X-ray microanalysis of airway surface liquid in the mouse

The ionic composition of airway surface liquid (ASL) has been debated, and, in particular for the mouse, a wide range of values has been published. Two techniques were developed to measure the elemental composition of the ASL. X-ray microanalysis of ASL was carried out at low temperature on trachea removed from isoflurane-anesthetized animals and shock-frozen. In the second technique, dextran beads were placed on top of the epithelium of the trachea removed from pentobarbital-anesthetized animals, left to equilibrate with the ASL, dried, and subjected to X-ray microanalysis. Both techniques showed that mouse tracheal ASL has significantly lower concentrations of Na and Cl (approximately 60-80 mM) than serum. Differences between the two techniques were due to different sampling of mucus. CFTR(-/-) mice had significantly higher concentrations of Na and Cl in their ASL than age-matched controls. Pilocarpine or isoproterenol stimulation significantly reduced the ion concentrations in tracheal ASL. ASL was also collected with the dextran bead method from the nasal cavity in situ in pentobarbital-anesthetized animals. In control animals, the elemental composition of nasal fluid was similar to that of tracheal ASL. Pilocarpine stimulation caused a significant increase in Na, Cl, and K; stimulation with isoproterenol or phenylephrine caused a significant increase only in K. It is concluded that mouse ASL under unstimulated conditions is hypotonic, which may be related to the relative paucity of submucosal glands in the mouse trachea.     

Transgenic hCFTR expression fails to correct β-ENaC mouse lung disease

The relationships between airway epithelial Cl(-) secretion-Na(+) absorption balance, airway surface liquid (ASL) homeostasis, and lung disease were investigated in selected transgenic mice. 1) To determine if transgenic overexpression of wild-type (WT) human CFTR (hCFTR) accelerated Cl(-) secretion and regulated Na(+) absorption in murine airways, we utilized a Clara cell secretory protein (CCSP)-specific promoter to generate mice expressing airway-specific hCFTR. Ussing chamber studies revealed significantly (～2.5-fold) elevated basal Cl(-) secretory currents in CCSP-hCFTR transgenic mouse airways. Endogenous murine airway Na(+) absorption was not regulated by hCFTR, and these mice exhibited no lung disease. 2) We tested whether hCFTR, transgenically expressed on a transgenic mouse background overexpressing the β-subunit of the epithelial Na(+) channel (β-ENaC), restored ion transport balance and ASL volume homeostasis and ameliorated lung disease. Both transgenes were active in CCSP-hCFTR/β-ENaC transgenic mouse airways, which exhibited an elevated basal Cl(-) secretion and Na(+) hyperabsorption. However, the airway disease characteristic of β-ENaC mice persisted. Confocal studies of ASL volume homeostasis in cultured tracheal cells revealed ASL autoregulation to a height of ～6 μm in WT and CCSP-hCFTR cultures, whereas ASL was reduced to <4 μm in β-ENaC and CCSP-hCFTR/β-ENaC cultures. We conclude that 1) hCFTR overexpression increases basal Cl(-) secretion but does not regulate Na(+) transport in WT mice and 2) transgenic hCFTR produces increased Cl(-) secretion, but not regulation of Na(+) channels, in β-ENaC mouse airways and does not ameliorate β-ENaC mouse lung disease.     

In vivo microdialysis for determination of nasal liquid ion composition

Airway surface liquid (ASL) contains substances important in mucociliary clearance and airway defense. Little is known about substance concentrations in ASL because of its small volume and sampling difficulties. We used in vivo microdialysis (IVMD) to sample liquid lining the nasal cavity without net volume removal and incorporated into IVMD a potential difference (PD) electrode to assess airway integrity. The cystic fibrosis (CF) mouse nasal epithelia exhibit ion transport defects identical to those in CF human airways and, thus, are a good model for CF disease. We determined that nasal liquid [Na+] (107 +/- 4 mM normal; 111 +/- 9 mM CF) and [Cl-] (120 +/- 6 mM normal; 122 +/- 4 mM CF) did not differ between genotypes. The nasal liquid [K+] (8.7 +/- 0.4 mM) was significantly less in normal than in CF mice (16.6 +/- 4 mM). IVMD accurately samples nasal liquid for ionic composition. The ionic composition of nasal liquid in the normal and CF mice is similar.     

