Cloning and characterization of a Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene of the moderately halophilic bacterium <Emphasis Type="Italic">Halobacillus aidingensis</Emphasis> AD-6<Superscript>T</Superscript>

A gene encoding a Na(+)/H(+) antiporter was obtained from the genome of Halobacillus aidingensis AD-6(T), which was sequenced and designated as nhaH. The deduced amino acid sequence of the gene was 91% identical to the NhaH of H. dabanensis, and shared 54% identity with the NhaG of Bacillus subtilis. The cloned gene enable the Escherichia coli KNabc cell, which lack all of the major Na(+)/H(+) antiporters, to grow in medium containing 0.2 M NaCl or 10 mM LiCl. The nhaH gene was predicted to encode a 43.5 kDa protein (403 amino acid residues) with 11 putative transmembrane regions. Everted membrane vesicles prepared from E. coli KNabc cells carrying NhaH exhibited Na(+)/H(+) as well as Li(+)/H(+) antiporter activity, which was pH-dependent with the highest activity at pH 8.0, and no K(+)/H(+) antiporter activity was detected. The deletion of hydrophilic C-terminal amino acid residues showed that the short C-terminal tail was vital for Na(+)/H(+) antiporter activity.     

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.     

Muscarinic Receptor Stimulation Activates a Ca<Superscript>2+</Superscript>-dependent Cl<Superscript>-</Superscript> Conductance in Rat Distal Colon

Recently, it was observed that the acetylcholine analogue carbachol induces a transient stimulation of an apical Cl(-) conductance in basolaterally depolarized rat distal colonic epithelium (Schultheiss et al., 2003). The further characterization of this conductance was the aim of the present study. All experiments were performed at basolaterally depolarized tissues (111.5 mmol.l(-1) KCl buffer at the serosal side); in the absence of a K(+) gradient, a Cl(-) current was driven across the apical membrane (107 mmol.l(-1) K gluconate/4.5 mmol.l(-1) KCl buffer on the mucosal side). Under these conditions, carbachol evoked an atropine-sensitive biphasic change in short-circuit current (I(SC)), consisting of a transient increase followed by a long-lasting decrease, suggesting a stimulation of apical Cl(-) conductance followed by an inhibition. This conductance was inhibited by SITS, but was resistant against glibenclamide, a blocker of CFTR. The carbachol-induced I(SC) was dependent on the presence of mucosal Ca(2+). Ionomycin, a Ca(2+) ionophore, mimicked the effect of carbachol. An antibody against bovine Ca(2+)-activated Cl(-) channel ClCa 1 stained rat colonic epithelial cells both at the cell membrane as well as intracellularly, suggesting that the action of Ca(2+) may be caused by a stimulation of a ClC a-type anion channel. The activation of apical Cl(-) conductance by carbachol was resistant against any blockers of the phospholipase C/IP3/protein kinase C pathway tested (e.g., U-73122, 2-ABP, Li(+), staurosporine), but was inhibited by the NO-synthase blocker L: -NNA. Vice versa, NO-donating compounds such as GEA 3162 or sodium nitroprusside evoked a transient increase of I(SC). Consequently, NO seems to be involved in the transient stimulation of apical Ca(2+)-dependent Cl(-) conductance after muscarinic receptor stimulation.     

Sepsis correlated with increased erythrocyte Na<Superscript>+</Superscript> content and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript> pump activity

The aims of the present study were twofold: (1) simultaneous determinations of Na(+) transport parameters of erythrocytes from 40 healthy donors and 28 septic patients as assessed by a score of severity of sepsis (SSS), and (2) examination of the correlation between the SSS and specific Na(+) transport abnormalities. Erythrocytes were obtained and loaded with different ionic compositions and cellular Na(+) contents before determination of the near-maximal Na(+) pump rate (Vmax), the physiological extrusion rate of Na(+) (v) and the number of ouabain-binding sites (Bmax). In erythrocytes from septic patients, the cellular Na(+) content was 28% higher (p < 0.001), with no differences in water content compared to erythrocytes from healthy donors. This elevated Na(+) content was accompanied by significantly higher values for Vmax (43%), v (24%) and Bmax (48%) of the Na(+) pump in septic erythrocytes. Moreover, significant positive correlations existed between Vmax and SSS (p = 0.028) and between cellular Na(+) content and SSS (p = 0.005). These data suggest that during sepsis, membrane alterations occur and result in an increased cellular Na(+) content. Active Na(+) transport (Vmax and v) was significantly stimulated, possibly as a consequence of a secondary response to the elevated Na(+) of cells. Both cellular Na(+) and Vmax correlated well with the severity of sepsis, suggesting that these altered transport parameters may reflect the progress of sepsis.     

KCl reabsorption by the lower malpighian tubule of rhodnius prolixus: inhibition by Cl(-) channel blockers and acetazolamide

Iono- and osmoregulation by the blood-feeding hemipteran Rhodnius prolixus involves co-ordinated actions of the upper and lower Malpighian tubules. The upper tubule secretes ions (Na(+), K(+), Cl(-)) and water, whereas the lower tubule reabsorbs K(+) and Cl(-) but not water. The extent of KCl reabsorption by the lower tubule in vitro was monitored by ion-selective microelectrode measurement of Cl(-) and/or K(+) concentration in droplets of fluid secreted by Malpighian tubules isolated under oil. An earlier study proposed that K(+) reabsorption involves an omeprazole-sensitive apical K(+)/H(+) ATPase and Ba(2+)-sensitive basolateral K(+) channels. This paper examines the effects acetazolamide and of compounds that inhibit chloride channels, Cl(-)/HCO(3)(-) exchangers and Na(+)/K(+)/2Cl(-) or K(+)/Cl(-) co-transporters. The results suggest that Cl(-) reabsorption is inhibited by acetazolamide and by Cl(-) channel blockers, including diphenylamine-2-carboxylate(DPC) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), but not by compounds that block Na(+)/K(+)/Cl(-) and K(+)/Cl(-) co-transporters. Measurements of transepithelial potential and basolateral membrane potential during changes in bathing saline chloride concentration indicate the presence of DPC- and NPPB-sensitive chloride channels in the basolateral membrane. A working hypothesis of ion movements during KCl reabsorption proposes that Cl(-) moves from lumen to cell through a stilbene-insensitive Cl(-)/HCO(3)(-) exchanger and then exits the cell through basolateral Cl(-) channels.     

Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes.

In a previous work, we have shown that overexpression of the Na(+)/K(+)/Cl(-) cotransporter (NKCC1) induces cell proliferation and transformation. We investigate in the present study the role of the NKCC1 in the mitogenic signal transduction. We show that overexpression of the cotransporter gene (NKCC1) in stablely transfected cells (Balb/c-NKCC1), resulted in enhanced phosphorylation of the extracellular regulated kinase (ERK) to produce double phosphorylated ERK (DP-ERK). Furthermore, the level of DP-ERK was reduced by 50-80% following the addition of bumetanide, a specific inhibitor of the Na(+)/K(+)/Cl(-) cotransporter, in quiescent as well as in proliferating cultures of the Balb/c-NKCC1 clone. In order to explore further the role of the Na(+)/K(+)/Cl(-) cotransporter in mitogenic signal transduction, we measured the effect of the two specific inhibitors of the cotransporter; bumetanide and furosemide, on DP-ERK level in immortalized non-transformed cells. In Balb/c 3T3 fibroblasts stimulated with FGF, bumetanide, and furosemide inhibited 50-60% of the ERK 1/2 phosphorylation. The inhibitor concentration needed for maximal inhibition of ERK 1/2 phosphorylation was similar to the concentration needed to block the K(+) influx mediated by the Na(+)/K(+)/Cl(-) cotransporter in these cells. To analyze whether the Na(+)/K(+)/Cl(-) cotransporter has a role in the mitogenic signal of normal cells, we measured the effect of bumetanide on ERK phosphorylation in human peripheral blood lymphocytes. The phosphorylation of ERK 1/2 in resting human lymphocytes, as well as in lymphocytes stimulated with phytohemagglutinin (PHA) was inhibited by bumetanide. The effect of bumetanide on ERK 2 phosphorylation was much lower than that of ERK 1 phosphorylation. The finding that the Na(+)/K(+)/Cl(-) cotransporter controls the ERK/MAPK (mitogen-activated protein kinase) signal transduction pathway, support our hypothesis that Na(+) and K(+) influxes mediated by this transporter plays a central role in the control of normal cell proliferation. Exploring the cellular ionic currents and levels, mediated by the Na(+)/K(+)/Cl(-) cotransporter, should lead to a better comprehension of cell proliferation and transformation machinery.     

Effects of palytoxin on cation occlusion and phosphorylation of the (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-ATPase

Palytoxin (PTX) inhibits the (Na(+) + K+)-driven pump and simultaneously opens channels that are equally permeable to Na+ and K+ in red cells and other cell membranes. In an effort to understand the mechanism by which PTX induces these fluxes, we have studied the effects of PTX on: 1) K+ and Na+ occlusion by the pump protein; 2) phosphorylation and dephosphorylation of the enzyme when a phosphoenzyme is formed from ATP and from P(i); and 3) p-nitro phenyl phosphatase (p-NPPase) activity associated with the (Na+, K+)-ATPase. We have found that palytoxin 1) increases the rate of deocclusion of K+(Rb+) in a time- and concentration-dependent manner, whereas Na+ occluded in the presence of oligomycin is unaffected by the toxin; 2) makes phosphorylation from P(i) insensitive to K+, and 3) stimulates the p-NPPase activity. The results are consistent with the notion that PTX produces a conformation of the Na+, K(+)-pump that resembles the one observed when ATP is bound to its low-affinity binding site. Further, they suggest that the channels that are formed by PTX might arise as a consequence of a perturbation in the ATPase structure, leading to the loss of control of the outside "gate" of the enzyme and hence to an uncoupling of the ion transport from the catalytic function of the ATPase.     

Hepatic versus gallbladder bile composition: in vivo transport physiology of the gallbladder in rainbow trout

Ion and water transport across the teleost Oncorhynchus mykiss gallbladder were studied in vivo by comparing flow and composition of hepatic bile, collected by chronic catheter, to volume and composition of terminally collected gallbladder bile. Differences in composition were comparable with those of other vertebrates, whereas bile flow (75 microl. kg(-1). h(-1)) was below values reported for endothermic vertebrates. The gallbladder concentrates bile acids five- to sevenfold and exhibits higher net Cl(-) than Na(+) transport in vivo, in contrast to the 1:1 transport ratio from gallbladders under saline/saline conditions. Transepithelial potential (TEP) in the presence of bile, at the apical surface, was -13 mV (bile side negative) but +1.5 mV in the presence of saline. Bile acid in the apical saline reversed the TEP, presumably by a Donnan effect. We propose that ion transport across the gallbladder in vivo involves backflux of Na(+) from blood to bile resulting in higher net Cl(-) than Na(+) flux. This Na(+) backflux is driven by a bile side negative TEP and low Na(+) activity in bile due to the complexing effects of bile acids.     

Restitution of the bullfrog gastric mucosa is dependent on a DIDS-inhibitable pathway not related to HCO3- ion transport

This study was conducted to determine the contribution of ion transport to restitution after injury in the gastric mucosa. For this, intact sheets of stomach from the bullfrog, Rana catesbeiana, were mounted in Ussing chambers. Restitution was evaluated in the presence or absence of ion transport inhibitors amiloride, DIDS, and bumetanide to block Na(+)/H(+) exchange, Cl(-)/HCO(3)(-) exchange and Na(+)/HCO(3)(-) co-transport, and Na(+)-K(+)-2Cl(-) cotransport, respectively. Ion substitution experiments with Na(+)-free, Cl(-)-free, and HCO(3)(-)-free solutions were also performed. Injury to the mucosa was produced with 1 M NaCl, and restitution was evaluated by recovery of transepithelial resistance (TER), mannitol flux, and morphology. Amiloride, bumetanide, Cl(-)-free, or HCO(3)(-)-free solutions did not affect restitution. In Na(+)-free solutions, recovery of TER and mannitol flux did not occur because surface cells did not attach to the underlying basement membrane. In contrast, all aspects of restitution were inhibited by DIDS, a compound that inhibits Na(+)-dependent HCO(3)(-) transport. Because HCO(3)(-)-free solutions did not inhibit restitution, it was concluded that DIDS must block a yet undefined pathway not involved in HCO(3)(-) ion transport but essential for cell migration after injury and restitution in the gastric mucosa.     

Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa

The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.     

Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.     

Electrogenic H<Superscript>+</Superscript> Transport and pH Gradients Generated by a V-H<Superscript>+</Superscript>-ATPase in the Isolated Perfused Larval <Emphasis Type="Italic">Drosophila</Emphasis> Midgut

A method for microperfusion of isolated segments of the midgut epithelium of Drosophila larvae has been developed to characterize cellular transport pathways and membrane transporters. Stereological ultrastructural morphometry shows that this epithelium has unusually long tight junctions, with little or no lateral intercellular volume normally found in most epithelia. Amplification of the apical and basal aspects of the cells, by ≈ 17-fold and ≈ 7-fold, respectively, predicts an almost exclusively transcellular transport system for solutes. This correlates with the high lumen-negative transepithelial potential (V_t) of 38 to 45 mV and high resistance (R_t) of 800 to 1400 Ω • cm² measured by terminated cable analysis, in contrast to other microperfused epithelia like the renal proximal tubule. Several blockers (amiloride 10⁻⁴ M, ouabain 10⁻⁴ M, bumetanide 10⁻⁴ M), K⁺-free solutions, or organic solutes such as D-glucose 10 mM or DL-alanine 0.5 mM failed to affect V_t or R_t. Bafilomycin-A₁ (3 to 5 μM) decreased V_t by ≈ 40% and short-circuit current (I_sc) by ≈ 50%, and decreased intracellular pH when applied from the basal side only, consistent with an inhibition of an electrogenic V-H⁺-ATPase located in the basal membrane. Gradients of H⁺ were detected by pH microelectrodes close to the basal aspect of the cells or within the basal extracellular labyrinth. The apical membrane is more conductive than the basal membrane, facilitating secretion of base (presumably HCO₃⁻), driven by the basal V-H⁺-ATPase.     

Na<Superscript>+</Superscript> gradient-dependent transport of hypoxanthine by calf intestinal brush border membrane vesicles

The properties of hypoxanthine transport were investigated in purified brush border membrane vesicles isolated from calf proximal and distal jejunum. Hypoxanthine uptake in the vesicles was stimulated by a transmembrane Na(+) gradient and an inside negative potential resulting in a transient accumulation of intravesicular hypoxanthine, especially in the proximal jejunum. Na(+)-dependent hypoxanthine uptake at this site seemed to occur by two saturable transport systems, a high affinity (K(m)=0.33 micromol/l) and a low affinity (K(m)=165 micromol/l) transporter. Guanine, hypoxanthine, thymine and uracil inhibited intravesicular hypoxanthine uptake, whereas adenine and the nucleosides inosine and thymidine were without effect. These findings represent the first demonstration of active Na(+) gradient-dependent nucleobase transport in intestinal brush border membrane vesicles.     

Overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene induces cell proliferation and phenotypic transformation in mouse fibroblasts

Na(+)/K(+)/Cl(-) cotransporter activity is stimulated in early G(1) phase of the cell cycle and this stimulation was shown to be an essential event in fibroblast cell proliferation. In order to elucidate further the role of the Na(+)/K(+)/Cl(-) cotransporter in cell proliferation, we overexpressed the gene encoding the Na(+)/K(+)/Cl(-) cotransporter in mouse fibroblasts, and analyzed cellular phenotypic changes. Mouse Balb/c 3T3 cells were stably transfected with the cDNA of the shark rectal gland Na(+)/K(+)/Cl(-) cotransporter gene (NKCC1), and expressed in a mammalian vector under the cytomegalovirus promoter (Balb/c-NKCC1 cells). The transfected cells exhibited up to 10-fold greater bumetanide-sensitive Rb(+) influx compared to the control cells. The Balb/c-NKCC1 cells have acquired a typical transformation phenotype indicated by: (1) Loss of contact inhibition exhibited by growth to a higher cell density in confluent cultures, and formation of cell foci; (2) proliferation in low serum concentrations; and (3) formation of cell colonies in soft agar. The control cells transfected with the NKCC1 gene inserted in the opposite orientation in the vector retained their normal phenotype. Furthermore, the two specific inhibitors of the Na(+)/K(+)/Cl(-) cotransporter activity; bumetanide and furosemide inhibited the clonogenic efficiency in the NKCC1 transfected cells. These control experiments indicate that the apparent transformation phenotype acquired by the Balb/c-NKCC1 cells was not merely associated with the process of transfection and selecting for the neomycin-resistant clones, but rather with the overexpression of the Na(+)/K(+)/Cl(-) cotransporter gene. In order to ascertain that the regulated and normal expression of the Na(+)/K(+)/Cl(-) cotransporter control cell proliferation, the effect of bumetanide a specific inhibitor of the cotransporter, was tested on Balb/c 3T3 cell proliferation, induced by fibroblasts growth factor (FGF) and fetal calf serum (FCS). Bumetanide inhibited synchronized Balb/c 3T3 cell exit from the G(0)/G(1) arrest and entering S-phase. The inhibition was reversible, as removal of bumetanide completely released cell proliferation. Taken together, these results propose that the NKCC1 gene is involved in the control of normal cell proliferation, while its overexpression results in apparent cell transformation, in a manner similar to some protooncogenes.     

Proton pump-driven cutaneous chloride uptake in anuran amphibia

Krogh introduced the concept of active ion uptake across surface epithelia of freshwater animals, and proved independent transports of Na(+) and Cl(-) in anuran skin and fish gill. He suggested that the fluxes of Na(+) and Cl(-) involve exchanges with ions of similar charge. In the so-called Krogh model, Cl(-)/HCO(3)(-) and Na(+)/H(+) antiporters are located in the apical membrane of the osmoregulatory epithelium. More recent studies have shown that H(+) excretion in anuran skin is due to a V-ATPase in mitochondria-rich (MR) cells. The pump has been localized by immunostaining and H(+) fluxes estimated by pH-stat titration and mathematical modelling of pH-profiles in the unstirred layer on the external side of the epithelium. H(+) secretion is voltage-dependent, sensitive to carbonic-anhydrase inhibitors, and rheogenic with a charge/ion-flux ratio of unity. Cl(-) uptake from freshwater is saturating, voltage independent, and sensitive to DIDS and carbonic-anhydrase inhibitors. Depending on anuran species and probably on acid/base balance of the animal, apical exit of protons is coupled to an exchange of Cl(-) with base (HCO(3)(-)) either in the apical membrane (gamma-type of MR cell) or in the basolateral membrane (alpha-type MR cell). The gamma-cell model accounts for the rheogenic active uptake of Cl(-) observed in several anuran species. There is indirect evidence also for non-rheogenic active uptake accomplished by a beta-type MR cell with apical base secretion and basolateral proton pumping. Several studies have indicated that the transport modes of MR cells are regulated via ion- and acid/base balance of the animal, but the signalling mechanisms have not been investigated. Estimates of energy consumption by the H(+)-ATPase and the Na(+)/K(+)-ATPase indicate that the gamma-cell accomplishes uptake of NaCl in normal and diluted freshwater. Under common freshwater conditions with serosa-positive or zero V(t), the K(+) conductance of the basolateral membrane would have to maintain the inward driving force for Na(+) uptake across the apical membrane. With the K(+) equilibrium potential across the basolateral membrane estimated to -105 mV, this would apply to external Na(+) concentrations down to 40-120 micromol/l. NaCl uptake from concentrations down to 10 micromol/l, as observed by Krogh, presupposes that the H(+) pump hyperpolarizes the apical membrane, which would then have to be associated with serosa-negative V(t). In diluted freshwater, exchange of cellular HCO(3)(-) with external Cl(-) seems to be possible only if the proton pump has the additional function of keeping the external concentration of HCO(3)(-) low. Quantitative considerations also lead to the conclusion that with the above extreme demand, at physiological intracellular pH of 7.2, the influx of Cl(-) via the apical antiporter and the passive exit of Cl(-) via basolateral channels would be possible within a common range of intracellular Cl(-) concentrations.     

