Ion-transporting Epithelium Research Articles

Stanniocalcin-1 controls ion regulation functions of ion-transporting epithelium other than calcium balance.

Stanniocalcin-1 (STC-1) was first identified to involve in Ca2+ homeostasis in teleosts, and was thought to act as a hypocalcemic hormone in vertebrate. Recent studies suggested that STC-1 exhibits broad effects on ion balance, not confines to Ca2+, but the mechanism of this regulation process remains largely unknown. Here, we used zebrafish embryos as an alternative in vivo model to investigate how STC-1 regulates transepithelial ion transport function in ion-transporting epithelium. Expression of stc-1 mRNA in zebrafish embryos was increased in high-Ca2+ environments but decreased by acidic and ion-deficient treatments while overexpression of stc-1 impaired the hypotonic acclimation by decreasing whole body Ca2+, Na+, and Cl- contents and H+ secretion ability. Injection of STC-1 mRNA also down-regulated mRNA expressions of epithelial Ca2+ channel, H+-ATPase, and Na+-Cl- cotransporter, suggesting the roles of STC-1 in regulation of ions other than Ca2+. Knockdown of STC-1 caused an increase in ionocyte progenitors (foxi3a as the marker) and mature ionocytes (ion transporters as the markers), but did not affect epithelium stem cells (p63 as the marker) in the embryonic skin. Overexpression of STC-1 had the corresponding opposite effect on ionocyte progenitors, mature ionocytes in the embryonic skin. Taken together, STC-1 negatively regulates the number of ionocytes to reduce ionocyte functions. This process is important for body fluid ionic homeostasis, which is achieved by the regulation of ion transport functions in ionocytes. The present findings provide new insights into the broader functions of STC-1, a hypocalcemic hormone.

Evolution of salinity tolerance from transcriptome to physiological system

The relationship between microevolution and macroevolution is a topic of fundamental importance in evolutionary biology. The increasing accessibility of genomic tools is making the hunt for genes that underlie evolutionary divergence more tractable and, when combined with physiological approaches, provides exceptional power to elucidate the causal mechanisms of the relationship. In this issue of Molecular Ecology, Whitehead et al. (2013) employ this strategy to show that common physiological and genomic mechanisms lead to divergence in salinity tolerance across micro- and macroevolutionary timescales. They compare two killifish species from the genus Fundulus, F. majalis, which inhabits primarily marine and brackish environments and represents the ancestral state of the genus, and F. heteroclitus, which has derived an osmotic niche that expands into freshwater. Corresponding to the differences in osmotic niche, the species differ strikingly in how the structure of the ion-transporting epithelium and the transcriptome of the gills respond to osmotic challenge. These inter-specific differences were similar to but more pronounced than the differences associated with the more subtle intra-specific variation in osmotic niche within each species. It appears that a progression of the same functional adjustments first allowed expansion of the osmotic niche of F. heteroclitus into freshwater and then further expanded the niche of select F. heteroclitus populations towards more dilute freshwater environments. The work of Whitehead et al. therefore emphasizes how the mechanisms of adaptive divergence between populations can be expanded over time to produce the more complex differences that can exist between species.

Identification of zebrafish FXYD11a protein that is highly expressed in ion-transporting epithelium of the gill and skin and its possible role in ion homeostasis

FXYD proteins, small single-transmembrane proteins, have been proposed to be auxiliary regulatory subunits of Na+–K+-ATPase and have recently been implied in ion osmoregulation of teleost fish. In freshwater (FW) fish, numerous ions are actively taken up through mitochondrion-rich cells (MRCs) of the gill and skin epithelia, using the Na+ electrochemical gradient generated by Na+–K+-ATPase. In the present study, to understand the molecular mechanism for the regulation of Na+–K+-ATPase in MRCs of FW fish, we sought to identify FXYD proteins expressed in MRCs of zebrafish. Reverse-transcriptase PCR studies of adult zebrafish tissues revealed that, out of eight fxyd genes found in zebrafish database, only zebrafish fxyd11 (zfxyd11) mRNA exhibited a gill-specific expression. Double immunofluorescence staining showed that zFxyd11 is abundantly expressed in MRCs rich in Na+–K+-ATPase (NaK-MRCs) but not in those rich in vacuolar-type H+-transporting ATPase. An in situ proximity ligation assay demonstrated its close association with Na+–K+-ATPase in NaK-MRCs. The zfxyd11 mRNA expression was detectable at 1 day postfertilization, and its expression levels in the whole larvae and adult gills were regulated in response to changes in environmental ionic concentrations. Furthermore, knockdown of zFxyd11 resulted in a significant increase in the number of Na+–K+-ATPase–positive cells in the larval skin. These results suggest that zFxyd11 may regulate the transport ability of NaK-MRCs by modulating Na+–K+-ATPase activity, and may be involved in the regulation of body fluid and electrolyte homeostasis.

