Energetics of potassium ion transport in Aplysia gut

Basolateral membranes of Aplysia californica foregut epithelia contain an ATP-dependent Na(+)/K(+) transporter (Na(+)/K(+) pump or Na(+)/K (+) -ATPase). This Na(+)/K(+) pump accounts for both the intracellular Na(+) electrochemical potential (micro) being less than the extracelluar Na(+) micro and the intracellular K(+) micro being more than the extracellular K(+ ) micro. Also, K(+) channel activity resides in both luminal and basolateral membranes of the Aplysia foregut epithelial cells. Increased activity of the Na(+)/K(+) pump, coupled to luminal and basolateral membrane depolarization altered the K(+) transport energetics across the basolateral membrane to a greater extent than the alteration in K(+) transport energetics across the luminal membrane. These results suggest that K(+) transport, either into or out of the Aplysia foregut epithelial cells, is rate-limiting at the basolateral membrane.     

Characterization of salicylate uptake across the basolateral membrane of the malpighian tubules of Drosophila melanogaster

The organic anion salicylate is a plant secondary metabolite that can protect plants against herbivores. Transport of salicylate across the basolateral membrane of the Malpighian tubules of Drosophila melanogaster was studied using a radioisotope tracer technique. The uptake of [(14)C]salicylate by the Malpighian tubules was active, saturable and Na(+)-dependent; the maximum uptake rate (J(max)) and the half saturation concentration (K(t)) were 12.6 pmoltubule(-1)min(-1) and 30.7micromoll(-1), respectively. In contrast to organic anion transport by vertebrate renal tissues, salicylate uptake was not trans-stimulated by glutarate (0.01-1.0 mmoll(-1)) or cis-inhibited by high concentrations (5 mmoll(-1)) of various alpha-keto acids (glutaric acid, alpha-ketoglutaric acid, succinic acid, and citric acid). Changes in basolateral membrane potential or physiologically relevant changes in bathing saline pH did not affect the rate of [(14)C]salicylate uptake. Ring-structure monocarboxylic acids (benzoic acid, nicotinic acid, gentisic acid, unlabelled salicylic acid, alpha-cyano-4-hydroxycinnamic acid, probenecid, fluorescein, and P-aminohippuric acid) strongly inhibited [(14)C]salicylate uptake rate. In contrast, short-chain monocarboxylic acids had little (butyric acid) or no effect (lactic acid, pyruvic acid, and propionic acid). Our results suggest that salicylate uptake across the basolateral membrane of D. melanogaster Malpighian tubules is mediated by a non-electrogenic, alpha-cyano-4-hydroxycinnamic acid-sensitive, Na(+):salicylate cotransport system.     

Basolateral potassium channels of rabbit colon epithelium: role in sodium absorption and chloride secretion

In order to assess the role of different classes of K(+) channels in recirculation of K(+) across the basolateral membrane of rabbit distal colon epithelium, the effects of various K(+) channel inhibitors were tested on the activity of single K(+) channels from the basolateral membrane, on macroscopic basolateral K(+) conductance, and on the rate of Na(+) absorption and Cl(-) secretion. In single-channel measurements using the lipid bilayer reconstitution system, high-conductance (236 pS), Ca(2+)-activated K(+) (BK(Ca)) channels were most frequently detected; the second most abundant channel was a low-conductance K(+) channel (31 pS) that exhibited channel rundown. In addition to Ba(2+) and charybdotoxin (ChTX), the BK(Ca) channels were inhibited by quinidine, verapamil and tetraethylammonium (TEA), the latter only when present on the side of the channel from which K(+) flow originates. Macroscopic basolateral K(+) conductance, determined in amphotericin-permeabilised epithelia, was also markedly reduced by quinidine and verapamil, TEA inhibited only from the lumen side, and serosal ChTX was without effect. The chromanol 293B and the sulphonylurea tolbutamide did not affect BK(Ca) channels and had no or only a small inhibitory effect on macroscopic basolateral K(+) conductance. Transepithelial Na(+) absorption was partly inhibited by Ba(2+), quinidine and verapamil, suggesting that BK(Ca) channels are involved in basolateral recirculation of K(+) during Na(+) absorption in rabbit colon. The BK(Ca) channel inhibitors TEA and ChTX did not reduce Na(+) absorption, probably because TEA does not enter intact cells and ChTX is 'knocked off' its extracellular binding site by K(+) outflow from the cell interior. Transepithelial Cl(-) secretion was inhibited completely by Ba(2+) and 293B, partly by quinidine but not by the other K(+) channel blockers, indicating that the small (<3 pS) K(V)LQT1 channels are responsible for basolateral K(+) exit during Cl(-) secretion. Hence different types of K(+) channels mediate basolateral K(+) exit during transepithelial Na(+) and Cl(-) transport.     

