungus-PA interaction. In general, the PA extract caused the largest inhibition. However, the fungi isolated from S. jacobaea were positively affected by the PA extract (7–9%). Retrorsine N oxide was as effective as retrorsine in its inhibition of mycelium growth.     

Sequestration, Maintenance, and Tissue Distribution of Pyrrolizidine Alkaloid N-Oxides in Larvae of Two Oreina Species

Oreina cacaliae and O. speciosissima are leaf beetles that, as larvae and adults, sequester pyrrolizidine alkaloid N-oxides (PAs) as defensive compounds from their host plants Adenostyles alliariae and Senecio nemorensis. As in most Oreina species, O. speciosissima is also defended by autogenously produced cardenolides (mixed defensive strategy), whereas O. cacaliae does not synthesize cardenolides and is exclusively dependent on host-plant-acquired PAs (host-derived defense). Adults of the two Oreina species were found to have the same PA storage capacity. The larvae, however, differ; larvae of O. speciosissima possess a significantly lower capability to store PAs than O. cacaliae. The ability of Oreina larvae to sequester PAs was studied by using tracer techniques with ¹⁴C-labeled senecionine N-oxide. Larvae of the two species efficiently take up [¹⁴C]senecionine N-oxide from their food plants and store the alkaloid as N-oxide. In O. cacaliae, there is a slow but continuous loss of labeled senecionine N-oxide. This effect may reflect the equilibrium between continuous PA uptake and excretion, resulting in a time-dependent tracer dilution. No noticeable loss of labeled alkaloid is associated with molting. Senecionine N-oxide is detectable in all tissues. The hemolymph is, with ca. 50–60% of total PAs, the major storage compartment, followed by the integument, with ca 30%. The alkaloid concentration in the hemolymph is approximately sixfold higher than in the solid tissues. The selectivity of PA sequestration in larvae is comparable to PA sequestration in the bodies of adult beetles.     

Direct Evidence for Membrane Transport of Host-Plant-Derived Pyrrolizidine Alkaloid N-Oxides in Two Leaf Beetle Genera

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae, and O. speciosissima sequester pyrrolizidine alkaloids from their asteracean host plants and store them as nontoxic N-oxides. Previous analyses showed that Longitarsus is able to N-oxidize protoxic tertiary PAs, but did not resolve in which form N-oxides are taken up. For Oreina, beetles seem able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of them in the gut yielding protoxic free bases. Here, we confirm the predicted direct uptake of PAs as N-oxides by Oreina, and elucidate the situation for Longitarsus by applying double-labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC, and submitted to ESI-MS, GC-MS, and analysis of the specific radioactivity. The 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC-purified senecionine N-oxide with a second compound, identified as a dihydrosenecionine N-oxide by high-resolution CID analysis. The dihydrosenecionine N-oxide, probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle. The recovered senecionine N-oxide retained 74% 18O-label. The remaining 25% is mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar nontoxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).     

MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug–drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP⁺. Uptake of MPP⁺ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP⁺ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP⁺ transporting solute carrier proteins (SLCs) in general.     

Pyrrolizidine Alkaloids Induce Cell Death in Human HepaRG Cells in a Structure-Dependent Manner

Pyrrolizidine alkaloids (PAs) are a group of secondary metabolites produced in various plant species as a defense mechanism against herbivores. PAs consist of a necine base, which is esterified with one or two necine acids. Humans are exposed to PAs by consumption of contaminated food. PA intoxication in humans causes acute and chronic hepatotoxicity. It is considered that enzymatic PA toxification in hepatocytes is structure-dependent. In this study, we aimed to elucidate the induction of PA-induced cell death associated with apoptosis activation. Therefore, 22 structurally different PAs were analyzed concerning the disturbance of cell viability in the metabolically competent human hepatoma cell line HepaRG. The chosen PAs represent the main necine base structures and the different esterification types. Open-chained and cyclic heliotridine- and retronecine-type diesters induced strong cytotoxic effects, while treatment of HepaRG with monoesters did not affect cell viability. For more detailed investigation of apoptosis induction, comprising caspase activation and gene expression analysis, 14 PA representatives were selected. The proapoptotic effects were in line with the potency observed in cell viability studies. In vitro data point towards a strong structure–activity relationship whose effectiveness needs to be investigated in vivo and can then be the basis for a structure-associated risk assessment.     

