Immunohistological localization of regulatory peptides in the midgut of the female mosquitoAedes aegypti

The midgut of the female mosquitoAedes aegypti was studied immunohistologically with antisera to various regulatory peptides. Endocrine cells immunoreactive with antisera to perisulfakinin, RFamide, bovine pancreatic polypeptide, urotensin 1, locustatachykinin 2 and allatostatins A1 and B2 were found in the midgut. Perisulfakinin, RFamide and bovine pancreatic polypeptide all react with the same, about 500 endocrine cells, which were evenly distributed throughout the posterior midgut, with the exception of its most frontal and caudal regions. In addition, these antisera recognized three to five neurons in each ingluvial ganglion and their axons, which ran longitudinally over the anterior midgut, as well as axons innervating the pyloric sphincter. The latter axons appear to be derived from neurons located in the abdominal ganglia. Antisera to two different allatostatins recognized about 70 endocrine cells in the most caudal area of the posterior midgut and axons in the anterior midgut whose cell bodies were probably located in either the brain or the frontal ganglion. Antiserum to locustatachykinin 2 recognized endocrine cells present in the anterior midgut and the most frontal part of the posterior midgut, as well as about 50 cells in the most caudal region of the posterior midgut. Urotensin 1 immunoreactivity was found in endocrine cells in the same region as the perisulfakinin-immunoreactive cells, but no urotensin-immunoreactive axons were found in the midgut. Double labeling experiments showed that the urotensin and perisulfakinin immunoreactivities were located in different cells. Such experiments also showed that the locustatachykinin and allatostatin immunoreactivities in the most caudal area of the posterior midgut were present in different cells. No immunoreactivity was found in the mosquito midgut when using antisera to corazonin, allatotropin or leucokinin IV. Since these peptides have either been isolated from, or can reasonably be expected to be present in mosquitoes, it was concluded that these peptides are not present in the mosquito midgut.     

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.     

A comparative review of short and long neuropeptide F signaling in invertebrates: Any similarities to vertebrate neuropeptide Y signaling?

Neuropeptides referred to as neuropeptide F (NPF) and short neuropeptide F (sNPF) have been identified in numerous invertebrate species. Sequence information has expanded tremendously due to recent genome sequencing and EST projects. Analysis of sequences of the peptides and prepropeptides strongly suggest that NPFs and sNPFs are not closely related. However, the NPFs are likely to be ancestrally related to the vertebrate family of neuropeptide Y (NPY) peptides. Peptide diversification may have been accomplished by different mechanisms in NPFs and sNPFs; in the former by gene duplications followed by diversification and in the sNPFs by internal duplications resulting in paracopies of peptides. We discuss the distribution and functions of NPFs and their receptors in several model invertebrates. Signaling with sNPF, however, has been investigated mainly in insects, especially in Drosophila. Both in invertebrates and in mammals NPF/NPY play roles in feeding, metabolism, reproduction and stress responses. Several other NPF functions have been studied in Drosophila that may be shared with mammals. In Drosophila sNPFs are widely distributed in numerous neurons of the CNS and some gut endocrines and their functions may be truly pleiotropic. Peptide distribution and experiments suggest roles of sNPF in feeding and growth, stress responses, modulation of locomotion and olfactory inputs, hormone release, as well as learning and memory. Available data indicate that NPF and sNPF signaling systems are distinct and not likely to play redundant roles.     

Processed short neuropeptide F peptides regulate growth through the ERK-insulin pathway in Drosophila melanogaster

The DrosophilasNPF gene regulates growth through the ERK-insulin pathway. sNPF encodes a precursor protein that is processed and produces biologically active sNPF peptides. However, the functions of these peptides are not known. In Drosophila neuronal cells in culture and in flies in vivo, sNPF1 and sNPF2 activated the ERK-insulin pathway and regulated body growth. In addition, the sNPF precursor and the processed sNPF peptide were co-localized in the neurons of the central nervous system. These results indicate that sNPF1 and sNPF2 peptides processed from the sNPF precursor are sufficient for regulating body growth through the ERK-insulin pathway in Drosophila.     