Abnormal ion content, hydration and granule expansion of the secretory granules from cystic fibrosis airway glandular cells

The absence or decreased expression of cystic fibrosis transmembrane conductance regulator (CFTR) induces increased Na(+) absorption and hyperabsorption of the airway surface liquid (ASL) resulting in a dehydrated and hyperviscous ASL. Although the implication of abnormal airway submucosal gland function has been suggested, the ion and water content in the Cystic Fibrosis (CF) glandular secretory granules, before exocytosis, is unknown. We analyzed, in non-CF and CF human airway glandular cell lines (MM-39 and KM4, respectively), the ion content in the secretory granules by electron probe X-ray microanalysis and the water content by quantitative dark field imaging on freeze-dried cryosections. We demonstrated that the ion content (Na(+), Mg(2+), P, S and Cl(-)) is significantly higher and the water content significantly lower in secretory granules from the CF cell line compared to the non-CF cell line. Using videomicroscopy, we observed that the secretory granule expansion was deficient in CF glandular cells. Transfection of CF cells with CFTR cDNA or inhibition of non-CF cells with CFTR(inh)-172, respectively restored or decreased the water content and granule expansion, in parallel with changes in ion content. We hypothesize that the decreased water and increased ion content in glandular secretory granules may contribute to the dehydration and increased viscosity of the ASL in CF.     

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.     

Kinetics of hyperosmotically stimulated Na-K-2Cl cotransporter in Xenopus laevis oocytes

A detailed study of hypertonically stimulated Na-K-2Cl cotransport (NKCC1) in Xenopus laevis oocytes was carried out to better understand the 1 K(+):1 Cl(-) stoichiometry of transport that was previously observed. In this study, we derived the velocity equations for K(+) influx under both rapid equilibrium assumptions and combined equilibrium and steady-state assumptions and demonstrate that the behavior of the equations and curves in Lineweaver-Burke plots are consistent with a model where Cl(-) binds first, followed by Na(+), a second Cl(-), and then K(+). We further demonstrate that stimulation of K(+) movement by K(+) on the trans side is an intrinsic property of a carrier that transports multiple substrates. We also demonstrate that K(+) movement through NKCC1 is strictly dependent upon the presence of external Na(+), even though only a fraction of Na(+) is in fact transported. Finally, we propose that the larger transport of K(+), as compared with Na(+), is a result of the return of partially unloaded carriers, which masks the net 1Na(+):1K(+):2Cl(-) stoichiometry of NKCC1. These data have profound implications for the physiology of Na-K-2Cl cotransport, since transport of K-Cl in some conditions seems to be uncoupled from the transport of Na-Cl.     

X-ray microanalysis of apical fluid in cystic fibrosis airway epithelial cell lines.

The ionic composition of the fluid lining the airways (airway surface liquid, ASL) in healthy subjects and patients with cystic fibrosis (CF) has been a matter of controversy. It has been attempted to resolve conflicting theories by using cell cultures, but published results show a wide variety of values for the ionic concentrations in the apical fluid in these cultures. To investigate CFTR-mediated HCO(3)(-) conductance and the role of HCO(3)(-) in regulating ASL pH we determined the pH of the fluid covering the apical surface of airway epithelial cells. A normal (16HBE14o (-)) and a CF (CFBE41o (-)) bronchial epithelial cell line were grown on membrane inserts in both a liquid-liquid interface culture system for 7 days, and in an air-liquid interface culture system for one month. The elemental composition of the fluid covering the apical surface was determined by X-ray microanalysis of frozen-hydrated specimens, or by X-ray microanalysis of Sephadex beads that had been equilibrated with the apical fluid. Analysis showed that the apical fluid had a Na(+) and Cl(-) concentration of about 80-100 mM and thus was slightly hypotonic. The ionic concentrations were somewhat higher in air-liquid interface than in liquid-liquid interface cultures. The apical fluid in CF cells had significantly higher concentrations of Na and Cl than that in control cultures. In control cultures, the concentrations of Na and Cl in the apical fluid increased if glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR) was added to the apical medium. Exposing the cells to the metabolic inhibitor NaCN also resulted in a significant increase of the Na and Cl concentrations in the apical fluid. The results agree with the notion that these cell cultures are mainly absorptive cells, and that ion absorption by the CF cells is reduced compared to that in normal cells. The pH measurements of the fluid covering the apical part of cell cultures support the notion that bicarbonate ions may be transported by CFTR, and that this can be inhibited by specific CFTR inhibitors.     