Left/right, up/down: the role of endogenous electrical fields as directional signals in development, repair and invasion.

A fundamental aspect of biological systems is their spatial organization. In development, regeneration and repair, directional signals are necessary for the proper placement of the components of the organism. Likewise, pathogens that invade other organisms rely on directional signals to target vulnerable areas. It is widely understood that chemical gradients are important directional signals in living systems. Less well recognized are electrical fields, which can also provide directional information. Small, steady electrical fields can directly guide cell movement and growth and can generate chemical gradients of charged macromolecules against the leveling action of diffusion. At the site of a lesion in an ion-transporting epithelium, for example, a substantial electrical field is instantly generated and may extend over many cell diameters. There are numerous other situations in which relatively long-range electrical fields have been shown to exist naturally. Recently, there has been substantial progress in identifying specific processes that are controlled, to some extent, by these endogenous electrical fields. This review highlights these recent data and discusses possible mechanisms by which the fields might affect biological processes.

Confocal Laser Scanning and Electron Microscopical Studies on Osmoregulatory Epithelia in the Branchial Cavity of the Lobster Homarus Gammarus

ABSTRACT The adult lobster Homarus gammarus is a weak hyper-regulator at low salinity. The objective of this study was to locate the ion-transporting tissues in the branchial chamber of this species, using electron microscopy and confocal laser scanning microscopy with a fluorescent vital stain for mitochondria, DASPMI, which is widely used to locate mitochondria-rich cells in ion-transporting epithelia of fish. A thick mitochondria-rich epithelium is present on the inner side of the branchiostegite and over the entire surface of the epipodites. Ultrastructural observations confirm that this tissue has features typical of an ion-transporting epithelium. When the lobster is transferred to low salinity, these epithelia undergo marked ultrastructural changes, such as an increase in thickness related to the development of basolateral infoldings, the appearance of numerous vesicles and an increase in height of the apical microvilli. In the gills, the branchial filaments are lined by a thin and poorly differentiated epithelium, containing numerous mitochondria; no significant ultrastructural changes were observed in the gills of animals acclimated to low salinity. In summary, in H. gammarus, no evidence of osmoregulatory structures was found in the gills. Differentiated ion-transporting epithelia are present in the branchial cavity, on the inner side of the branchiostegite and on the epipodites; these organs are probably involved in osmoregulation.

Activation by Membrane Stretch and Depolarization of an Epithelial Monovalent Cation Channel From Teleost Intestine

ABSTRACT The intestine of euryhaline teleosts is an important osmoregulatory organ which actively absorbs Na+, Cl− and water from the lumen. This ion-transporting epithelium experiences a variety of physical stimuli resulting from variations in luminal osmolality and distension and from peristaltic contractions. Using patchclamp techniques in the inside-out configuration, single stretch-activated channels (SA channels) were identified and characterized. These SA channels had a conductance of about 67 pS in symmetrical solutions containing 140 mmol l−1 NaCl and were permeable to both Na+ and K+ (PNa/PK≈0.83) but not to anions. In excised, inside-out membrane patches, channel activity could be enhanced in the absence of membrane tension by strong depolarization of the membrane potential (Vm) to between 0 mV and +90 mV, with Vo [Vm at which the single-channel open probability (Po)=0.5] at +25.7mV. In the presence of membrane tension, the voltage-dependence of channel activity was shifted into the physiological range of Vm. Each kPa (10cmH2O) of applied pressure (ΔP) generated the same effect on Po as a membrane depolarization of 49 mV. Membrane tension also increased the single-channel current and single-channel conductance in a dose-dependent manner. The kinetic data suggest that this channel has two open states and three closed states. Both stretch- and depolarization-induced increases in Po were attributed to prolongation of the lifetime of the longer open state. Possible physiological roles for this channel include the cellular uptake of Na+ from the lumen as part of the salt and water absorptive process or a yet undefined involvement in cell volume regulation.

Energy consumption in the perfused gills of the euryhaline crab Eriocheir sinensis [H. Miln. Edw. ] adapted to freshwater

AbstractThe euryhaline crab Eriocheir sinensis adapted to freshwater shows an ion transporting‐type epithelium in the three posterior gills and a respiratory‐type epithelium in the three anterior gills. When they are perfused in freshwater conditions, there is an increased transport of ions principally in the posterior gills.We also measure an increased degradation of the branchial glycogen. This polysaccharide is present in an extraepithelial lamina located in the hemal space of the lamellae. Its localization outside the ion‐transporting epithelium and its higher consumption by the posterior gills submitted to hypo‐osmotic conditions suggest a well‐suited glucose transport mechanism through their epithelial membrane. Accordingly, the uptake of glucose by the epithelial cells is slightly higher in the posterior gills as compared with the anterior.

Ultrastructural and electrophysiological maturation of the chick tegmentum vasculosum

We have examined the ultrastructural and electrophysiological events associated with the embryonic development of the tegmentum vasculosum, the ion-transporting epithelium in the chick cochlear duct. The cytodifferentiation of the light and dark cells in the epithelium from embryonic day 6 through post-hatching day 7 was studied with transmission electron microscopy. The predominant ultrastructural change in the developing tegmentum vasculosum was the elaboration of the complex basolateral infoldings on the dark cells from embryonic day 11 through post-hatching day 7. The relationship between the development of the endocochlear transepithelial electrical potential difference (PD) and the cytodifferentiation of the tegmentum vasculosum was examined with in vitro studies. Microelectrode impalements of the scala media showed that the positive endocochlear PD was first detectable on embryonic day 20 but did not reach a mature value of +16 mV until post-hatching day 7. Maturation of the endocochlear PD paralleled the time during which the dark cells in the tegmentum vasculosum displayed the most extensive increase in basolateral infoldings. This correlation suggests that the development of the endocochlear PD may result from an increase in the number of Na +-K + pump sites located on the basolateral infoldings of the dark cells.

Isolation and functional characterization of crustacean larval salt gland.

A batch method for isolating viable salt glands from the naupliar brine shrimp (Artemia salina) has been developed. This protocol produces a final preparation consisting of approximately 185 isolated salt glands, representing 1 X 10(4) secretory cells/g wet wt nauplii, with a final purity of 88%. Assays of cell integrity and function indicate good retention of in situ characteristics. Vital dye was excluded by 95% of the cells for at least 24 h. The O2 consumption rate was 22.7 nM O2 X min-1 X mg protein-1 and could be altered predictably by compounds known to affect oxidative phosphorylation and ion transport. The specific activity of the Na+-K+-ATPase in the salt gland, measured here for the first time, was 9.1 mM Pi X h-1 X mg protein-1. This is a substantial proportion of the body total, 17%, as expected for an active ion-transporting epithelium.

A freeze-fracture study of the papillary layer of the rat incisor enamel organ

After tooth enamel has been secreted it undergoes maturation or hardening. This process is mediated by ruffled and smooth-ended ameloblasts and associated papillary layer cells. The cells of the papillary layer are characterized by large numbers of mitochondria, coated vesicles, microvilli, and gap junctions. These features have led numerous investigators to speculate that the papillary layer is an ion-transporting epithelium. We have conducted freeze-fracture studies of the rat papillary layer in order to better characterize the surface features of these cells. The cell membranes of the papillary cells contained large numbers of intramembrane particles of various sizes ranging from 4 to 9 nm in diameter. Gap junctions were present at the cell surface and in the cytoplasm in the form of annular gap junctions. The intramembrane particles or connexons of both types of gap junctions were about 8–9 nm wide and were either packed randomly or present in the so-called ‘crystallized’ state. At the interface between smooth-ended ameloblasts and papillary layer cells, a well-developed zonula occludens was present along the basal surfaces of the ameloblasts and several large gap junctions were formed between the two cell types. The capillary network associated with the papillary layer was characterized by a thin endothelium containing large numbers of fenestrations.

Effect of the polymeric cryoprotectant dextran on fluid secretion in the isolated rabbit pancreas.

Physiological effects of the polymeric cryoprotectant dextran on an ion-transporting epithelium were investigated. In the isolated rabbit pancreas, dextran caused inhibition of fluid secretion and an increase of the concentrations of Na+, K+ and Cl-in the secreted fluid. Dextran did not affect the basal or pancreozymin-stimulated enzyme secretion. These effects of dextran can partially be explained by the fact that it is osmotically active and does not permeate through the epithelium. The effect of dextran on water transport can be compensated by lowering the ion concentrations in the solvent of the cryoprotectant. It is concluded that in cryoprotected ion-transporting epithelia the absolute ion concentration values obtained by X-ray microanalysis of frozen-hydrated specimens may not be completely correct, but that valid conclusions about intracellular ion distribution may still be drawn.

Differential effects of trypsin on the epidermis of Rana catesbeiana

The filamentous cytoskeletons of epidermal cells of the bullfrog (Rana catesbeiana) were investigated by electron microscopy. Following treatment with trypsin, sheets of epithelium were removed from swatches of abdominal skin. Trypsinization produces differential effects on the ultrastructure of the various cell layers. The desmosomes of all layers, except those of the stratum corneum, are split by trypsinization and the resulting desmosomal plaques fastened to tonofilaments are retracted into cells to form deep "inpouchings" of the plasma membranes, while tonofilament bundles become diffuse. Epidermal sheets were gently homogenized to form a suspension of cell remnants with damaged plasma membranes as indicated by vital dye exclusion tests and electron microscopy. Cytoskeletons retain their shapes, yet the lateral distances between individual tonofilaments within bundles appear to increase, thus forming diffuse lacelike structures. These observations support the suggestion that tonofilament bundles, when fastened to desmosomes, have elastic properties. The possible role of the cytoskeletons in the maintenance of cell size and shape in an ion-transporting epithelium is discussed.