Metabolic Stress Responses in Drosophila Are Modulated by Brain Neurosecretory Cells That Produce Multiple Neuropeptides

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. We show here that in adult flies the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted us to initiate analysis of the function of the ten neurosecretory cells. We investigated the role of peptide signaling from large ipc-1 and 2a cells in stress responses by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system we targeted the peptide knockdown specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. Our data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells.     

Ontogeny of endocrine cells in the gut of the insect Periplaneta americana

Summary The ontogeny of the endocrine cells of the gut of the cockroach Periplaneta americana was studied by immunohistochemistry. During embryogenesis, the midgut begins to be formed as an outgrowth of the foregut and hindgut invaginations. Gut endocrine cells with pancreatic polypeptide (PP)-like immunoreactivity begin to appear at the anterior and posterior ends of the forming midgut. These cells are restricted to the midgut epithelium, and no mitotic cells with PP-like immunoreactivity are observed. These results strongly suggest that the gut endocrine cells, at least those with PP-like immunoreactivity, are derived from precursor cells they have in common with other epithelial cells of the midgut.     

Lineage analysis of transplanted individual cells in embryos of Drosophila melanogaster

Summary In this paper experiments concerning some aspects of the development of pole cells and midgut progenitors in Drosophila are reported. Cells were labelled by injecting horseradish-peroxidase (HRP) in embryos before pole bud formation and transplanted at different stages into unlabelled embryos, where the transplanted cells developed together with the unlabelled cells of the host. The hosts were then fixed and stained at different ages in order to demonstrate the presence of HRP in the progenies of transplanted cells. The main conlusions of the study are as follows. The gonads are the only organ to the formation of which pole cells normally contribute; those pole cells which do not participate in the formation of the gonads are finally eliminated or degenerate. Since the number of primordial germ cells in the gonads is the same irrespective of the number of pole cells present in the embryo, an (unknown) mechanism must exist regulating the final number of pole cells in each of the gonads. After their formation and before reaching the gonads, pole cells have been found to divide only up to two times. With respect to the midgut progenitors, the cells of both anlagen have been found to be committed to develop into midgut, although they behave as equivalent in that they do not apparently distinguish between the anterior and posterior anlage. Midgut progenitors have been found to divide a maximum of three times and to produce two different types of cells, epithelial cells of the midgut wall and spindle-like cells located internally in the gut.     

Light and electron microscopy studies of the midgut and salivary glands of second and third instars of the horse stomach bot,Gasterophilus intestinalis

A morphological study of the midgut and salivary glands of second and third instars of Gasterophilus intestinalis (De Geer) (Diptera: Oestridae) was conducted by light, scanning and transmission electron microscopy. The midgut is anteriorly delimited by a proventriculus, without caeca, and is composed of posterior foregut and anterior midgut tissue from which a double‐layered peritrophic matrix is produced. The midgut can be divided into anterior, median and posterior regions on the basis of the structural and physiological variations of the columnar cells which occur along its length. Two other types of cell were identified: regenerative cells scattered throughout the columnar cells, and, more rarely, endocrine cells of two structural types (closed and open). Different secretion mechanisms (merocrine, apocrine and microapocrine) occur along the midgut epithelium. Abundant microorganisms are observed in the endoperitrophic space of the anterior midgut. The origin and nature of these microorganisms remain unknown. No structural differences are observed between the second and third instar midguts. The salivary glands of G. intestinalis second and third instars consist of a pair of elongated tubular structures connected to efferent ducts which unite to form a single deferent duct linked dorsally to the pharynx. Several intermediate cells, without cuticle, make the junction with the salivary gland epithelium layer. Cytological characteristics of the gland epithelial cells demonstrate high cellular activity and some structural variations are noticed between the two larval stages.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Distribution and functional significance of Leu-callatostatins in the blowfly Calliphora vomitoria