Allelochemical Activities of Pyrrolizidine Alkaloids: Interactions with Neuroreceptors and Acetylcholine Related Enzymes

Thirteen pyrrolizidine alkaloids (PAs) 3′-acetylheliosupine, echihumiline, echihumiline N-oxide, echimidine, heliosupine, heliosupine N-oxide, heliotrine, monocrotaline, pycnanthine, retronecine, riddeline, senecionine, and seneciphylline) were analyzed for their interactions with acetylcholine-related enzymes, such as acetylcholine esterase (AChE), butyrylcholinesterase (BChE), choline acetyl transferase (ChAT), and neuroreceptors, such as α₁- and α₂-adrenergic, nicotinergic (nACh), muscarinergic (mACh) and serotonin₂ (5-HT₂) receptors. Whereas most PAs did not affect the enzymes, they show significant binding activities to mACh and 5-HT₂ receptors: Twelve PAs exhibited a 50% inhibition of the specific binding of the radioligand [³H]quinuclidinyl benzilate (QNB) at the mAChR, i.e., IC₅₀ values were between 8.7 μM and 512.5 μM, and 10 PAs exerted a 50% inhibition of the specific binding of the radioligand [³H]ketanserine at the 5-HT₂R with IC₅₀ values between 23.2 μM and 608.6 μM. The most active compound was 3′-acetylheliosupine, which was able to bind to all of the studied receptors with IC₅₀ values in the range between 2.9 μM and 159.7 μM. The data imply that free PAs and PA N-oxides can affect several molecular targets: Besides long-term toxicity through DNA alkylation (by PA metabolites generated in the liver), liver and pneumotoxicity, neuroreceptors (among other molecular targets) may be modulated. The interference of PAs with neuronal signal transduction could mediate adverse physiological responses in herbivores and could thus contribute to chemical defense in plants and animals against herbivores and predators.     

Acquired and Partially De Novo Synthesized Pyrrolizidine Alkaloids in Two Polyphagous Arctiids and the Alkaloid Profiles of Their Larval Food-Plants

The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A-C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1beta,2beta-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40-70% (E. acrea) and 17-37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.     

Taste receptors for pyrrolizidine alkaloids in a monophagous caterpillar

Caterpillars of Utetheisa ornatrix are monophagous on species of Crotalaria from which they obtain pyrrolizidine alkaloids (PAs) for defense and which the males convert to a pheromone. We show that a taste receptor neuron in each of the lateral and medial galeal styloconic sensilla responds to PAs of three different types. Monocrotaline, commonly present in Crotalaria species, is the most strongly stimulating with thresholds of response below 10^–11 and 10^–9 M in the two sensilla. These are among the lowest known taste thresholds in any insect and are similar to the thresholds for PAs in a polyphagous arctiid caterpillar that also sequesters PAs and uses them as the source of male pheromone. The receptors also respond to heliotrine, a type of PA that is probably never encountered by the insects. Monocrotaline and senecionine N-oxide are shown in behavioral assays to be phagostimulants. The data show that there is no tight link between taste receptor sensitivity to specific PAs and hostplant selection in these caterpillars. Perhaps the adults are primarily responsible for hostplant selection.     

In Vitro Characterization of Renal Drug Transporter Activity in Kidney Cancer

The activity of drug transporters is central to the secretory function of the kidneys and a defining feature of renal proximal tubule epithelial cells (RPTECs). The expression, regulation, and function of these membrane-bound proteins is well understood under normal renal physiological conditions. However, the impact of drug transporters on the pathophysiology of kidney cancer is still elusive. In the present study, we employed different renal cell carcinoma (RCC) cell lines and a prototypical non-malignant RPTEC cell line to characterize the activity, expression, and potential regulatory mechanisms of relevant renal drug transporters in RCC in vitro. An analysis of the uptake and efflux activity, the expression of drug transporters, and the evaluation of cisplatin cytotoxicity under the effects of methylation or epidermal growth factor receptor (EGFR) inhibition showed that the RCC cells retained substantial drug transport activity. In RCC cells, P-glycoprotein was localized in the nucleus and its pharmacological inhibition enhanced cisplatin toxicity in non-malignant RPTECs. On the other hand, methylation inhibition enhanced cisplatin toxicity by upregulating the organic cation uptake activity in RCC cells. Differential effects of methylation and EGFR were observed in transporter expression, showing regulatory heterogeneity in these cells. Interestingly, the non-malignant RPTEC cell line that was used lacked the machinery responsible for organic cation transport, which reiterates the functional losses that renal cells undergo in vitro.     