Basolateral membrane K+ channels in renal epithelial cells

The major function of epithelial tissues is to maintain proper ion, solute, and water homeostasis. The tubule of the renal nephron has an amazingly simple structure, lined by epithelial cells, yet the segments (i.e., proximal tubule vs. collecting duct) of the nephron have unique transport functions. The functional differences are because epithelial cells are polarized and thus possess different patterns (distributions) of membrane transport proteins in the apical and basolateral membranes of the cell. K(+) channels play critical roles in normal physiology. Over 90 different genes for K(+) channels have been identified in the human genome. Epithelial K(+) channels can be located within either or both the apical and basolateral membranes of the cell. One of the primary functions of basolateral K(+) channels is to recycle K(+) across the basolateral membrane for proper function of the Na(+)-K(+)-ATPase, among other functions. Mutations of these channels can cause significant disease. The focus of this review is to provide an overview of the basolateral K(+) channels of the nephron, providing potential physiological functions and pathophysiology of these channels, where appropriate. We have taken a "K(+) channel gene family" approach in presenting the representative basolateral K(+) channels of the nephron. The basolateral K(+) channels of the renal epithelia are represented by members of the KCNK, KCNJ, KCNQ, KCNE, and SLO gene families.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

Electrolyte Transport in the Mouse Trachea: No Evidence for a Contribution of Luminal K+ Conductance

Recent studies on frog skin acini have challenged the question whether Cl(-) secretion or Na(+) absorption in the airways is driven by luminal K(+) channels in series to a basolateral K(+) conductance. We examined the possible role of luminal K(+) channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl(-) secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+2)Cl(-)K(+) cotransporter azosemide. Similarly, the compound 293B, a blocker of basolateral KCNQ1/KCNE3 K(+) channels effectively blocked Cl(-) secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K(+) channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K(+) channels in mouse airways, using luminal 293B, clotrimazole and Ba(2+) or different K(+) channel toxins such as charybdotoxin, apamin and a-dendrotoxin. Thus, the present study demonstrates Cl(-) secretion in mouse airways, which depends on basolateral Na(+2)Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl(-) channels. Cl(-) secretion is maintained by the activity of basolateral K(+) channels, while no clear evidence was found for the presence of a luminal K(+) conductance.     

Properties and regulation of organic cation transport in freshly isolated human proximal tubules.

The kidney, and more specifically the proximal tubule, is the main site of elimination of cationic endogenous metabolites and xenobiotics. Although numerous studies exist on renal organic cation transport of rat and rabbit, no information is available from humans. Therefore, we examined organic cation transport and its regulation across the basolateral membrane of isolated human proximal tubules. mRNA for the cation transporters hOCT1 and hOCT2 as well as hOCTN1 and hOCTN2 was detected in these tubules. Organic cation transport across the basolateral membrane of isolated collapsed proximal tubules was recorded with the fluorescent dye 4-(4-dimethylamino)styryl-N-methylpyridinium (ASP(+)). Depolarization of the cells by rising extracellular K(+) concentration to 145 mm reduced ASP(+) uptake by 20 +/- 5% (n = 15), indicating its electrogeneity. The substrates of organic cation transport tetraethylammonium (K(i) = 63 microm) and cimetidine (K(i) = 11 microm) as well as the inhibitor quinine (K(i) = 2.9 microm) reduced ASP(+) uptake concentration dependently. Maximal inhibition reached with these substances was approximately 60%. Stimulation of protein kinase C with 1,2-dioctanoyl-sn-glycerol (DOG, 1 microm) or ATP (100 microm) inhibited ASP(+) uptake by 30 +/- 3 (n = 16) and 38 +/- 13% (n = 6), respectively. The effect of DOG could be reduced with calphostin C (0.1 microm, n = 7). Activation of adenylate cyclase by forskolin (1 microm) decreased ASP(+) uptake by 29 +/- 3% (n = 10). hANP (10 nm) or 8-bromo-cGMP (100 microm) also decreased ASP(+) uptake by 17 +/- 3 (n = 9) or 32 +/- 5% (n = 10), respectively. We show for the first time that organic cation transport across the basolateral membrane of isolated human proximal tubules, most likely mediated via hOCT2, is electrogenic and regulated by protein kinase C, the cAMP- and the cGMP-dependent protein kinases.     

Evidence for the presence of GPRC6A receptors in rat mesenteric arteries

In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.     