The Leu-callatostatins are a series of four neuropeptides isolated from nervous tissues of the blowfly Calliphora vomitoria that show C-terminal sequence homology to the allatostatins of cockroaches. The allatostatins have an important role in the reproductive processes of insects as inhibitors of the synthesis and release of juvenile hormone from the corpus allatum. In this study, the distribution of the Leu-callatostatin-immunoreactive neurones and endocrine cells has been mapped in C. vomitoria and, in contrast to the cockroach allatostatins, it has been shown that there is no cytological basis to suggest that the dipteran peptides act as regulators of juvenile hormone. Although occurring in various neurones in the brain and thoracico-abdominal ganglion, there is no evidence of Leu-callatostatin-immunoreactive pathways linking the brain to the corpus allatum, or of immunoreactive terminals in this gland. Three different types of functions for the Leu-callatostatins are suggested by the occurrence of immunoreactive material in cells and by the pathways that have been identified. (1) A role in neurotransmission or neuromodulation appears evident from immunoreactive neurones in the medulla of the optic lobes, and from immunoreactive material in the central body and in descending interneurones in the suboesophageal ganglion that project to the neuropile of the thoracico-abdominal ganglion. (2) Leu-callatostatin neurones directly innervate muscles of the hindgut and the heart. Immunoreactive fibres from neurones of the abdominal ganglion pass by way of the median abdominal nerve to ramify extensively over several areas of the hindgut. Physiological experiments with synthetic peptides show that the Leu-callatostatins are potent inhibitors of peristaltic movements of the ileum. Leu-callatostatin 3 is active at 10^-16 to 10^-13 M. This form or regulatory control over gut motility appears to be highly specific since the patterns of contraction in other regions are unaffected by these peptides. (3) Evidence that the Leu-callatostatins act as neurohormones comes from the presence of varicosities in axons passing through the corpus cardiacum (but not the corpus allatum) and also from material in extraganglionic neurosecretory cells in the thorax. Fibres from these peripheral neurones are especially prominent over the large nerve bundles supplying the legs. There are also a considerable number of Leu-callatostatin-immunoreactive endocrine cells in a specific region of the midgut. The conclusion from this study is that although conservation of the structure of the allatostatin-type of peptides is evident through a long period of evolution it cannot be assumed that all of their functions have also been conserved. Several different types of functions for the Leu-callatostatins of the blowfly are proposed in this study, but there is no evidence to suggest a role in the regulation of juvenile hormone synthesis and release.     

Distribution of short neuropeptide F and its receptor in neuronal circuits related to feeding in larval Drosophila

Four forms of short neuropeptide F (sNPF1–4), derived from the gene snpf, have been identified in Drosophila and are known to act on a single G-protein-coupled receptor (sNPFR). Several functions have been suggested for sNPFs in Drosophila, including the regulation of feeding and growth in larvae, the control of insulin signalling and the modulation of neuronal circuits in adult flies. Furthermore, sNPF has been shown to act as a nutritional state-dependent neuromodulator in the olfactory system. The role of sNPF in the larval nervous system is less well known. To analyse sites of action of sNPF in the larva, we mapped the distribution of sNPF- and sNPFR-expressing neurons. In particular, we studied circuits associated with chemosensory inputs and systems involved in the regulation of feeding, including neurosecretory cell systems and the hypocerebral ganglion. We employed a combination of immunocytochemistry and enhancer trap and promoter Gal4 lines to drive green fluorescent protein. We found a good match between the distribution of the receptor and its ligand. However, several differences between the larval and adult systems were observed. Thus, neither sNPF nor its receptor was found in the olfactory (or other sensory) systems in the larva and cells producing insulin-like peptides did not co-express sNPFR, as opposed to results from adults. Moreover, sNPF was expressed in a subpopulation of Hugin cells (second-order gustatory neurons) only in adult flies. We propose that the differences in sNPF signalling between the developmental stages is explained by differences in their feeding behaviour.     

Neuropeptide F and the corn earworm, Helicoverpa zea: A midgut peptide revisited

The neuropeptide Y family of peptides is implicated in the regulation of feeding across a broad range of animals, including insects. Among vertebrates, neuropeptide Y exerts its actions mainly centrally, whereas peptide YY and pancreatic polypeptide arise from digestive tissues. Among invertebrates, neuropeptide F (NPF) is the sole counterpart of the NPY family. Shared features of NPF sequences derived for Lepidoptera indicate that the midgut peptide (Hez-MP-I) of the corn earworm, Helicoverpa zea, characterized more than a decade ago, is a carboxyl fragment of a full-length NPF. An antibody to Hez-MP-I was used to characterize the peptide's distribution in tissues of larvae, pupae, and adults. Immunostaining demonstrated NPF-related material both in nervous tissues and in abundant endocrine cells of the midgut. Radioimmunoassay of Hez-MP-I in the head, midgut and hemolymph of fifth instar larvae revealed concentration changes corresponding to development and feeding state. As with the vertebrate homologs, NPF may arise both centrally and peripherally to modulate the physiology of feeding and digestion of Lepidoptera.     

Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by electron-microscopic gold-labelling immunocytochemistry

An immunogold-labelling electron-microscopic study of the frontal ganglion of two noctuids, Lacanobia oleracea and Helicoverpa armigera, has been carried out with antisera directed against three neuropeptides; allatostatins of the Y/FXFGL-NH₂ type, Manduca sexta allatostatin (Mas-AS) and M. sexta allatotropin. The ganglion of both noctuids has two pairs of large peptidergic neurones with many clusters of electron-dense granules, one pair being situated anteriorly and the other posteriorly. By means of a double-labelling (flip-flop) technique, with different sizes of gold particles, all possible paired combinations of the three different types of peptide have been visualised within granules of the anterior neurones, leading to the conclusion that the three peptides are co-packaged and co-stored in these cells. Within the posterior neurones of L. oleracea, gold labelling of granules is only linked to the Y/FXFGL-NH₂ allatostatin antisera and, in contrast to the anterior cells of this species in which double gold labelling results in a sparse accumulation of gold particles for any one peptide type, single labelling gives a more intense, uniform pattern of gold particles. In contrast to L. oleracea, the gold-labelling pattern seen in the posterior neurones of H. armigera reflects the co-localisation of allatostatins of the Y/FXFGL-NH₂ type with Mas-AS in this species. Allatotropin is absent in the posterior neurones of both species.Grant funding was from the Wellcome Trust: grant no. 068105 (A.T.)     

Cytoplasmic factors determining anteroposterior polarity in Drosophila embryos

Summary Cytoplasm removal/transplant techniques applied to Drosophila cleavage-stage embryos induced changes in anteroposterior polarity. Removal of anterior cytoplasm or anterior transplantation of posterior cytoplasm caused the anterior formation of posterior (telson) structures, and the replacement of anterior cytoplasm with posterior cytoplasm induced double-abdomen embryos, as reported by Frohnhöfer et al. [J Embryol Exp Morphol 97 (suppl):169–179 (1986)]. Changing the conditions of anterior cytoplasm removal we showed that greater volumes, earlier stages, and removal from the periphery were efficient. In addition we found that double-cephalon embryos are induced by replacing posterior cytoplasm with anterior cytoplasm, while removal of posterior cytoplasm or the posterior transplantation of anterior cytoplasm was without effect. However, introduction of anterior cytoplasm into the posterior of nanos embryos, which are mutants not developing abdominal segments, caused the formation of double-cephalon embryos. Similarly, double-abdomen embryos are produced by introducing posterior cytoplasm into the anterior of bicoid embryos, which are mutants not forming cephalic and thoracic structures. These results are compatible with the initial involvement of separate anterior, posterior and terminal cytoplasmic factors deduced from mutant analysis (Nüsslein-Volhard and Roth 1989).     

Widely distributed Drosophila G‐protein‐coupled receptor (CG7887) is activated by endogenous tachykinin‐related peptides

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G‐protein‐coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. We determined that HEK‐293 cells transfected with DTKR displayed dose‐dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR‐green fluorescent protein (GFP) fusion constructs in HEK‐293 cells. We generated specific antireceptor antisera and showed that DTKR is widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. Our findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand‐receptor system plays multiple functional roles. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006     

Peptidomics of the Agriculturally Damaging Larval Stage of the Cabbage Root Fly Delia radicum (Diptera: Anthomyiidae)