Interaction Profiles of Central Nervous System Active Drugs at Human Organic Cation Transporters 1–3 and Human Plasma Membrane Monoamine Transporter

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1–3; hOCT1–3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.     

Incorporation of Sulfonamide Moiety into Biguanide Scaffold Results in Apoptosis Induction and Cell Cycle Arrest in MCF-7 Breast Cancer Cells

Metformin, apart from its glucose-lowering properties, has also been found to demonstrate anti-cancer properties. Anti-cancer efficacy of metformin depends on its uptake in cancer cells, which is mediated by plasma membrane monoamine transporters (PMAT) and organic cation transporters (OCTs). This study presents an analysis of transporter mediated cellular uptake of ten sulfonamide-based derivatives of metformin in two breast cancer cell lines (MCF-7 and MDA-MB-231). Effects of these compounds on cancer cell growth inhibition were also determined. All examined sulfonamide-based analogues of metformin were characterized by greater cellular uptake in both MCF-7 and MDA-MB-231 cells, and stronger cytotoxic properties than those of metformin. Effective intracellular transport of the examined compounds in MCF-7 cells was accompanied by high cytotoxic activity. For instance, compound 2 with meta-methyl group in the benzene ring inhibited MCF-7 growth at micromolar range (IC₅₀ = 87.7 ± 1.18 µmol/L). Further studies showed that cytotoxicity of sulfonamide-based derivatives of metformin partially results from their ability to induce apoptosis in MCF-7 and MDA-MB-231 cells and arrest cell cycle in the G0/G1 phase. In addition, these compounds were found to inhibit cellular migration in wound healing assay. Importantly, the tested biguanides are more effective in MCF-7 cells at relatively lower concentrations than in MDA-MB-231 cells, which proves that the effectiveness of transporter-mediated accumulation in MCF-7 cells is related to biological effects, including MCF-7 cell growth inhibition, apoptosis induction and cell cycle arrest. In summary, this study supports the hypothesis that effective transporter-mediated cellular uptake of a chemical molecule determines its cytotoxic properties. These results warrant a further investigation of biguanides as putative anti-cancer agents.     

Synergistic Control of Transmitter Turnover at Glycinergic Synapses by GlyT1, GlyT2, and ASC-1

In addition to being involved in protein biosynthesis and metabolism, the amino acid glycine is the most important inhibitory neurotransmitter in caudal regions of the brain. These functions require a tight regulation of glycine concentration not only in the synaptic cleft, but also in various intracellular and extracellular compartments. This is achieved not only by confining the synthesis and degradation of glycine predominantly to the mitochondria, but also by the action of high-affinity large-capacity glycine transporters that mediate the transport of glycine across the membranes of presynaptic terminals or glial cells surrounding the synapses. Although most cells at glycine-dependent synapses express more than one transporter with high affinity for glycine, their synergistic functional interaction is only poorly understood. In this review, we summarize our current knowledge of the two high-affinity transporters for glycine, the sodium-dependent glycine transporters 1 (GlyT1; SLC6A9) and 2 (GlyT2; SLC6A5) and the alanine–serine–cysteine-1 transporter (Asc-1; SLC7A10).     

Relationships between Inhibition,Transport and Enhanced Transport via the Organic Cation Transporter 1

