Physiological screening for target site insensitivity and localization of Na(+)/K(+)-ATPase in cardenolide-adapted Lepidoptera

Cardenolides are toxic plant compounds which specifically inhibit Na(+)/K(+)-ATPase, an animal enzyme which is essential for many physiological processes, such as the generation of action potentials. Several adapted insects feeding on cardenolide-containing plants sequester these toxins for their own defence. Some of these insects were shown to possess Na(+)/K(+)-ATPases with a reduced sensitivity towards cardenolides (target site insensitivity). In the present study we screened five species of arctiid moths feeding on cardenolide-containing plants for target site insensitivity towards cardenolides using an in vitro enzyme assay. The derived dose response curves of the respective Na(+)/K(+)-ATPases were compared to the insensitive Na(+)/K(+)-ATPase of the monarch butterfly (Danaus plexippus). Na(+)/K(+)-ATPases of all arctiid species tested were highly sensitive to ouabain, a water-soluble cardenolide which is most widely used in laboratory studies. Nevertheless, we detected substantial amounts of cardenolides in the haemolymph of two of the arctiid species. In caterpillars of the sequestering arctiid Empyreuma pugione and of D. plexippus we localized Na(+)/K(+)-ATPase by immunohistochemistry and western blot (in D. plexippus). Both techniques revealed strong expression of the enzyme in the nervous tissue and indicated weak expression or even absence in other tissues tested. We conclude that instead of target site insensitivity the investigated arctiid species use a different strategy to tolerate cardenolides. Most plausibly, the perineurium surrounding the nervous tissue functions as a barrier which prevents cardenolides from reaching Na(+)/K(+)-ATPase in the ventral nerve cord.     

Host use evolution in Chrysochus milkweed beetles: evidence from behaviour, population genetics and phylogeny

In two sister species of leaf beetles with overlapping host associations, Chrysochus auratus and C. cobaltinus, we established diet breadth and food preference of local populations for evaluation together with genetic differentiation between populations. While C. auratus turned out to be monophagous on the same plant wherever we collected the beetles, the studied populations of C. cobaltinus fed on three different plant species in the field. Plant preference and ranking of the potential host plants significantly differed between these populations. The amount of genetic differentiation between populations was measured by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay of a 1300 bp mitochondrial DNA (mtDNA) sequence. In addition, the dominant genotypes of all populations were sequenced. No genetic differentiation between the populations of C. auratus could be detected in the RFLP assay and sequence divergence was low (= 0.3%). In C. cobaltinus, on the other hand, genetic differentiation between populations was high, revealing a lack of gene flow over a much smaller scale and a maximum of 1.3% sequence divergence. C. cobaltinus thereby has the prerequisites for host race formation on different plants from the original host spectrum. Our sequence-based phylogeny estimate allows us to reconstruct historical diet evolution in Chrysochus. Starting from an original association with Asclepiadaceae, the common ancestor of C. auratus and C. cobaltinus included Apocynaceae in its diet. The strict specialization on Apocynum and the loss of acceptance of Asclepiadaceae observed in C. auratus could have resulted from a process similar to that displayed by C. cobaltinus populations.     

Molecular basis for the insensitivity of the Monarch (Danaus plexippus) to cardiac glycosides.

The Monarch (Danaus plexippus) sequesters cardiac glycosides for its chemical defence against predators. Larvae and adults of this butterfly are insensitive towards dietary cardiac glycosides, whereas other Lepidoptera, such as Manduca sexta and Creatonotos transiens are sensitive and intoxicated by ouabain. Ouabain inhibits the Na+,K(+)-ATPase by binding to its alpha-subunit. We have amplified and cloned the DNA sequence encoding the respective ouabain binding site. Instead of the amino acid asparagine at position 122 in ouabain-sensitive insects, the Monarch has a histidine in the putative ouabain binding site, which consists of about 12 amino acids. This change may explain the ouabain insensitivity.     

