V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 immunoreactivity in the gill epithelium of the Pacific hagfish (Epatretus stoutii)

We report the presence of the ion transporting proteins V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in the gill epithelium of the Pacific hagfish Epatretus stoutii. Heterologous antibodies recognized single bands of the appropriate sizes for the three transporters in western blots. Immunohistochemical staining revealed that the distribution of labeled cells in the gill epithelium was identical for the three proteins. Immunopositive cells were most abundant in the primary filament from the afferent side of the gill pouch, and their number diminished towards the lamella. Na(+)/K(+)-ATPase-like immunoreactivity (L-IR) occurred throughout the cell cytoplasm, probably associated to the basolateral tubular system. V-H(+)-ATPase L-IR was similar to Na(+)/K(+)-ATPase, although some cells had slightly heavier staining in either the supra- or infra-nuclear region. NHE2 L-IR was also generally cytoplasmic, but a minority of the cells had stronger immunoreactivity in the apical region. In general, all three ion transporting proteins were localized in the same cells, as estimated from 4-microm immunostained consecutive sections. We hypothesize that these putative ion-transporting cells are involved in systemic acid/base regulation and discuss other possible roles. This is the first report of V-H(+)-ATPase in myxinoids, and the first NHE2 report in the Pacific hagfish.     

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.     

Immunolocalization of Na+,K+-ATPase in the organs of the branchial cavity of the European lobster Homarus gammarus (Crustacea, Decapoda)

The localization of Na+,K(+)-ATPase in epithelia of the organs of the branchial cavity of Homarus gammarus exposed to seawater and dilute seawater was examined by immunofluorescence microscopy and immunogold electron microscopy with a monoclonal antibody IgG alpha 5 raised against the avian alpha-subunit of the Na-,K(+)-ATPase. In juveniles held in seawater, fluorescent staining was observed only in the epithelial cells of epipodites. In juveniles held in dilute seawater, heavier immunoreactivity was observed in the epithelial cells of epipodites, and positive immunostaining was also observed along the inner-side epithelial layer of the branchiostegites. No fluorescent staining was observed in the gill epithelia. At the ultrastructural level, the Na+,K(+)-ATPase was localized in the basolateral infolding systems of the epipodite and inner-side branchiostegite epithelia of juveniles held in dilute seawater, mostly along the basal lamina. The expression of Na+,K(+)-ATPase therefore differs within tissues of the branchial cavity and according to the external salinity. These and previous ultrastructural observations suggest that the epipodites, and to a lesser extent the inner-side epithelium of the branchiostegites, are involved in the slight hyper-regulation displayed by lobsters at low salinity. Enhanced Na+,K(+)-ATPase activity and de novo synthesis of Na+,K(+)-ATPase within the epipodite and branchiostegite epithelia may be key points enabling lobsters to adapt to low salinity environments.     

The putative mechanism of Na(+) absorption in euryhaline elasmobranchs exists in the gills of a stenohaline marine elasmobranch, Squalus acanthias

We recently cloned an NHE3 orthologue from the gills of the euryhaline Atlantic stingray (Dasyatis sabina), and generated a stingray NHE3 antibody to unequivocally localize the exchanger to the apical side of epithelial cells that are rich with Na(+)/K(+)-ATPase (A MRC). We also demonstrated an increase in NHE3 expression when stingrays are in fresh water, suggesting that NHE3 is responsible for active Na(+) absorption. However, the vast majority of elasmobranchs are only found in marine environments. In the current study, immunohistochemistry with the stingray NHE3 antibody was used to localize the exchanger in the gills of the stenohaline marine spiny dogfish shark (Squalus acanthias). NHE3 immunoreactivity was confined to the apical side of cells with basolateral Na(+)/K(+)-ATPase and was excluded from cells with high levels of vacuolar H(+)-ATPase. Western blots detected a single protein of 88 kDa in dogfish gills, the same size as NHE3 in stingrays and mammals. These immunological data demonstrate that the putative cell type responsible for active Na(+) absorption in euryhaline elasmobranchs is also present in stenohaline marine elasmobranchs, and suggest that the inability of most elasmobranchs to survive in fresh water is not due to a lack of the gill ion transporters for Na(+) absorption.     