The organic cation transporter 1 (OCT1, SLC22A1) transports a large number of structurally diverse endogenous and exogenous substrates. There are numerous known competitive and non-competitive inhibitors of OCT1, but there are no studies systematically analyzing the relationship between transport, stimulation, and inhibition. Here, we tested in vitro OCT1 inhibition by OCT1 substrates and transport of OCT1 inhibitors under uniform analytical conditions. Beyond inhibition testing with two model substrates, we tested nine additional OCT1 substrates for their mutual inhibition. Inhibition of ASP⁺ uptake by most OCT1 substrates was weak. The model substrate sumatriptan, with its moderately stronger inhibitability, was used to confirm this. Interestingly, OCT1 substrates exhibiting stronger OCT1 inhibition were mainly biaromatic β-agonistic drugs, such as dobutamine, fenoterol, ractopamine and ritodrine. Biaromatic organic cations were both, strong inhibitors and good substrates, but many OCT1 substrates showed little pairwise inhibition. Surprisingly, sumatriptan did significantly enhance dobutamine uptake. This effect was concentration dependent and additional experiments indicated that efflux inhibition may be one of the underlying mechanisms. Our data suggests, that OCT1 substrates are mainly weak OCT1 inhibitors and among those inhibiting well, noncompetitive inhibition could be responsible. Weak competitive inhibition confirms that OCT1 inhibition screenings poorly predict OCT1 substrates. Additionally, we showed that the OCT1 substrate sumatriptan can enhance uptake of some other OCT1 substrates. OCT1 transport stimulation was already observed earlier but is still poorly understood. Low OCT1 uptake inhibition and strong OCT1 efflux inhibition could be mechanisms exploitable for enhancing transport.     

Magnesium Isoglycyrrhizinate Reduces Hepatic Lipotoxicity through Regulating Metabolic Abnormalities

The excessive accumulation of lipids in hepatocytes induces a type of cytotoxicity called hepatic lipotoxicity, which is a fundamental contributor to liver metabolic diseases (such as NAFLD). Magnesium isoglycyrrhizinate (MGIG), a magnesium salt of the stereoisomer of natural glycyrrhizic acid, is widely used as a safe and effective liver protectant. However, the mechanism by which MGIG protects against NAFLD remains unknown. Based on the significant correlation between NAFLD and the reprogramming of liver metabolism, we aimed to explore the beneficial effects of MGIG from a metabolic viewpoint in this paper. We treated HepaRG cells with palmitic acid (PA, a saturated fatty acid of C16:0) to induce lipotoxicity and then evaluated the antagonistic effect of MGIG on lipotoxicity by investigating the cell survival rate, DNA proliferation rate, organelle damage, and endoplasmic reticulum stress (ERS). Metabolomics, lipidomics, and isotope tracing were used to investigate changes in the metabolite profile, lipid profile, and lipid flux in HepaRG cells under different intervention conditions. The results showed that MGIG can indeed protect hepatocytes against PA-induced cytotoxicity and ERS. In response to the metabolic abnormality of lipotoxicity, MGIG curtailed the metabolic activation of lipids induced by PA. The content of total lipids and saturated lipids containing C16:0 chains increased significantly after PA stimulation and then decreased significantly or even returned to normal levels after MGIG intervention. Lipidomic data show that glycerides and glycerophospholipids were the two most affected lipids. For excessive lipid accumulation in hepatocytes, MGIG can downregulate the expression of the metabolic enzymes (GPATs and DAGTs) involved in triglyceride biosynthesis. In conclusion, MGIG has a positive regulatory effect on the metabolic disorders that occur in hepatocytes under lipotoxicity, and the main mechanisms of this effect are in lipid metabolism, including reducing the total lipid content, reducing lipid saturation, inhibiting glyceride and glycerophospholipid metabolism, and downregulating the expression of metabolic enzymes in lipid synthesis.     

Sequestration of ingested [14C]senecionineN-oxide in the exocrine defensive secretions of chrysomelid beetles

Oreina cacaliae (Chrysomelidae) sequesters in its elytral and pronotal defensive secretion theN-oxides of pyrrolizidine alkaloids (PAN-oxides) from its food plantAdenostyles alliariae (Asteraceae). [¹⁴C]SenecionineN-oxide was applied for detailed studies of PAN-oxide sequestration. An average of 11.4% of total radioactivity is taken up by individual beetles which had received [¹⁴C]senecionineN-oxide with their food leaves 8 days before. An average of 28.9% of the ingested radioactivity could be recovered from the defensive secretions collected twice, i.e., 5 and 8 days after tracer feeding. The tracer transfer into the secretion seems to be a slow but progressive process as indicated by the high percentage of tracer still recovered from the secretion sampled after 8 days. Chromatographic analysis revealed that [¹⁴C]senecionineN-oxide is the only labeled compound in the defensive secretion. Beetles that fed on tertiary [¹⁴C]senecionine sequestered only trace amounts of radioactivity (exclusively present as labeled IV-oxide) in their secretions.O. speciosissima, a species also adapted to PA containing food plants, was shown to sequester [¹⁴C]senecionineN-oxide with the same efficiency asO. cacaliae. O. bifrons, a specialist feeding onChaerophyllum hirsutum (Apiaceae), rejected PA treated leaf samples already at very low PA concentrations (10 nmol/leaf piece). In bothO. cacaliae andO. speciosissima, [¹⁴C]senecionineN-oxide applied by injection into the hemolymph is rapidly transferred into the glands.O. bifrons, not adapted to pyrrolizidine alkaloid containing plants was unable to sequester [¹⁴C]-senecionineN- oxide in the secretion but rapidly eliminated the tracer with the frass. Again, only traces of labeled [¹⁴C]senecionineN-oxide were found in the defensive secretions of the two PA adapted species if labeled senecionine was injected. It is suggested that the beetles are adapted to theN-oxide form of PAs, similarly as their food plants, and that they lack the ability to efficientlyN-oxidize tertiary PAs. No indication forde novo PA synthesis by the beetles was found in tracer feeding experiments with the biogenetic PA precursor putrescine.     