Rates of efflux and intracellular concentrations of potassium, sodium and chloride ions in isolated fat-cells from the rat

1. The metabolism of K(+), Na(+) and Cl(-) has been investigated in isolated fat-cells prepared from the epididymal adipose tissue of rats. 2. Methods are described for measuring the intracellular water space, the rates of loss of intracellular (42)K(+), (22)Na(+) and (36)Cl(-) and the intracellular concentrations of K(+), Na(+) and Cl(-) in isolated fat-cells. 3. The intracellular water space, measured as the [(3)H]water space minus the [carboxylic acid-(14)C]inulin space, was 3.93+/-0.38mul./100mg. cell dry wt. 4. The first-order rate constants for radioisotope effluxes from isolated fat-cells were 0.029min.(-1) for (42)K(+), 0.245min.(-1) for (22)Na(+) and 0.158min.(-1) for (36)Cl(-). 5. The intracellular concentrations of K(+), Na(+) and Cl(-) were 146m-equiv./l., 18.6+/-2.9m-equiv./l. and 43+/-2.4m-equiv./l. respectively. 6. The total intracellular K(+) content of isolated fat-cells was determined by atomic-absorption spectrophotometry to confirm the value obtained from the radioisotope-efflux data. 7. The ion effluxes from isolated fat-cells were: K(+), 1.5pmoles/cm.(2)/sec., Na(+), 1.6pmoles/cm.(2)/sec., and Cl(-), 2.4pmoles/cm.(2)/sec. 8. The membrane potential of isolated fat-cells calculated from the Cl(-) distribution ratio was -28.7mv.     

Effect of divalent cations on ion fluxes and leaf photochemistry in salinized barley leaves

Photosynthetic characteristics, leaf ionic content, and net fluxes of Na(+), K(+), and Cl(-) were studied in barley (Hordeum vulgare L) plants grown hydroponically at various Na/Ca ratios. Five weeks of moderate (50 mM) or high (100 mM) NaCl stress caused a significant decline in chlorophyll content, chlorophyll fluorescence characteristics, and stomatal conductance (g(s)) in plant leaves grown at low calcium level. Supplemental Ca(2+) enabled normal photochemical efficiency of PSII (F(v)/F(m) around 0.83), restored chlorophyll content to 80-90% of control, but had a much smaller (50% of control) effect on g(s). In experiments on excised leaves, not only Ca(2+), but also other divalent cations (in particular, Ba(2+) and Mg(2+)), significantly ameliorated the otherwise toxic effect of NaCl on leaf photochemistry, thus attributing potential targets for such amelioration to leaf tissues. To study the underlying ionic mechanisms of this process, the MIFE technique was used to measure the kinetics of net Na(+), K(+), and Cl(-) fluxes from salinized barley leaf mesophyll in response to physiological concentrations of Ca(2+), Ba(2+), Mg(2+), and Zn(2+). Addition of 20 mM Na(+) as NaCl or Na(2)SO(4) to the bath caused significant uptake of Na(+) and efflux of K(+). These effects were reversed by adding 1 mM divalent cations to the bath solution, with the relative efficiency Ba(2+)>Zn(2+)=Ca(2+)>Mg(2+). Effect of divalent cations on Na(+) efflux was transient, while their application caused a prolonged shift towards K(+) uptake. This suggests that, in addition to their known ability to block non-selective cation channels (NSCC) responsible for Na(+) entry, divalent cations also control the activity or gating properties of K(+) transporters at the mesophyll cell plasma membrane, thereby assisting in maintaining the high K/Na ratio required for optimal leaf photosynthesis.     