The larvae of the cabbage root fly induce serious damage to cultivated crops of the family Brassicaceae. We here report the biochemical characterisation of neuropeptides from the central nervous system and neurohemal organs, as well as regulatory peptides from enteroendocrine midgut cells of the cabbage maggot. By LC-MALDI-TOF/TOF and chemical labelling with 4-sulfophenyl isothiocyanate, 38 peptides could be identified, representing major insect peptide families: allatostatin A, allatostatin C, FMRFamide-like peptides, kinin, CAPA peptides, pyrokinins, sNPF, myosuppressin, corazonin, SIFamide, sulfakinins, tachykinins, NPLP1-peptides, adipokinetic hormone and CCHamide 1. We also report a new peptide (Yamide) which appears to be homolog to an amidated eclosion hormone-associated peptide in several Drosophila species. Immunocytochemical characterisation of the distribution of several classes of peptide-immunoreactive neurons and enteroendocrine cells shows a very similar but not identical peptide distribution to Drosophila. Since peptides regulate many vital physiological and behavioural processes such as moulting or feeding, our data may initiate the pharmacological testing and development of new specific peptide-based protection methods against the cabbage root fly and its larva.     

Distribution of peptidyl-glycine alpha-amidating monooxygenase immunoreactivity in the brain,pituitary and islet organ of the anglerfish (Lophius americanus)

Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is an enzyme that catalyzes conversion of glycine-extended peptides to alpha-amidated bioactive peptides. Two peptides that are processed at their carboxyl-termini by this enzyme are neuropeptide Y and anglerfish peptide Y, both of which possess a C-terminal glycine that is used as a substrate for amidation. Results from previous reports have demonstrated that neuropeptide Y-like and anglerfish peptide Y-like immunoreactivities are present in the brain of anglerfish (Lophius americanus). Furthermore, neuropeptide Y-like peptides, namely anglerfish peptide Y and anglerfish peptide YG (the homologues of pancreatic polypeptide) are present in the islet organ of this species. Neuropeptide Y has also been localized in the anterior, intermediated and posterior lobes of the pituitary gland in a variety of species. In order to learn more about the distribution of the enzyme responsible for alpha amidation of these peptides in the brain and pituitary and to specifically investigate the relationship of this enzyme to peptide synthesizing endocrine cells of the anglerfish islet, we performed an immunohistochemical study using several antisera generated against different peptide sequences of the enzyme. PAM antisera labeled cells in the islet organ, pituitary and brain, and fibers in the brain and pituitary gland. The PAM staining pattern in the brain was remarkably similar to the distribution of neuropeptide Y immunoreactivity reported previously. Clusters of cells adjacent to vessels in the anterior pituitary displayed punctate PAM immunoreactivity while varicose fibers were observed in the pituitary stalk and neurohypophysis. Endocrine cells of the islet organ were differentially labeled with different PAM antisera. Comparison of the staining patterns of insulin, glucagon, and anglerfish peptide Y in the islet organ to PAM immunoreactivity suggests a distribution of forms of PAM enzyme in insulin and anglerfish peptide Y-containing cells, but no overlap with glucagon-producing cells. The results also indicate that PAM immunoreactivity is widely distributed in the brain, pituitary and islet organ of anglerfish in cells that contain peptides that require presence of a C-terminal glycine for amidation.     

The morphology and fine structure of the larval midgut of a moth (Manduca sexta) in relation to active ion transport.

Light and electron microscopic examination of the midgut of Manduca sexta has shown that the organization of this tissue is more complex than was originally believed. The midgut can be divided into anterior, middle and posterior regions on the basis of the pattern of folding of the epithelial sheet, and variations in the structure of goblet and columnar cells which occur along its length. The columnar cells show gradual structural changes form the anterior to the posterior end of the midgut. For example, the microvilli in the anterior region form a dense, interconnecting network from which vesicles break off. This organization becomes less obvious through the middle region, until by the posterior region each microvillus is unconnected to adjacent microvilli along its entire length and vesicles are no longer produced. Two distinct types of goblet cells are found. In the anterior and middle regions the goblet cells have a large basally located cavity, but in the posterior region the cavity occupies only the apical half of the cell. In both cases the cavity is formed by invagination of the apical membrane, which is studded with small particles implicated in active ion transport. In the anterior and middle regions this membrane is closely associated with mitochondria, but not in the posterior region. The significance of the observed structural differences is discussed in relation to active ion transport.