The evolution of cardenolide-resistant forms of Na⁺,K⁺ -ATPase in Danainae butterflies

Cardenolides are a class of plant secondary compounds that inhibit the proper functioning of the Na⁺, K⁺‐ATPase enzyme in susceptible animals. Nonetheless, many insect species are able to sequester cardenolides for their own defence. These include butterflies in the subfamily Danainae (Family: Nymphalidae) such as the monarch (Danaus plexippus). Previous studies demonstrated that monarchs harbour an asparagine (N) to histidine (H) substitution (N122H) in the α subunit of Na⁺, K⁺‐ATPase (ATPα) that reduces this enzyme’s sensitivity to cardenolides. More recently, it has been suggested that at ATPα position 111, monarchs may also harbour a leucine (L)/glutamine (Q) polymorphism. This later amino acid could also contribute to cardenolide insensitivity. However, here we find that incorrect annotation of the initially reported DNA sequence for ATPα has led to several erroneous conclusions. Using a population genetic and phylogenetic analysis of monarchs and their close relatives, we show that an ancient Q111L substitution occurred prior to the radiation of all Danainae, followed by a second substitution at the same site to valine (V), which arose before the diversification of the Danaus genus. In contrast, N122H appears to be a recent substitution specific to monarchs. Surprisingly, examination of a broader insect phylogeny reveals that the same progression of amino acid substitutions (Q111L → L111V + N122H) has also occurred in Chyrsochus beetles (Family: Chrysomelidae, Subfamily: Eumolpinae) that feed on cardenolide‐containing host plants. The parallel pattern of amino acid substitution in these two distantly related lineages is consistent with an adaptive role for these substitutions in reducing cardenolide sensitivity and suggests that their temporal order may be limited by epistatic interactions.     

Cytonuclear disequilibrium in chrysochus hybrids is not due to patterns of mate choice

We investigated patterns of cytonuclear disequilibrium between nuclear allozyme loci and partial mitochondrial COI and COII restriction fragment length polymorphism patterns within a population of hybridizing chrysomelid beetles and assessed to what degree the genotype frequencies of F1 hybrids were consistent with patterns of mate choice or endosymbiont infection. We document that in this population, > or = 50% of the heterospecific pairs at a given time are composed of Chrysochus auratus females and Chrysochus cobaltinus males, suggesting that at least half of the F1 hybrids should possess the C. auratus mitochondrial genotype. However, we found that the majority (89%) of F1 hybrids possessed C. cobaltinus mtDNA (P < 0.001). The lack of evidence for Wolbachia infection in these highly promiscuous beetles, coupled with the fact that F1 hybrids of both cross types do exist, indicates that endosymbionts are an unlikely explanation for the discrepancy between cytonuclear genotype frequencies and behavior. We argue that cytonuclear disequilibrium at this focal Chrysochus hybrid site is likely due to a strong directional bias in postmating prezygotic barriers in this system. The results presented here underscore the importance of combining both field and molecular data in studies of cytonuclear disequilibrium and point to the dangers inherent in attributing patterns of cytonuclear disequilibrium to assortative mating.     

Toxic cardenolides: chemical ecology and coevolution of specialized plant-herbivore interactions

Cardenolides are remarkable steroidal toxins that have become model systems, critical in the development of theories for chemical ecology and coevolution. Because cardenolides inhibit the ubiquitous and essential animal enzyme Na?/K?-ATPase, most insects that feed on cardenolide-containing plants are highly specialized. With a huge diversity of chemical forms, these secondary metabolites are sporadically distributed across 12 botanical families, but dominate the Apocynaceae where they are found in > 30 genera. Studies over the past decade have demonstrated patterns in the distribution of cardenolides among plant organs, including all tissue types, and across broad geographic gradients within and across species. Cardenolide production has a genetic basis and is subject to natural selection by herbivores. In addition, there is strong evidence for phenotypic plasticity, with the biotic and abiotic environment predictably impacting cardenolide production. Mounting evidence indicates a high degree of specificity in herbivore-induced cardenolides in Asclepias. While herbivores of cardenolide-containing plants often sequester the toxins, are aposematic, and possess several physiological adaptations (including target site insensitivity), there is strong evidence that these specialists are nonetheless negatively impacted by cardenolides. While reviewing both the mechanisms and evolutionary ecology of cardenolide-mediated interactions, we advance novel hypotheses and suggest directions for future work.     

Predicted alterations in tertiary structure of the N-terminus of Na(+)/K(+)-ATPase alpha-subunit caused by phosphorylation or acidic replacement of the PKC phosphorylation site Ser-23