HCO3(-) secretion by murine nasal submucosal gland serous acinar cells during Ca2+-stimulated fluid secretion

Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3)(-) during Ca(2+)-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl(-) channel, with HCO(3)(-) secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na(+)-dependent pH(i) regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na(+)-free media.     

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.     

Gastrointestinal processing of Na+, Cl-, and K+ during digestion: implications for homeostatic balance in freshwater rainbow trout

The role of the gastrointestinal tract in maintaining ionic homeostasis during digestion, as well as the relative contribution of the diet for providing electrolytes, has been generally overlooked in many aquatic species. An experimental diet that contained an inert reference marker (lead-glass beads) was used to quantify the net transport of Na(+), K(+), and Cl(-) during the digestion and absorption of a single meal (3% ration) by freshwater rainbow trout (Oncorhynchus mykiss). Secretion of Cl(-) into the stomach peaked at 8 and 12 h following feeding at a rate of 1.1 mmol.kg(-1).h(-1), corresponding to a theoretical pH of 0.6 in the secreted fluid (i.e., 240 mmol/l HCl). The majority ( approximately 90%) of dietary Na(+) and K(+) was absorbed in the stomach, whereas subsequent large fluxes of Na(+) and Cl(-) into the anterior intestine corresponded to a large flux of water previously observed. The estimated concentration of Na(+) in fluids secreted into the anterior intestine was approximately 155 mmol/l, equivalent to reported hepatic bile values, whereas the estimated concentration of Cl(-) ( approximately 285 mmol/l) suggested seepage of HCl acid from the stomach in advance of the chyme front. Net absorption of K(+) in the stomach occurred following the cessation of Cl(-) secretion, providing indirect evidence of K(+) involvement with HCl acid production. Overall, 80-90% of the K(+) and Cl(-) contents of the meal were absorbed on a net basis, whereas net Na(+) absorption was negligible. Chyme-to-plasma ion concentration gradients were often opposed to the direction of ion transport, especially for Na(+) and Cl(-).     

The sodium-activated potassium channel is encoded by a member of the Slo gene family

Na(+)-activated potassium channels (K(Na)) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. We now report that K(Na) is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native K(Na) including activation by intracellular Na(+), high conductance, and prominent subconductance states. In addition to activation by Na(+), we report that rSLO-2 channels are cooperatively activated by intracellular Cl(-), similar to C. elegans SLO-2 channels. Since intracellular Na(+) and Cl(-) both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis revealed that the SLO-2 current is a major component of the delayed rectifier. We demonstrate in C. elegans that slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily.     

Heat stress preconditioning does not protect renal epithelial Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from their modulation by severe heat stress

This study compares the effects of heat and osmotic stress on heat stress protein (HSP) production while examining the putative protective action of HSPs on modulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters in Madin-Darby canine kidney (MDCK) epithelial cells by severe heat stress (46 degrees C, 15 min). Preconditioning heat stress (43 degrees C, 20 min) followed by 4 h recovery at 37 degrees C led to a 35-fold increase of HSP70 mRNA expression measured by Northern blot analysis. The protein content of HSP70 and HSP27, assessed by Western blots, was augmented by 5- and 2-fold, respectively, after 6 h of recovery. In contrast to preconditioning heat stress, hyperosmotic stress (520 vs. 320 mosm) elevated HSP70 mRNA content only by 7-fold and did not significantly affect the protein content of HSP70 or HSP27. Neither cell survival, assessed as lactate dehydrogenase (LDH) release, nor the basal activities of the ion transporters and their modulation by protein kinase C, P(2)-purinoceptor and cell volume were altered by preconditioning heat stress. Severe heat stress increased extracellular LDH content from 3+/-2 to 23+/-5% and enhanced Na(+),K(+),Cl(-) and Na(+),P(i) cotransport activity by 2-3-fold. The volume- and protein kinase C-dependent regulation of these carriers was abolished by severe heat stress while regulation by P(2)-purinoceptors was preserved. Preconditioning heat stress diminished severe heat stress-induced LDH release to 11+/-4% but did not protect Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters from activation by severe heat stress and did not prevent severe heat stress-induced inactivation of protein kinase C- and volume-dependent signaling pathways. These results show that in MDCK cells, preconditioning heat stress-induced HSPs are not involved in the regulation of Na(+),K(+),Cl(-) and Na(+),P(i) cotransporters and do not protect them from modulation by severe heat stress.     