Structure-activity relationships of pyrrolizidine alkaloids in insect chemical defense against the orb-weaving spider Nephila clavipes

Pyrrolizidine alkaloids (PAs) are known to protect Arctiidae moths and Danainae and Ithomiinae butterflies against the orb-weaving spider Nephila clavipes (Araneae, Araneidae, Tetragnathinae), which liberates adults of these insects unharmed from its web. We tested against this spider the role of eight PAs and one derived structure [an 89:11 mixture of the 12-membered macrocyclic diester free base integerrimine and senecionine and the respective N-oxide; two hydrolysis products from this mixture (the necine base retronecine, its respective N-oxide, and a mixture of integerriminic and senecionic necic acids); the 12-membered macrocyclic senkirkine; the 9-O-monoester free base senecioylretronecine and its respective N-oxide; and the 9-O-monoester free base callimorphine (a PA biosynthesized only by insects from retronecine)]. The mixture integerrimine–senecionine N-oxide seems to be more active than the respective free base [LibD₅₀ (liberation dose 50) = 0.042 and 0.153 g/dry weight of prey, respectively], but the difference in activity between the N-oxide and free base of the 9-O-monoester senecioylretronecine was slight (LibD₅₀ = 0.167 and 0.104, respectively). Senkirkine, an otonecine base PA that does not form N-oxide and is not found in insects, was the less active, showing the highest LibD₅₀ (0.354). The difference in antipredator activity between N-oxides and free bases from macrocyclic diesters and monoesters may be correlated with physicochemical properties of these molecules in interaction with the Nephila receptors. For the active structures, there was a significant correlation between dosage and antipredator activity. Both forms of retronecine and a necic acid mixture were inactive, supporting the hypothesis that PAs biosynthesized by insects from retronecine were originally produced and stored in order to optimize chemical defense. Comparison of dose/activity data with reported amounts of PAs in butterflies suggested that, in general, PA-specialist insects are protected against predation by Nephila.     

A Review on the Role of Bicarbonate and Proton Transporters during Sperm Capacitation in Mammals

Alkalinization of sperm cytosol is essential for plasma membrane hyperpolarization, hyperactivation of motility, and acrosomal exocytosis during sperm capacitation in mammals. The plasma membrane of sperm cells contains different ion channels implicated in the increase of internal pH (pH_i) by favoring either bicarbonate entrance or proton efflux. Bicarbonate transporters belong to the solute carrier families 4 (SLC4) and 26 (SLC26) and are currently grouped into Na⁺/HCO₃⁻ transporters and Cl⁻/HCO₃⁻ exchangers. Na⁺/HCO₃⁻ transporters are reported to be essential for the initial and fast entrance of HCO₃⁻ that triggers sperm capacitation, whereas Cl⁻/HCO₃⁻ exchangers are responsible for the sustained HCO₃⁻ entrance which orchestrates the sequence of changes associated with sperm capacitation. Proton efflux is required for the fast alkalinization of capacitated sperm cells and the activation of pH-dependent proteins; according to the species, this transport can be mediated by Na⁺/H⁺ exchangers (NHE) belonging to the SLC9 family and/or voltage-gated proton channels (HVCN1). Herein, we discuss the involvement of each of these channels in sperm capacitation and the acrosome reaction.