Distribution of sodium-potassium ATPase in the rat testis and epididymis.

Sodium-potassium ATPase (Na+K(+)-ATPase) is a ubiquitous plasma membrane enzyme which uses the hydrolysis of ATP to regulate cellular Na+ and K+ levels and fluid volume. This ion pumping action is also thought to be involved in fluid movement across certain epithelia. There are several different genes for this enzyme, some of which are tissue specific. Using an antibody specific for the catalytic subunit of canine kidney Na+K(+)-ATPase, we have localized immunoreactivity in the seminiferous and epididymal epithelium of rats of various ages. There was no specific staining of 10-day-old rat testis. Faint staining was detected at 13 days and appeared to be associated with the borders of Sertoli cells. At 16 days prominent apical and lateral staining but no basal staining of Sertoli cell membranes was observed. This type of distribution continued until spermatids were present in the epithelium. In the adult rat testis, specific staining was detected in Sertoli cell crypts associated with elongating spermatids, and on the apical and lateral Sertoli cell membrane. In some instances immunoreactivity was concentrated at presumed sites of junctional specializations. In the excurrent ducts of immature and mature rats, Na+K(+)-ATPase staining was heavy in the efferent ducts and somewhat lighter in the epididymis. In all regions, the staining was basolateral although there were variations in intensity among the different parts of the epididymis. These results show 1) that rat testis and epididymal Na+K(+)-ATPase share some immunological determinants with the canine enzyme; 2) that the epididymal enzyme is located in the conventional basolateral position; and 3) that the distribution of Sertoli cell Na+K(+)-ATPase is probably apical and lateral rather than basal.     

Immunocytochemical localization of Na+,K(+)-ATPase in rat retinal pigment epithelial cells

We investigated quantitatively the ultrastructural localization of the alpha-subunit of Na+,K(+)-ATPase in rat retinal pigment epithelial cells by the protein A-gold technique, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. Immunoblot analysis showed that the antibody bound specifically to the alpha- and alpha(+)-subunits of Na+,K(+)-ATPase in the whole retina [the sensory retina plus retinal pigment epithelium (RPE)]. Rat eyes were fixed by perfusion with 4% paraformaldehyde containing 1% glutaraldehyde and embedded in Lowicryl K4M. Ultra-thin sections were incubated with affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase and subsequently with protein A-gold complex. Light microscopy with a silver enhancement procedure revealed Na+,K(+)-ATPase localized to both the apical and the basal plasma membrane domains of the RPE. Quantitative immunocytochemical analysis by electron microscopy showed a higher density of gold particles on the apical surface than on the basolateral one. Microvilli are so well developed on the apical surface of the RPE that the apical surface profile is much longer than the basolateral one. This means that Na+,K(+)-ATPase is mainly located on the apical surface of the RPE cells.     

Distribution of aquaporins in the colon of Octodon degus,a South American desert rodent

Octodon degus is a desert rodent of northern Chile, adapted to survive with a limited supply of water. This rodent has a high degree of fecal dehydration, related to colon water absorption. With the hypothesis that aquaporins (AQPs) might be present in the colon epithelium of O. degus and involved in fluid absorption, we studied colon water absorption in vivo and the distribution of AQPs and Na(+) transporters by immunocytochemistry. AQP-1 was found in apical and basolateral membranes of surface-absorptive and crypt epithelial cells. AQP-8 was found in the cytoplasm of enterocytes of surface colon. AQP-3 immunolabeling, on the other hand, was absent from the epithelium but present in a subepithelial fibroblast layer, pericryptal cells, and muscularis mucosae. The hydration state did not modify the amount of immunostaining for any of the AQPs. Colon water absorption was markedly decreased by the mercurial agent p-chloromercuribenzenesulfonic acid and was not affected by water deprivation. The NHE3 isoform of Na(+)/H(+) exchanger and alpha-1 subunit of the Na(+)-K(+)-ATPase were found in apical and basolateral membranes of surface-absorptive cells, respectively. These results suggest that colon water absorption is mostly transcellular and mediated by water channels like AQP-1. Apical Na(+)/H(+) exchanger and basolateral Na(+)-K(+)-ATPase in surface cells could be part of the Na(+) absorption pathway. It is hypothesized that this transport is necessary to provide an osmotic gradient for water absorption. The roles of AQP-8 and AQP-3 in water absorption remain to be established.     

