Christian Lytle

Immunohistochemical Localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear:

We mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.

Activation of the Avian Erythrocyte Na-K-Cl Cotransport Protein by Cell Shrinkage, cAMP, Fluoride, and Calyculin-A Involves Phosphorylation at Common Sites

Na-K-Cl cotransport activity in duck erythrocytes increases ∼10-fold in response to osmotic cell shrinkage, norepinephrine, fluoride, or calyculin-A (an inhibitor of type-1 and -2a phosphatases). To assess whether all four stimuli promote phosphorylation of the cotransport protein and whether this phosphorylation is catalyzed by the same kinase, the cotransporter was isolated from erythrocytes by immunoprecipitation and its pattern of phosphorylation was evaluated. Each stimulus evoked proportionate increases in cotransporter activity and phosphorylation. No two stimuli in combination evoked greater activation and phosphorylation than did the more potent of the two stimuli acting alone. Phosphoamino acid analysis of the cotransport protein indicated that phosphorylation occurs at serine and threonine residues. Phosphopeptide mapping revealed a distinctive pattern of 8 major tryptic phosphopeptides, none of which were significantly phosphorylated in the unstimulated state. Maps of cotransporters activated by the four different stimuli were indistinguishable. Measurements of phosphorylation stoichiometry indicated that each cotransporter acquires ∼5 phosphates on going from an inactive state in swollen cells to an active state in shrunken cells. Staurosporine, a kinase inhibitor with broad selectivity, inhibited each stimulus equipotently (IC50 ∼ 0.7 μm). Staurosporine promptly reversed cotransporter activity and phosphorylation when added to shrinkage-stimulated but not to calyculin-stimulated cells, indicating that it enters the cell rapidly and blocks phosphorylation. These results suggest that cell shrinkage, cAMP, fluoride, and calyculin-A promote the phosphorylation of the Na-K-Cl cotransport protein at a similar constellation of serine and threonine residues. It is proposed that all modes of stimulation ultimately involve the same protein kinase.

[Cl−]i-dependent Phosphorylation of the Na-K-Cl Cotransport Protein of Dog Tracheal Epithelial Cells

Basolateral Na-K-Cl cotransport activity in primary cultures of dog tracheal epithelial cells is stimulated by β-adrenergic agents, such as isoproterenol, and by apical UTP, which acts through an apical P2-purinergic receptor. While at least part of the stimulatory effect of isoproterenol appears to involve direct activation of the cotransporter via cAMP-dependent protein kinase, cotransport stimulation by apical UTP is entirely secondary to apical Cl− efflux and a resultant decrease in intracellular [Cl−] ([Cl−]i) and/or cell shrinkage (Haas, M., and McBrayer, D. G.(1994) Am. J. Physiol. 266, C1440-C1452). In the secretory epithelia of the shark rectal gland and avian salt gland, Na-K-Cl cotransport activation by both cAMP-dependent and cAMP-independent secretagogues has been shown to be accompanied by phosphorylation of the cotransport protein itself (Lytle, C., and Forbush, B., III(1992) J. Biol. Chem. 267, 25438-25443; Torchia, J., Lytle, C., Pon, D. J., Forbush, B., III, and Sen, A. K.(1992) J. Biol. Chem. 267, 25444-25450). In the present study, we immunoprecipitate the ∼170-kDa Na-K-Cl cotransport protein of dog tracheal epithelial cells with a monoclonal antibody against the cotransporter of the intestinal cell line T84. Incubation of confluent primary cultures of tracheal cells with isoproterenol and apical UTP increases basolateral-to-apical 36Cl− flux 3.4- and 2.6-fold, respectively, and produces similar increases (3.2- and 2.8-fold, respectively) in 32P incorporation into the ∼170-kDa cotransport protein. Decreasing [Cl−]i (without concomitant cell shrinkage) by incubating cultures with apical nystatin and reduced apical [Cl−] ([Cl−]a) likewise increases both cotransport activity and cotransport protein phosphorylation. These effects become more pronounced with greater reductions in [Cl−]a; after 20 min of incubation with nystatin and 32 mM [Cl−]a, cotransport activity and 32P incorporation into the cotransport protein are increased 2.8- and 2.7-fold, respectively, similar to increases seen with apical UTP. 2-3-fold increases in cotransporter activity and phosphorylation are also seen in nystatin-treated cells under hypertonic conditions (50 mM sucrose added apically and basolaterally). These findings suggest a close correlation between Na-K-Cl cotransport activity and phosphorylation of the ∼170-kDa cotransport protein. The latter is phosphorylated in response to both reduced [Cl−]i and cell shrinkage, either or both of which are likely to be involved in secondary cotransport activation in response to apical UTP.

The peroxisome proliferator-activated receptor γ ligand rosiglitazone delays the onset of inflammatory bowel disease in mice with interleukin 10 deficiency

Aims: To test whether the peroxisome proliferator‐activated receptor &ggr; (PPAR&ggr;) ligand rosiglitazone (Ro) has therapeutic activity in the IL‐10−/− mouse model of inflammatory bowel disease (IBD), and to identify the cellular targets and molecular mechanisms of Ro action. Methods: The progression of spontaneous chronic colitis in IL‐10−/− mice was compared in 5‐week‐old mice fed a standard diet with or without Ro for 12 weeks. The possible therapeutic effect of Ro was also tested over a 6‐week interval in older IL‐10−/− mice with established IBD. Results: Treatment with Ro slowed the onset of spontaneous IBD in IL‐10−/− mice. Crypt hyperplasia, caused by increased mitotic activity of crypt epithelial cells, was also delayed by Ro. Treatment with Ro significantly decreased expression of interferon &ggr; (IFN&ggr;), interleukin 17 (IL‐17), tumor necrosis factor &agr;, and the inducible nitric oxide synthase mRNA in the colon, whereas expression of IL‐12p40 was unchanged. PPAR&ggr; was detected in epithelial cells throughout the crypts and surface. Ro increased expression of PPAR&ggr; protein in these cells, suggesting the existence of a positive feedback loop that would potentiate its action in these cells. Ro also specifically increased expression of a novel PPAR target, aquaporin‐8 (AQP8), in differentiated colonic epithelial surface cells, demonstrating that PPAR&ggr; is not only present but also regulates gene expression in these cells in vivo. Finally, Ro was ineffective in improving disease activity in older IL‐10−/− mice with established IBD. Conclusions: PPAR&ggr; is expressed, and the PPAR&ggr; ligand Ro regulates gene expression in colonic epithelial cells. As a single agent, Ro works best for disease prevention in the IL‐10−/− mouse model for IBD.

Na:K:2Cl Cotransporter (NKCC) of Intestinal Epithelial Cells SURFACE EXPRESSION IN RESPONSE TO cAMP

During intestinal chloride secretion, epithelial uptake of salts is accomplished largely by a bumetanide-sensitive Na:K:2Cl cotransporter designated here as NKCC. Using monoclonal antibodies directed against NKCC from the human crypt epithelial cell line, T84, we define its surface localization as a function of cotransporter activation. Immunoelectron microscopy, confocal localization, and selective surface biotinylation studies revealed that the 195-kDa NKCC protein is polarized to the basolateral domain. Following immunoprecipitation, several polypeptides coprecipitated with the 195-kDa cotransporter including two prominent proteins of molecular mass 160 and 130 kDa. Immunoblotting with three distinct anti-NKCC monoclonal antibodies in conjunction with deglycosylation experiments suggested that the 160- and 130-kDa bands represented novel proteins unrelated to the cotransporter. Stimulation of T84 monolayers with cAMP agonists, a condition which elicits chloride secretion and leads to microfilament-dependent NKCC activation, did not significantly increase the number of bumetanide-binding sites and only marginally increased surface expression of the 195-kDa cotransporter available for surface biotinylation. In contrast, cAMP agonist stimulation increased the surface expression of the coprecipitating 160- and 130-kDa proteins ∼6-fold. The increase in surface 160- and 130-kDa proteins was attenuated by phalloidin preloading the cells, a condition which also prevents activation of NKCC without influencing the activity of other membrane transporters participating in chloride secretion. These studies define the polarized distribution of the NKCC protein on intestinal epithelia, indicate that NKCC may be associated with two other previously unidentified membrane proteins and such association is influenced by the F-actin cytoskeleton.

Na-K-Cl cotransporter expression in the developing and senescent gerbil cochlea

Changes in the cellular expression pattern of the Na-K-Cl cotransporter (NKCC) were investigated during postnatal development and with advancing age in the gerbil cochlea. At birth, faint immunostaining for NKCC was discernable in the developing stria vascularis (StV), Reissners membrane, interdental cells and some relatively undifferentiated cells lining the cochlear partition. Between 2 and 4 days after birth (DAB) immunostaining persisted and increased in the future interdental, inner and outer sulcus and claudius cells but then disappeared from these sites by 8 DAB. In contrast, NKCC immunoreactivity in the StV increased progressively during development and approached adult levels by 12 DAB. Immunostaining for NKCC in subpopulations of fibrocytes in the inferior portion of the spiral ligament, the suprastrial region and the spiral limbus was first detectable between 10 and 12 DAB and staining intensity reached adult levels around 16 DAB. Changes in NKCC expression with advancing age generally mimicked those previously observed for Na,K-ATPase in focal regions of atrophic lateral wall. Diminished immunostaining was first seen in the StV, presumably associated with the involution of the marginal cells basolateral processes. Further atrophy culminated in complete loss of immunostaining in the StV and an associated down-regulation of NKCC expression in spiral ligament transport fibrocytes. The marked similarities in the developmental and age-related expression patterns of NKCC and Na,K-ATPase point to a high level of functional cooperativity between these two ion transport mediators, which together provide an efficient mechanism for generating and maintaining high K+ levels in endolymph and the endocochlear potential.

A model of Na-K-2Cl cotransport based on ordered ion binding and glide symmetry

In the duck red blood cell, Na-K-2Cl cotransport exhibits two modes of ion movement: net cotransport and obligate cation exchange. In high-K cells, the predominant exchange is K/K (or K/Rb). In high-Na cells, it becomes Na/Na (or Na/Li). Both represent partial reactions in which a fully loaded carrier releases part of its cargo, rebinds fresh ions, and returns back across the membrane fully loaded. Net cotransport occurs when the carrier unloads completely and returns empty. This mode has a fixed stoichiometry of 1Na:1K:2Cl under all conditions tested. The ion requirements of the two exchanges differ: K/K exchange requires only K and Cl outside but all three ions inside. Na/Na exchange requires all three ions outside but only Na inside. We propose a simple model in which the carrier can only move when either fully loaded or completely empty and in which the ions bind in a strictly ordered sequence. For example, externally, a Na binds first and then a Cl, followed by a K and a second Cl. Internally, the first on is the first off (glide symmetry), so the Na is released first and then the first Cl, followed by the K and finally by the second Cl. Only then can the empty form return to the outside to start a new cycle.

A volume-sensitive protein kinase regulates the Na-K-2Cl cotransporter in duck red blood cells

When Na-K-2Cl cotransport is activated in duck red blood cells by either osmotic cell shrinkage, norepinephrine, fluoride, or calyculin A, phosphorylation of the transporter occurs at a common set of serine/threonine sites. To examine the kinetics and regulation of the activating kinase, phosphatase activity was inhibited abruptly with calyculin A and the subsequent changes in transporter phosphorylation and activity were determined. Increases in fractional incorporation of 32P into the transporter and uptake of 86Rb by the cells were closely correlated, suggesting that the phosphorylation event is rate determining in the activation process. Observed in this manner, the activating kinase was 1) stimulated by cell shrinkage, 2) inhibited by cell swelling, staurosporine, or N-ethylmaleimide, and 3) unaffected by norepinephrine or fluoride. The inhibitory effect of swelling on kinase activity was progressively relieved by calyculin A, suggesting that the kinase itself is switched on by phosphorylation. The kinetics of activation by calyculin A conformed to an autocatalytic model in which the volume-sensitive kinase is stimulated by a product of its own reaction (e.g., via autophosphorylation).

Segregation of Na/H exchanger-3 and Cl/HCO3 exchanger SLC26A3 (DRA) in rodent cecum and colon

The colon is believed to absorb NaCl via the coupled operation of apical Na/H exchanger-3 (NHE3) and Cl/HCO(3) exchanger SLC26A3 (DRA). Efficient coupling requires that NHE3 and DRA operate in close proximity within common luminal and cytosolic microenvironments. Thus we examined whether these proteins coexist along the apical margin of surface enterocytes by quantitative immunofluorescence microscopy in consecutive colon segments from nonfasted mice and rats. The cecocolonic profiles of NHE3 and DRA expression were roughly inverse; NHE3 was highest in proximal colon (PC) and negligible in distal colon, whereas DRA was absent in early PC and highest in the late midcolon, and DRA was prominent in the cecum whereas NHE3 was not. NHE3 and DRA coexisted only in the middle third of the colon. The consequences of unpaired NHE3/DRA expression on mucosal surface (subscript MS) pH and Na(+) concentration ([Na(+)]) were assessed in nonfasted rats in situ using miniature electrodes. In the cecum, where only DRA is expressed, pH(MS) was approximately 7.5, markedly higher than underlaying stool (6.3), consistent with net HCO(3)(-) secretion. In the early PC, where NHE3 is not expressed with DRA, pH(MS) was acidic (6.2), consistent with unopposed H(+) secretion. [Na(+)](MS) was approximately 60 mM in the cecum, decreased along the PC to approximately 20 mM, and declined further to approximately 10 mM distally. Cl(-) was secreted into the PC, then reabsorbed distally. Our results suggest a model in which 1) unpaired DRA activity in the cecum maintains an alkaline mucosal surface that could neutralize fermentative H(+); 2) unpaired NHE3 activity in the early PC preserves an acidic mucosal surface that could energize short-chain fatty acid absorption; and 3) coupled NHE3/DRA activities in the midcolon allow for vigorous NaCl absorption at a neutral pH(MS).

Intranasal M Cell Uptake of Nanoparticles Is Independently Influenced by Targeting Ligands and Buffer Ionic Strength

In mucosal tissues, epithelial M cells capture and transport microbes across the barrier to underlying immune cells. Previous studies suggested that high affinity ligands targeting M cells may be used to deliver mucosal vaccines; here, we show that particle composition and dispersion buffer ionic strength can independently influence their uptake in vivo. First, addition of a poloxamer 188 to nanoparticle formulations increased uptake of intranasally administered nanoparticles in vivo, but the effect was dependent on the presence of the M cell-targeting ligand. Second, solvent ionic strength is known to effect electrostatic interactions; accordingly, reduced ionic strength increased the electrostatic potential between the epithelium and the particles. Interestingly, below a critical ionic strength, intranasal particle uptake in vivo significantly was increased even when controlled for osmolarity. Similar results were obtained for uptake of bacterial particles. Surprisingly, at low ionic strength, the specific enhancement effect by the targeting peptide was negligible. Modeling of the electrostatic forces predicted that the enhancing effects of the M cell-targeting ligand only are enabled at high ionic strength, as particle electrostatic forces are reduced through Debye screening. Thus, electrostatic forces can have a dramatic effect on the in vivo M cell particle uptake independent of the action of targeting ligands. Examination of these forces will be helpful to optimizing mucosal vaccine and drug delivery.