MAGI-1a functions as a scaffolding protein for the distal renal tubular basolateral K+ channels

As the K(+) recycling pathway for renal Na(+) reabsorption, renal tubular K(+) channels participate in the fluid and electrolyte homeostasis. Previously, we showed that the Kir5.1/Kir4.1 heteromer, which is a heteromeric assembly of two inwardly rectifying K(+) channels, composes the principal basolateral K(+) channels in distal renal tubules and that two motifs in the carboxyl-terminal portion of the Kir4.1 subunit regulate its functional expression. In this study, by using yeast two-hybrid screening, we identified a new isoform of membrane-associated guanylate kinase with inverted domain structure 1 (MAGI-1a-long) as a scaffolding protein for the basolateral K(+) channels. MAGI-1a-long interacted with the PSD-95/Dlg/ZO-1 (PDZ)-binding motif of Kir4.1 by its fifth PDZ domain, and a high salt diet, which could suppress mineralocorticoid secretion, facilitated the interaction. The phosphorylation of serine 377 in the PDZ-binding motif disrupted the interaction, and the disruption of the interaction altered the intracellular localization of the channels from the basolateral side to perinuclear components. These results demonstrate that the phosphorylation-dependent scaffolding of the basolateral K(+) channels by MAGI-1a-long participates in the renal regulation of the fluid and electrolyte homeostasis.     

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.     

Potassium channels in primary cultures of seawater fish gill cells. II. Channel activation by hypotonic shock

Previous studies performed on apical membranes of seawater fish gills in primary culture have demonstrated the existence of stretch-activated K(+) channels with a conductance of 122 pS. The present report examines the involvement of K(+) channels in ion transport mechanisms and cell swelling. In the whole cell patch-clamp configuration, K(+) currents were produced by exposing cells to a hypotonic solution or to 1 microM ionomycin. These K(+) currents were inhibited by the addition of quinidine and charybdotoxin to the bath solution. Isotopic efflux measurements were performed on cells grown on permeable supports using (86)Rb(+) as a tracer to indicate potassium movements. Apical and basolateral membrane (86)Rb effluxes were stimulated by the exposure of cells to a hypotonic medium. During the hypotonic shock, the stimulation of (86)Rb efflux on the apical side of the monolayer was inhibited by 500 microM quinidine or 100 microM gadolinium but was insensitive to scorpion venom [Leirus quinquestriatus hebraeus (LQH)]. An increased (86)Rb efflux across the basolateral membrane was also reduced by the addition of quinidine and LQH venom but was not modified by gadolinium. Moreover, basolateral and apical membrane (86)Rb effluxes were not modified by bumetanide or thapsigargin. There is convincing evidence for two different populations of K(+) channels activated by hypotonic shock. These populations can be separated according to their cellular localization (apical or basolateral membrane) and as a function of their kinetic behavior and pharmacology.     

Recovery from blood alkalosis in the Pacific hagfish (Eptatretus stoutii): involvement of gill V-H+-ATPase and Na+/K+-ATPase

To investigate the base secretory mechanisms in the Pacific hagfish (Eptatretus stoutii), we injected animals with NaHCO3 into the subcutaneous sinus. In the first series of experiments, hagfish were injected with 6000 micromol kg(-1) NaHCO3 (base-infused hagfish, BIH) or NaCl (controls). Blood pH increased significantly 1 h after injection in BIH (8.05+/-0.05 vs. 7.82+/-0.03 pH units), but returned to control values by t=6 h. Plasma total CO2 (TCO2) followed the same pattern. Immunolabeled sections revealed that Na+/K+-ATPase and V-H+-ATPase were usually located in the same cells. Western blotting revealed that the abundance of both proteins remained unchanged in whole gill homogenates and in a fraction enriched in cell membranes 6 h after the injections. The second experimental series was to induce long-term alkalosis by serially injecting 6000 micromol kg(-1) NaHCO3 every 6 h for 24 h. Blood pH completely recovered from the base loads within 6 h after each injection. Moreover, plasma TCO2 was not elevated 3 h after the second infusion, suggesting that HCO3(-) secreting mechanisms had been upregulated by that time. Na+/K+-ATPase and V-H+-ATPase cellular localizations did not change in the 24 h base infusion protocol. Na+/K+-ATPase abundance was similar in gill homogenates from fish from both treatments. However, Na+/K+-ATPase abundance in the membrane fraction was significantly lower in BIH, while V-H+-ATPase was greater both in whole gill and membrane fractions. Our results suggest that differential insertion of V-H+-ATPase and Na+/K+-ATPase into the basolateral membrane is involved in recovering from alkalotic stress in hagfish.     

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.     