Ultracytochemical location of Na(+)/K(+)-atpase activity and effect of high salinity acclimation in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii (Crustacea, Decapoda).

Accumulation sites of lead phosphate reaction product consequent to Na(+)/K(+)-ATPase activity in gill and renal epithelia of the freshwater shrimp Macrobrachium olfersii were located ultracytochemically by para-nitrophenyl-phosphate hydrolysis and lead precipitation, and quantified per unit membrane area and cytoplasmic volume. In shrimps in freshwater (<0.5 per thousand S, 20 mOsm/kg H(2)O, 0.7 mEq Na(+)/liter), numerous sites of electron-dense, Na(+)/K(+)-ATPase reaction product accumulation were demonstrated in the membrane invaginations of the mitochondria-rich, intralamellar septal cells (12.5 +/- 1.7 sites/microm(2) membrane, 179 +/- 22 sites/microm(3) cytoplasm, mean+/- SEM, N 相似文献   

Acid-base regulation in fishes: cellular and molecular mechanisms

The mechanisms underlying acid-base transfers across the branchial epithelium of fishes have been studied for more than 70 years. These animals are able to compensate for changes to internal pH following a wide range of acid-base challenges, and the gill epithelium is the primary site of acid-base transfers to the water. This paper reviews recent molecular, immunohistochemical, and functional studies that have begun to define the protein transporters involved in the acid-base relevant ion transfers. Both Na(+)/H(+) exchange (NHE) and vacuolar-type H(+)-ATPase transport H(+) from the fish to the environment. While NHEs have been thought to carry out this function mainly in seawater-adapted animals, these proteins have now been localized to mitochondrial-rich cells in the gill epithelium of both fresh and saltwater-adapted fishes. NHEs have been found in the gill epithelium of elasmobranchs, teleosts, and an agnathan. In several species, apical isoforms (NHE2 and NHE3) appear to be up-regulated following acidosis. In freshwater teleosts, H(+)-ATPase drives H(+) excretion and is indirectly coupled to Na(+) uptake (via Na(+) channels). It has been localized to respiratory pavement cells and chloride cells of the gill epithelium. In the marine elasmobranch, both branchial NHE and H(+)-ATPase have been identified, suggesting that a combination of these mechanisms may be utilized by marine elasmobranchs for acid-base regulation. An apically located Cl(-)/HCO(3)(-) anion exchanger in chloride cells may be responsible for base excretion in fresh and seawater-adapted fishes. While only a few species have been examined to date, new molecular approaches applied to a wider range of fishes will continue to improve our understanding of the roles of the various gill membrane transport processes in acid-base balance.     

Intestinal carbonic anhydrase, bicarbonate, and proton carriers play a role in the acclimation of rainbow trout to seawater

Abrupt transfer of rainbow trout from freshwater to 65% seawater caused transient disturbances in extracellular fluid ionic composition, but homeostasis was reestablished 48 h posttransfer. Intestinal fluid chemistry revealed early onset of drinking and slightly delayed intestinal water absorption that coincided with initiation of NaCl absorption and HCO(3)(-) secretion. Suggestive of involvement in osmoregulation, relative mRNA levels for vacuolar H(+)-ATPase (V-ATPase), Na(+)-K(+)-ATPase, Na(+)/H(+) exchanger 3 (NHE3), Na(+)-HCO(3)(-) cotransporter 1, and two carbonic anhydrase (CA) isoforms [a general cytosolic isoform trout cytoplasmic CA (tCAc) and an extracellular isoform trout membrane-bound CA type IV (tCAIV)], were increased transiently in the intestine following exposure to 65% seawater. Both tCAc and tCAIV proteins were localized to apical regions of the intestinal epithelium and exhibited elevated enzymatic activity after acclimation to 65% seawater. The V-ATPase was localized to both basolateral and apical regions and exhibited a 10-fold increase in enzymatic activity in fish acclimated to 65% seawater, suggesting a role in marine osmoregulation. The intestinal epithelium of rainbow trout acclimated to 65% seawater appears to be capable of both basolateral and apical H(+) extrusion, likely depending on osmoregulatory status and intestinal fluid chemistry.     