Cl- uptake mechanism in freshwater-adapted tilapia (Oreochromis mossambicus)

In this study, the correlation between Cl(-) influx in freshwater tilapia and various transporters or enzymes, the Cl(-)/HCO(3)(-) exchanger, Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase were examined. The inhibitors 2x10(-4) M ouabain (a Na(+),K(+)-ATPase inhibitor), 10(-5) M NEM (a V-type H(+)-ATPase inhibitor), 10(-2) M ACTZ (acetazolamide, a carbonic anhydrase inhibitor), and 6x10(-4) M DIDS (a Cl(-)/HCO(3)(-) exchanger inhibitor) caused 40%, 60%-80%, 40%-60%, and 40%-60% reduction in Cl(-) influx of freshwater tilapia, respectively. The inhibitor 2x10(-4) M ouabain also caused 50%-65% inhibition in gill Na(+),K(+)-ATPase activity. Western blot results showed that protein levels of gill Na(+),K(+)-ATPase, V-type H(+)-ATPase, and carbonic anhydrase in tilapia acclimated in low-Cl(-) freshwater were significantly higher than those acclimated to high-Cl(-) freshwater. Based on these data, we conclude that Na(+),K(+)-ATPase, V-H(+)-ATPase, the Cl(-)/HCO(3)(-) exchanger, and carbonic anhydrase may be involved in the active Cl(-) uptake mechanism in gills of freshwater-adapted tilapia.     

Ion regulatory patterns of mosquito larvae collected from breeding sites in the Amazon rain forest

We examined the ion composition of mosquito breeding sites located in the Amazon rain forest and the ion regulatory patterns of larvae from these habitats. We found larvae of Toxorhynchites haemorroidalis, Limatus durhamii, Culex (Carrollia) bonnei, and Culex (Culex) sp. residing in fallen palm bracts, leaves, and tree holes that were filled with water. These breeding sites had micromolar levels of Na(+) (1.6-99 micromol L(-1)), but K(+) and Cl(-) concentrations were higher and varied over a large range (231-17,615 micromol L(-1) K(+); 355-2,700 micromol L(-1) Cl(-)). Despite the variability in environmental ion levels and ratios, all four species maintain high hemolymph NaCl levels (80-120 mmol L(-1) Na(+); 60-80 mmol L(-1) Cl(-)). However, the species differed in the means by which they maintain hemolymph ion balance, as indicated by the range of unidirectional Na(+) and Cl(-) uptake rates. Toxorhynchites haemorroidalis had extremely low rates of Na(+) uptake and undetectable Cl(-) uptake, whereas L. durhamii had high rates of uptake for both ions. This variability in rates of uptake may reflect species differences in rates of diffusive ion loss (i.e., permeability). We observed the same curious pattern of Na(+) inhibition and Cl(-) stimulation by low-pH exposure in all four species of mosquitoes, as has been documented in other mosquitoes and aquatic insects. Kinetic analyses of Na(+) and Cl(-) uptake in C. bonnei larvae revealed an unusual pattern of Na(+) uptake that increases linearly (nonsaturable) to extremely high rates, while Cl(-) uptake is a low-affinity, low-capacity system. This pattern contrasts with L. durhamii and Culex (Culex) sp. larvae, which had large increases in both Na(+) and Cl(-) uptake when external NaCl levels were increased. Our results suggest that although these rain forest mosquito larvae are residing in habitats with similar low Na(+), high Cl(-) composition and maintain similar hemolymph NaCl levels, the underlying mechanisms of ion regulation differ among the species.     

Osmoregulatory effects of hypophysectomy and homologous prolactin replacement in hybrid striped bass

The effects of ovine prolactin (oPRL) and striped bass prolactin (sbPRL; Morone saxatilis) on plasma osmolality, electrolyte balance, and gill Na(+),K(+)-ATPase activity were investigated in hypophysectomized (Hx), freshwater (FW)-acclimated, hybrid striped bass (M. saxatilisxMorone chrysops). They were kept in dilute (isoosmotic) seawater for about 10 days after surgery. Seven days after transfer to FW, Hx fish had lower plasma osmolality and lower levels of Na(+), Cl(-), and Ca(2+) than sham-operated and intact fish. Fish were injected four times with oPRL (1, 5, or 20 microg/g body mass), sbPRL (10 or 100 ng/g), or hormone vehicle (0.9% NaCl) at 48-h intervals (days 0, 2, 4, and 6) in FW and then sampled for blood plasma 24 h after the fourth injection (day 7). In Hx fish, oPRL (5 and 20 microg/g) and sbPRL (10 and 100 ng/g) were effective in maintaining plasma osmolality and levels of Na(+), Cl(-), and Ca(2+) above values seen in saline-injected controls. Hypophysectomy did not affect branchial Na(+),K(+)-ATPase activity, but enzyme activity was significantly reduced in Hx fish receiving oPRL (20 mug/g) or sbPRL (10 or 100 ng/g). These results indicate that PRL acts to maintain plasma osmotic and ionic balance in FW-adapted hybrid striped bass, and that this may involve downregulation of branchial Na(+),K(+)-ATPase activity.     

Role of the choline exchanger in Na(+)-independent Mg(2+) efflux from rat erythrocytes

Two types of Na(+)-independent Mg(2+) efflux exist in erythrocytes: (1) Mg(2+) efflux in sucrose medium and (2) Mg(2+) efflux in high Cl(-) media such as KCl-, LiCl- or choline Cl-medium. The mechanism of Na(+)-independent Mg(2+) efflux in choline Cl medium was investigated in this study. Non-selective transport by the following transport mechanisms has been excluded: K(+),Cl(-)- and Na(+),K(+),Cl(-)-symport, Na(+)/H(+)-, Na(+)/Mg(2+)-, Na(+)/Ca(2+)- and K(+)(Na(+))/H(+) antiport, Ca(2+)-activated K(+) channel and Mg(2+) leak flux. We suggest that, in choline Cl medium, Na(+)-independent Mg(2+) efflux can be performed by non-selective transport via the choline exchanger. This was supported through inhibition of Mg(2+) efflux by hemicholinum-3 (HC-3), dodecyltrimethylammonium bromide (DoTMA) and cinchona alkaloids, which are inhibitors of the choline exchanger. Increasing concentrations of HC-3 inhibited the efflux of choline and efflux of Mg(2+) to the same degree. The K(d) value for inhibition of [(14)C]choline efflux and for inhibition of Mg(2+) efflux by HC-3 were the same within the experimental error. Inhibition of choline efflux and of Mg(2+) efflux in choline medium occurred as follows: quinine>cinchonine>HC-3>DoTMA. Mg(2+) efflux was reduced to the same degree by these inhibitors as was the [(14)C]choline efflux.     

Scaling of Na+,K+-ATPase molecular activity and membrane fatty acid composition in mammalian and avian hearts

We have examined Na(+),K(+)-ATPase molecular activity and membrane fatty acid composition in the heart of six mammalian and eight avian species ranging in size from 30 g in mice to 280 kg in cattle and 13 g in zebra finches to 35 kg in emus, respectively. Na(+),K(+)-ATPase activity scaled negatively with body mass in both mammals and birds. In small mammals, the elevated enzyme activity was related to allometric changes in both the concentration and molecular activity (turnover rate) of Na(+),K(+)-ATPase enzymes, while in small birds, higher Na(+),K(+)-ATPase activity appeared to result primarily from an increased molecular activity of individual enzymes. The unsaturation index of cardiac phospholipids scaled negatively with body mass in both groups, while a significant allometric increase in monounsaturate content was observed in the larger mammals and birds. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid (22:6n-3) displayed the greatest variation, scaling negatively with body mass and varying greater than 40-fold in both mammals and birds. Membrane fatty acid profile was correlated with Na(+),K(+)-ATPase molecular activity in both mammals and birds, suggesting a potential association between membrane lipid composition and the activity of membrane-bound enzymes in the hearts of endotherms.