Mechanism of substance P-induced liquid secretion across bronchial epithelium

The present study was undertaken to identify and determine the mechanism of noncholinergic pathways for the induction of liquid secretion across airway epithelium. Excised porcine bronchi secreted substantial and significant quantities of liquid when exposed to acetylcholine, substance P, or forskolin but not to isoproterenol, norepinephrine, or phenylephrine. Bumetanide, an inhibitor of Na(+)-K(+)-2Cl(-) cotransport, reduced the liquid secretion response to substance P by 69%. Approximately two-thirds of bumetanide-insensitive liquid secretion was blocked by dimethylamiloride (DMA), a Na(+)/H(+) exchange inhibitor. Substance P responses were preserved in airways after surface epithelium removal, suggesting that secreted liquid originated from submucosal glands. The anion channel blockers diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) inhibited >90% of substance P-induced liquid secretion, whereas DIDS had no effect. DMA, DPC, and NPPB had greater inhibitory effects on net HCO(3)(-) secretion than on liquid secretion. Although preserved relative to liquid secretion, net HCO(3)(-) secretion was reduced by 39% in the presence of bumetanide. We conclude that substance P induces liquid secretion from bronchial submucosal glands of pigs through active transport of Cl(-) and HCO(3)(-). The pattern of responses to secretion agonists and antagonists suggests that the cystic fibrosis transmembrane conductance regulator mediates this process.     

Recent experiments towards a model for fluid secretion in Rhodnius Upper Malpighian Tubules (UMT)

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na(+) concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na(+)-K(+)-2Cl(-) cotransporter; of intermediate importance are the Na(+)-K(+)-ATPase and a ouabain-insensitive Na(+)-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H(+)-ATPase; and a K(+) and/or Na(+)-H(+) exchanger.     

Potassium-chloride promiscuous channels in mitochondrial membranes

Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and channel currents were measured in 250/50 mmol/l KCl cis/trans solutions. The channel currents measured from -40 to +40 mV had various linear voltage-current relationships and K(+)/Cl(-) permeability ratios at distinct voltage ranges. The channels possessed K(+)-Cl(-) promiscuous property. Depending on voltage, membrane permeability suddenly switched from K(+) over Cl(-) to Cl(-) over K(+) and back. The channels had Cl(-)/K(+) > 1 permeability at potentials around 0 mV and the permeability was switched to K(+)/Cl(-) > 1 at more negative and positive potentials. The chloride channel blocker, 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 5 x 10(-5) mol/l), influenced properties of the promiscuous channels - it activated potassium conductance of the channels.     

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.