Branchial chamber tissues in two caridean shrimps: the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata

The structure of the epithelia of the branchial chamber organs (gills, branchiostegites, epipodites) and the localization of the Na(+),K(+)-ATPase were investigated in two caridean shrimps, the epibenthic Palaemon adspersus and the deep-sea hydrothermal Rimicaris exoculata. The general organization of the phyllobranchiate gills, branchiostegites and epipodites is similar in P. adspersus and in R. exoculata. The gill filaments are formed by a single axial epithelium made of H-shaped cells with thin lateral expansions and a basal lamina limiting hemolymph lacunae. In P. adspersus, numerous ionocytes are present in the epipodites and in the inner-side of the branchiostegites; immunofluorescence reveals their high content in Na(+),K(+)-ATPase. In R. exoculata, typical ionocytes displaying a strong Na(+),K(+)-ATPase specific fluorescence are observed in the epipodites only. While the epipodites and the branchiostegites appear as the main site of osmoregulation in P. adspersus, only the epipodites might be involved in ion exchanges in R. exoculata. In both species, the gill filaments are mainly devoted to respiration.     

Upregulation of apical NHE3 in renal OK cells overexpressing the rodent alpha(1)-subunit of the Na(+) pump

Vectorial Na(+) reabsorption across the proximal tubule is mediated by apical entry of Na(+), primarily via Na(+)/H(+) exchanger isoform 3 (NHE3), and basolateral extrusion via the Na(+) pump (Na(+)-K(+)-ATPase). We hypothesized that regulation of Na(+) reabsorption should involve not only the activity of the basolateral Na(+)-K(+)-ATPase, but also the apical NHE3, in a concerted manner. To generate a cell line that overexpresses Na(+)-K(+)-ATPase, opossum kidney (OK) cells were transfected with the rodent Na(+)-K(+)-ATPase alpha(1)-subunit (pCMV ouabain vector), and native cells were used as a control. The existence of distinct functional classes of Na(+)-K(+)-ATPase in wild-type and transfected cells was confirmed by the inhibition profile of Na(+)-K(+)-ATPase activity by ouabain. In contrast to wild-type cells, transfected cells exhibited two IC(50) values for ouabain: the first value was similar to the IC(50) of control cells, and the second value was 2 log units greater than the first, consistent with the presence of rat and opossum alpha(1)-isozymes. It is shown that transfection of OK cells with Na(+)-K(+)-ATPase increased Na(+)-K(+)-ATPase and NHE3 activities. This was associated with overexpression of the Na(+)-K(+)-ATPase alpha(1)-subunit and NHE3 in transfected OK cells. The abundance of the Na(+)-K(+)-ATPase beta(1)-subunit was slightly lower in transfected OK cells. In conclusion, the increase in expression and function of Na(+)-K(+)-ATPase in cells transfected with the rodent Na(+) pump alpha(1)-subunit cDNA is expected to stimulate apical Na(+) influx into the cells, thereby accounting for the observed stimulation of the apical NHE3 activity.     

Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus

The role of aquaporins in cerebrospinal fluid (CSF) secretion was investigated in this study. Western analysis and immunocytochemistry were used to examine the expression of aquaporin 1 (AQP1) and aquaporin 4 (AQP4) in the rat choroid plexus epithelium. Western analyses were performed on a membrane fraction that was enriched in Na(+)/K(+)-ATPase and AE2, marker proteins for the apical and basolateral membranes of the choroid plexus epithelium, respectively. The AQP1 antibody detected peptides with molecular masses of 27 and 32 kDa in fourth and lateral ventricle choroid plexus. A single peptide of 29 kDa was identified by the AQP4 antibody in fourth and lateral ventricle choroid plexus. Immunocytochemistry demonstrated that AQP1 is expressed in the apical membrane of both lateral and fourth ventricle choroid plexus epithelial cells. The immunofluorescence signal with the AQP4 antibody was diffusely distributed throughout the cytoplasm, and there was no evidence for AQP4 expression in either the apical or basolateral membrane of the epithelial cells. The data suggest that AQP1 contributes to water transport across the apical membrane of the choroid plexus epithelium during CSF secretion. The route by which water crosses the basolateral membrane, however, remains to be determined.     