The protein kinase C (PKC)-mediated phosphorylation of the Na(+)/K(+)-ATPase alpha-subunit has been shown to play an important role in regulation of the Na(+)/K(+)-ATPase activity. In the rat alpha1-subunit, phosphorylation occurs at Ser-23 and results in inhibition of the transport function of the Na(+)/K(+)-ATPase, which is mimicked by replacing the Ser-23 by the negatively charged glutamic acid or by aspartic acid. Using comparative molecular modeling, we investigated whether phosphorylation or acidic replacement at position 23 causes a dramatic change in the molecular electrostatic potential at position 23 as a result of insertion of a negative charge of the phosphoryl group or Glu per se, or whether, alternatively, the modification causes larger-scale conformational changes in the N-terminus of the alpha-subunit. The results predict a considerable conformational change of the 30-residue stretch around Ser-23 when mutated to the residues carrying a net negative charge or being phosphorylated. The structural rearrangements occur within the N-terminal helix-loop-helix motif with a set of charged residues. This motif has structural homology with one in the Ca(2+)-ATPase and may form a function-related structural site in the P-type ATPases. Comparative molecular modeling indicates a lengthening of the interhelical loop and an order-to-disorder transition by disrupting a helix at position 23 because of posphorylation.     

Fine‐tuning of defences and counter‐defences in a specialised plant–herbivore system

1. The plant–herbivore arms race has been postulated to be a major driver for generating biological and biochemical diversity on Earth. Herbivore feeding is reduced by the production of chemical and physical barriers, but increases plant resistance against subsequent attack. Accordingly, specialisation is predicted to be an outcome of herbivores being able to circumvent plant‐induced defences. 2. Using a specialised plant–herbivore system, in which adult chrysomelid beetles (Chrysochus auratus) feed on leaves and larvae feed on roots of dogbane (Apocynum spp.), this study investigated whether root latex and cardenolides are effective against the soil‐dwelling larvae, and whether such defences could be circumvented by the herbivore. 3. Across two Apocynum species, C. auratus larvae were not affected by latex production or cardenolide amounts and diversity. By contrast, cardenolide apolarity was detrimental to larval growth. Yet larval feeding decreased average root cardenolide apolarity in A. cannabinum and larvae performed better on those plants. Finally, above‐ground induction rendered the plants more toxic by increasing root cardenolide apolarity and maintaining it, even during subsequent larval herbivory. 4. Therefore, the intimate relationship and interaction between Chrysochus and Apocynum are maintained by a delicate balance of herbivore manipulation and plant chemical induction.     

Insect Na(+)/K(+)-ATPase

Na(+)/K(+)-ATPase (sodium/potassium pump) is a P-type ion-motive ATPase found in the plasma membranes of animal cels. In vertebrates, the functions of this enzyme in nerves, heart and kidney are well characterized and characteristics a defined by different isoforms. In contrast, despite different tissue distributions, insects possess a single isoform of the alpha-subunit. A comparison of insect and vertebrate Na(+)/K(+)-ATPases reveals that although the mode of action and structure are very highly conserved, the specific roles of the enzyme in most tissues varies. However, the enzyme is essential for the function of nerve cells, and in this respect Na(+)/K(+)-ATPase appears to be fundamental in metazoan evolution.     

Cryptic gametic interactions confer both conspecific and heterospecific advantages in the Chrysochus (Coleoptera: Chrysomelidae) hybrid zone

Most species pairs are isolated through the collective action of a suite of barriers. Recent work has shown that cryptic barriers such as conspecific sperm precedence can be quite strong, suggesting that they evolve quickly. However, because the strength of multiple barriers has been formally quantified in very few systems, the relative speed with which conspecific sperm precedence evolves remains unclear. Here, we measure the strength of both conspecific sperm precedence and cryptic non-competitive isolation between the hybridizing sister species, Chrysochus auratus and C. cobaltinus (Coleoptera: Chrysomelidae), and compare the strength of those barriers to the strength of other known reproductive barriers in this system. Overall, cryptic barriers in this system are weaker than other barriers, indicating that they have not evolved rapidly. Furthermore, their evolution has been asymmetric. Non-competitive barriers substantially reduce the production of hybrid offspring by C. auratus females but not by C. cobaltinus females. In multiply-mated C. cobaltinus females, heterospecific sperm outcompete conspecific sperm, as evidenced by the fact that heterospecific males sired disproportionately more offspring than predicted from the results for singly-mated females. In C. auratus females, neither sperm type has a competitive advantage. Such asymmetries explain why nearly all F1 hybrids in the field are from crosses between C. cobaltinus females and C. auratus males. We discuss these findings in terms of understanding the cost of mating 'mistakes' in the Chrysochus hybrid zone. In addition, our discovery that 95% confidence intervals for commonly-used isolation statistics can be very wide has important implications for speciation research. Specifically, to avoid biases in the interpretation of such isolation metrics, we suggest that studies should routinely include error estimates in their analyses of reproductive isolation.     

Gene duplications circumvent trade‐offs in enzyme function: Insect adaptation to toxic host plants

Herbivorous insects and their adaptations against plant toxins provide striking opportunities to investigate the genetic basis of traits involved in coevolutionary interactions. Target site insensitivity to cardenolides has evolved convergently across six orders of insects, involving identical substitutions in the Na,K‐ATPase gene and repeated convergent gene duplications. The large milkweed bug, Oncopeltus fasciatus, has three copies of the Na,K‐ATPase α‐subunit gene that bear differing numbers of amino acid substitutions in the binding pocket for cardenolides. To analyze the effect of these substitutions on cardenolide resistance and to infer possible trade‐offs in gene function, we expressed the cardenolide‐sensitive Na,K‐ATPase of Drosophila melanogaster in vitro and introduced four distinct combinations of substitutions observed in the three gene copies of O. fasciatus. With an increasing number of substitutions, the sensitivity of the Na,K‐ATPase to a standard cardenolide decreased in a stepwise manner. At the same time, the enzyme's overall activity decreased significantly with increasing cardenolide resistance and only the least substituted mimic of the Na,K‐ATPase α1C copy maintained activity similar to the wild‐type enzyme. Our results suggest that the Na,K‐ATPase copies in O. fasciatus have diverged in function, enabling specific adaptations to dietary cardenolides while maintaining the functionality of this critical ion carrier.     

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation

Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation.     

Are the states that occlude rubidium obligatory intermediates of the Na(+)/K(+)-ATPase reaction?

In the Albers-Post model, occlusion of K(+) in the E(2) conformer of the enzyme (E) is an obligatory step of Na(+)/K(+)-ATPase reaction. If this were so the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na(+)/K(+)-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K(+) congener Rb(+). Assuming that always two Rb(+) are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb(+)]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na(+)/K(+)-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb(+)] <10 mM. This points to the presence of an extra ATP hydrolysis that is not Na(+)-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb(+) increases ATP hydrolysis without occlusion.     

Functional role of protein kinase B/Akt in gastric acid secretion

Epidermal growth factor (EGF) stimulates gastric acid secretion and H(+)/K(+)-ATPase alpha-subunit gene expression. Because EGF activates the serine-threonine protein kinase Akt, we explored the role of Akt in gastric acid secretion. Akt phosphorylation and activation were measured by kinase assays and by Western blots with an anti-phospho-Akt antibody, using lysates of purified (>95%) canine gastric parietal cells in primary culture. EGF induced Akt phosphorylation and activation, whereas carbachol had no effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely blocked EGF induction of Akt phosphorylation, whereas the MEK1 inhibitor PD98059 and the protein kinase C inhibitor GF109203X had no effect. We examined the role of Akt in H(+)/K(+)-ATPase gene expression by Northern blotting using a canine H(+)/K(+)-ATPase alpha-subunit cDNA probe. The parietal cells were transduced with a multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt, which overexpresses a constitutively active Akt gene, or with the control vector Ad.CMV-beta-gal, which expresses beta-galactosidase. Ad.Myr-Akt induced H(+)/K(+)-ATPase alpha-subunit gene expression 3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which was potently induced by carbachol in the same parietal cells. Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed to induce changes in either parietal cell actin content, measured by Western blots with an anti-actin antibody or in the organization of the actin cellular cytoskeleton, visualized by fluorescein phalloidin staining and confocal microscopy. Transduction of the parietal cells with a multiplicity of infection of 100 of the adenoviral vector Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the stimulatory effect of EGF on both aminopyrine uptake and H(+)/K(+)-ATPase production, measured by Western blots with an anti-H(+)/K(+)-ATPase alpha-subunit antibody. Thus, EGF induces a cascade of events in the parietal cells that results in the activation of Akt. The functional role of Akt appears to be stimulation of gastric acid secretion through induction of H(+)/K(+)-ATPase expression.     

Simultaneous phosphorylation of Ser11 and Ser18 in the alpha-subunit promotes the recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane

Renal sodium homeostasis is a major determinant of blood pressure and is regulated by several natriuretic and antinatriuretic hormones. These hormones, acting through intracellular second messengers, either activate or inhibit proximal tubule Na(+),K(+)-ATPase. We have shown previously that phorbol ester (PMA) stimulation of endogenous PKC leads to activation of Na(+),K(+)-ATPase in cultured proximal tubule cells (OK cells) expressing the rodent Na(+), K(+)-ATPase alpha-subunit. We have now demonstrated that the treatment with PMA leads to an increased amount of Na(+),K(+)-ATPase molecules in the plasmalemma, which is proportional to the increased enzyme activity. Colchicine, dinitrophenol, and potassium cyanide prevented the PMA-dependent stimulation of activity without affecting the increased level of phosphorylation of the Na(+), K(+)-ATPase alpha-subunit. This suggests that phosphorylation does not directly stimulate Na(+),K(+)-ATPase activity; instead, phosphorylation may be the triggering mechanism for recruitment of Na(+),K(+)-ATPase molecules to the plasma membrane. Transfected cells expressing either an S11A or S18A mutant had the same basal Na(+),K(+)-ATPase activity as cells expressing the wild-type rodent alpha-subunit, but PMA stimulation of Na(+),K(+)-ATPase activity was completely abolished in either mutant. PMA treatment led to phosphorylation of the alpha-subunit by stimulation of PKC-beta, and the extent of this phosphorylation was greatly reduced in the S11A and S18A mutants. These results indicate that both Ser11 and Ser18 of the alpha-subunit are essential for PMA stimulation of Na(+), K(+)-ATPase activity, and that these amino acids are phosphorylated during this process. The results presented here support the hypothesis that PMA regulation of Na(+),K(+)-ATPase is the result of an increased number of Na(+),K(+)-ATPase molecules in the plasma membrane.     

Ouabain assembles signaling cascades through the caveolar Na+/K+-ATPase

Based on the observation that the Na(+)/K(+)-ATPase alpha subunit contains two conserved caveolin-binding motifs, we hypothesized that clustering of the Na(+)/K(+)-ATPase and its partners in caveolae facilitates ouabain-activated signal transduction. Glutathione S-transferase pull-down assay showed that the Na(+)/K(+)-ATPase bound to the N terminus of caveolin-1. Significantly, ouabain regulated the interaction in a time- and dose-dependent manner and stimulated tyrosine phosphorylation of caveolin-1 in LLC-PK1 cells. When added to the isolated membrane fractions, ouabain increased tyrosine phosphorylation of proteins from the isolated caveolae but not other membrane fractions. Consistently, ouabain induced the formation of a Na(+)/K(+)-ATPase-Src-caveolin complex in the isolated caveolae preparations as it did in live cells. Finally, depletion of either cholesterol by methyl beta-cyclodextrin or caveolin-1 by siRNA significantly reduced the caveolar Na(+)/K(+)-ATPase and Src. Concomitantly, cholesterol depletion abolished ouabain-induced recruitment of Src to the Na(+)/K(+)-ATPase signaling complex. Like depletion of caveolin-1, it also blocked the effect of ouabain on ERKs, which was restored after cholesterol repletion. Clearly, the caveolar Na(+)/K(+)-ATPase represents the signaling pool of the pump that interacts with Src and transmits the ouabain signals.     

Comparative properties of caveolar and noncaveolar preparations of kidney Na+/K+-ATPase

To evaluate previously proposed functions of renal caveolar Na(+)/K(+)-ATPase, we modified the standard procedures for the preparation of the purified membrane-bound kidney enzyme, separated the caveolar and noncaveolar pools, and compared their properties. While the subunits of Na(+)/K(+)-ATPase (α,β,γ) constituted most of the protein content of the noncaveolar pool, the caveolar pool also contained caveolins and major caveolar proteins annexin-2 tetramer and E-cadherin. Ouabain-sensitive Na(+)/K(+)-ATPase activities of the two pools had similar properties and equal molar activities, indicating that the caveolar enzyme retains its ion transport function and does not contain nonpumping enzyme. As minor constituents, both caveolar and noncaveolar pools also contained Src, EGFR, PI3K, and several other proteins known to be involved in stimulous-induced signaling by Na(+)/K(+)-ATPase, indicating that signaling function is not limited to the caveolar pool. Endogenous Src was active in both pools but was not further activated by ouabain, calling into question direct interaction of Src with native Na(+)/K(+)-ATPase. Chemical cross-linking, co-immunoprecipitation, and immunodetection studies showed that in the caveolar pool, caveolin-1 oligomers, annexin-2 tetramers, and oligomers of the α,β,γ-protomers of Na(+)/K(+)-ATPase form a large multiprotein complex. In conjunction with known roles of E-cadherin and the β-subunit of Na(+)/K(+)-ATPase in cell adhesion and noted intercellular β,β-contacts within the structure of Na(+)/K(+)-ATPase, our findings suggest that interacting caveolar Na(+)/K(+)-ATPases located at renal adherens junctions maintain contact of two adjacent cells, conduct essential ion pumping, and are capable of locus-specific signaling in junctional cells.