Characteristics of the K(+)-ion transport in the rat intestine following the use of natural zeolites as food additives

Effects of zeolites as a food supplement have been studied on Wistar rats both in vivo perfusion experiments in the jejunum and distal colon and Rb fluxes through intestinal wall in the Ussing chamber. It has been found that zeolites decrease the K+ absorption and stimulate K+ secretion in the gut. This effect was due to inhibition of the apical N(+)-K(+)-ATPase and ouabain-sensitive Na(+)-independent K(+)-ATPase as well as the activation of the basolateral N(+)-K(+)-ATPase.     

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.     

The small conductance K+ channel, KCNQ1: expression, function, and subunit composition in murine trachea

The gene KCNQ1 encodes a K(+) channel alpha-subunit important for cardiac repolarization, formerly known as K(v)LQT1. In large and small intestine a channel complex consisting of KCNQ1 and the beta-subunit KCNE3 (MiRP2) is known to mediate the cAMP-activated basolateral K(+) current, which is essential for luminal Cl(-) secretion. Northern blot experiments revealed an expression of both subunits in lung tissue. However, previous reports suggested a role of KCNE1 (minK, Isk) but not KCNE3 in airway epithelial cells. Here we give evidence that KCNE1 is not detected in murine tracheal epithelial cells and that Cl(-) secretion by these cells is not reduced by the knock-out of the KCNE1 gene. In contrast we show that a complex consisting of KCNQ1 and KCNE3 probably forms a basolateral K(+) channel in murine tracheal epithelial cells. As described for colonic epithelium, the current through KCNQ1 complexes in murine trachea is specifically inhibited by the chromanol 293B. A 293B-sensitive current was present after stimulation with forskolin and agonists that increase Ca(2+) as well as after administration of the pharmacological K(+) channel activator, 1-EBIO. A 293B-inhibitable current was already present under control conditions and reduced after administration of amiloride indicating a role of this K(+) channel not only for Cl(-) secretion but also for Na(+) reabsorption. We conclude that at least in mice a KCNQ1 channel complex seems to be the dominant basolateral K(+) conductance in tracheal epithelial cells.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

Chlorzoxazone or 1-EBIO increases Na(+) absorption across cystic fibrosis airway epithelial cells

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl(-) secretion by increasing basolateral K(+) conductance (G(K)) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778-787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across Delta F508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na(+) absorption. The addition of chlorzoxazone (400 microM) or 1-EBIO (2 mM) increased the amiloride-sensitive I(sc) approximately 2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G(K) with a K(+) gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an approximately 10-fold increase in clotrimazole-sensitive G(K). In contrast, chlorzoxazone did not alter epithelial Na(+) channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G(K) alone can modulate epithelial Na(+) transport.     

Role of inwardly rectifying K(+) channels in K(+)-induced cerebral vasodilatation in vivo

We tested whether activation of inwardly rectifying K(+) (Kir) channels, Na(+)-K(+)-ATPase, or nitric oxide synthase (NOS) play a role in K(+)-induced dilatation of the rat basilar artery in vivo. When cerebrospinal fluid [K(+)] was elevated from 3 to 5, 10, 15, 20, and 30 mM, a reproducible concentration-dependent vasodilator response was elicited (change in diameter = 9 +/- 1, 27 +/- 4, 35 +/- 4, 43 +/- 12, and 47 +/- 16%, respectively). Responses to K(+) were inhibited by approximately 50% by the Kir channel inhibitor BaCl(2) (30 and 100 microM). In contrast, neither ouabain (1-100 microM, a Na(+)-K(+)-ATPase inhibitor) nor N(G)-nitro-L-arginine (30 microM, a NOS inhibitor) had any effect on K(+)-induced vasodilatation. These concentrations of K(+) also hyperpolarized smooth muscle in isolated segments of basilar artery, and these hyperpolarizations were virtually abolished by 30 microM BaCl(2). RT-PCR experiments confirmed the presence of mRNA for Kir2.1 in the basilar artery. Thus K(+)-induced dilatation of the basilar artery in vivo appears to partly involve hyperpolarization mediated by Kir channel activity and possibly another mechanism that does not involve hyperpolarization, activation of Na(+)-K(+)-ATPase, or NOS.     

Quantitative immunogold localization of Na+,K(+)-ATPase alpha-subunit in the tympanic wall of rat cochlear duct

Ultrastructural localization of Na+,K(+)-ATPase was quantitatively investigated in the tympanic wall of rat cochlear duct by use of the protein A-gold method, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. A moderate number of gold particles were found on the basolateral membrane of the interdental cells of the spiral limbus. A small number of gold particles were found on the basolateral surfaces of the border cells and Hensen's cells. On the inner and outer sensory hair cells, however, the plasma membranes were rarely labeled by gold particles. The general pattern of labeling densities in cochlear structures determined here and in a previous communication from our laboratory shows good correlation with the distribution of Na+,K(+)-ATPase activity as previously estimated biochemically, cytochemically, and autoradiographically.