Ouabain-insensitive acidification by dopamine in renal OK cells: primary control of the Na(+)/H(+) exchanger

The present study was aimed at evaluating the role of D(1)- and D(2)-like receptors and investigating whether inhibition of Na(+) transepithelial flux by dopamine is primarily dependent on inhibition of the apical Na(+)/H(+) exchanger, inhibition of the basolateral Na(+)-K(+)-ATPase, or both. The data presented here show that opossum kidney cells are endowed with D(1)- and D(2)-like receptors, the activation of the former, but not the latter, accompanied by stimulation of adenylyl cyclase (EC(50) = 220 +/- 2 nM), marked intracellular acidification (IC(50) = 58 +/- 2 nM), and attenuation of amphotericin B-induced decreases in short-circuit current (28.6 +/- 4.5% reduction) without affecting intracellular pH recovery after CO(2) removal. These results agree with the view that dopamine, through the activation of D(1)- but not D(2)-like receptors, inhibits both the Na(+)/H(+) exchanger (0.001933 +/- 0.000121 vs. 0.000887 +/- 0.000073 pH unit/s) and Na(+)-K(+)-ATPase without interfering with the Na(+)-independent HCO transporter. It is concluded that dopamine, through the action of D(1)-like receptors, inhibits both the Na(+)/H(+) exchanger and Na(+)-K(+)-ATPase, but its marked acidifying effects result from inhibition of the Na(+)/H(+) exchanger only, without interfering with the Na(+)-independent HCO transporter and Na(+)-K(+)-ATPase.     

Distinguishing roles of the membrane-cytoskeleton and cadherin mediated cell-cell adhesion in generating different Na+,K(+)-ATPase distributions in polarized epithelia

In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)- ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal- lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)- dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B- cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation,and effects of ouabain and bumetamide

Isolated salivary glands of Periplaneta americana were used to measure secretion rates and, by quantitative capillary electrophoresis, Na(+), K(+), and Cl(-) concentrations in saliva collected during dopamine (1 micro M) and serotonin (1 micro M) stimulation in the absence and presence of ouabain (100 micro M) or bumetanide (10 micro M). Dopamine stimulated secretion of a NaCl-rich hyposmotic saliva containing (mM): Na(+) 95 +/- 2; K(+) 38 +/- 1; Cl(-) 145 +/- 3. Saliva collected during serotonin stimulation had a similar composition. Bumetanide decreased secretion rates induced by dopamine and serotonin; secreted saliva had lower Na(+), K(+) and Cl(-) concentrations and osmolarity. Ouabain caused increased secretion rates on a serotonin background. Saliva secreted during dopamine but not serotonin stimulation in the presence of ouabain had lower K(+) and higher Na(+) and Cl(-) concentrations, and was isosmotic. We concluded: The Na(+)-K(+)-2Cl(-) cotransporter is of cardinal importance for electrolyte and fluid secretion. The Na(+)/K(+)-ATPase contributes to apical Na(+) outward transport and Na(+) and K(+) cycling across the basolateral membrane in acinar P-cells. The salivary ducts modify the primary saliva by Na(+) reabsorption and K(+) secretion, whereby Na(+) reabsorption is energized by the basolateral Na(+)/K(+)-ATPase which imports also some of the K(+) needed for apical K(+) extrusion.     

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.     

Theoretical considerations underlying Na(+) uptake mechanisms in freshwater fishes

Ion and acid-base regulating mechanisms have been studied at the fish gill for almost a century. Original models proposed for Na(+) and Cl(-) uptake, and their linkage with H(+) and HCO(3)(-) secretion have changed substantially with the development of more sophisticated physiological techniques. At the freshwater fish gill, two dominant mechanisms for Na(+) uptake from dilute environments have persisted in the literature. The use of an apical Na(+)/H(+) exchanger driven by a basolateral Na(+)/K(+)-ATPase versus an apical Na(+) channel electrogenically coupled to an apical H(+)-ATPase have been the source of debate for a number of years. Advances in molecular biology have greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This review will focus on the recent molecular advances for Na(+) uptake in freshwater fish. Emphasis will be placed on thermodynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermodynamic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner.