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C Lytle

Publications and source records attributed to C Lytle.

At least 19 recordsLinked to original sources
PubMed2001

Immunolocalization of the Na-K-Cl cotransporter in bovine ciliary epithelium.

PURPOSE: Recent evidence suggests that Na-K-Cl cotransport plays a major role in blood-to-aqueous anion transport across the ciliary body epithelium. The present study was undertaken to determine the location of the Na-K-Cl cotransporter in fixed sections of bovine eye. METHODS: Sections of paraformaldehyde-fixed adult and calf bovine eyes were treated with a monoclonal antibody to mammalian Na-K-Cl cotransporter and a fluorescent secondary antibody and examined under a fluorescent microscope. Na-K-Cl cotransporter protein was detected on immunoblots of dissected tissue and purified nonpigmented ciliary epithelial (NPE) and pigmented ciliary epithelial (PE) cells. RESULTS: Cotransporter immunofluorescence was most intense along the basolateral surfaces of the PE cells. Anterior pars plicata possessed the greatest PE immunofluorescence, and this diminished posteriorly toward the pars plana. Quantitation of immunofluorescence images indicated 7- to 10-fold more cotransporter protein in pars plicata PE than in pars plana PE. Diffuse cytoplasmic fluorescence was seen in the NPE cells, which was also brightest in anterior pars plicata. Immunoblots of separated PE and NPE cells from anterior pars plicata showed that PE contain four times more 170-kDa cotransporter protein than NPE. This confirmed fluorescence quantitation estimates. Cotransporter was barely detectable in cells isolated from pars plana in either cell layer. Immunoblots of the Na,K-ATPase catalytic (alpha) subunit in separated NPE and PE cells showed that NPE cells possessed approximately eight times more alpha subunit protein than PE. Immunofluorescence indicated a similar distribution of alpha subunits and indicated a basolateral membrane location for the subunit on both cell types. Na-K-Cl cotransporter fluorescence patterns showed more variability in adult animals than in calves, which may be related to aging and/or disease. Distinctive patterns of cotransporter fluorescence were also seen in the cornea, iris, and retina. CONCLUSIONS: Localization of the Na-K-Cl cotransporter to the plasma membrane on the blood side of the ciliary epithelium tight junctions supports a role for the Na-K-Cl cotransporter in ciliary epithelium as a chloride entry mechanism involved in blood-to-aqueous chloride transport. The concentration of Na,K-ATPase catalytic subunits on NPE basolateral membranes could provide net Na(+) efflux into the aqueous humor.

Animals↗
PubMed2000

Identification of two new translocations that disrupt the AML1 gene.

The AML1 gene, located at chromosome 21q22, encodes a component (CBFalpha2) of a heterodimeric transcription factor complex termed core binding factor (CBF), which binds to DNA and activates gene expression. Chromosomal rearrangements may lead to disruption of this gene and development of acute leukemia. Twelve AML1 translocations have been identified to date, and include sites on chromosomes 1, 2, 3, 5, 8, 12, 14, 15, 16, 17, 18, and 19. Here we report two new translocations involving AML1 in acute myeloid leukemia, in which the disruption of the AML1 gene was documented by GTG banding cytogenetic studies and metaphase and interphase FISH analysis. These chromosomal breakpoints identified as harboring new fusion partners for AML1 are at 2p11.2 and 20q13.1. The two patients in who these translocation were identified were elderly males with newly diagnosed AML. These patients shared the same poor outcomes reported for other rare AML1 translocations.

Acute Disease↗
PubMed2000

Prenatal diagnosis of inverted duplicated 8p.

The phenotype of inverted duplicated 8p, region 8p11.2-p23, reported in children and adults, includes: severe mental retardation, minor facial anomalies, agenesis of corpus callosum, and other malformations including those of heart and kidneys. We report on the prenatal diagnosis of 2 cases of inverted duplication 8p. Both cases were ascertained by abnormal level 2 ultrasound findings. Case 1 presented at 16.5 weeks of gestation with massive distention of the fetal bladder, bilateral hydronephrosis, abnormality of the lower lumbar spine, absence of the sacral spine and a Dandy-Walker variant (interhemispheric cyst and enlarged third ventricle). Case 2 presented at 30 weeks of gestation with agenesis of corpus callosum, slightly enlarged lateral ventricles, interhemispheric cyst and enlarged third ventricle, and possible coarctation of the aorta. The intracranial and cardiac anomalies were confirmed and further defined after delivery. Cytogenetic analysis in both cases showed additional material on 8p. In both cases, fluorescence in situ hybridization (FISH) defined the abnormal chromosome, as a pseudodicentric chromosome with duplication of the short arm from centromere to p23 and deletion from p23 to pter. Our findings support those of prior reports of the inverted duplicated 8p chromosome with multiple anomalies and add prenatal findings to our knowledge.

Adult↗
PubMed2000

p21-activated protein kinase gamma-PAK is translocated and activated in response to hyperosmolarity. Implication of Cdc42 and phosphoinositide 3-kinase in a two-step mechanism for gamma-PAK activation.

A member of the family of p21-activated protein kinases, gamma-PAK, has cytostatic properties and is activated during apoptosis and in response to DNA damage. To determine whether gamma-PAK is activated by other types of cell stress and to assess its mechanism of activation, the response of gamma-PAK to hyperosmotic stress was examined. In 3T3-L1 mouse fibroblasts, there are two pools of gamma-PAK: the majority of the protein kinase is soluble and has low specific activity, whereas gamma-PAK associated with the particulate fraction has significantly higher specific activity. Hyperosmolarity promotes translocation of gamma-PAK from the soluble to the particulate fraction; this parallels activation of the protein kinase. Activation but not translocation of gamma-PAK is wortmannin-sensitive, suggesting the involvement of a phosphoinositide 3-kinase-related activity. gamma-PAK translocation in response to hyperosmolarity parallels Cdc42 translocation to the particulate fraction in vivo and can be induced in vitro by guanosine 5'-3-O-(thio)triphosphate. Cotransfection of gamma-PAK with constitutively active Cdc42 induces gamma-PAK activation and translocation, whereas inactive Cdc42 inhibits both processes in response to hyperosmotic stress, suggesting that Cdc42 has a role in the translocation and activation of gamma-PAK. alpha-PAK is not activated in response to hyperosmolarity in 3T3-L1 cells. A two-step model of gamma-PAK activation is presented.

3T3 Cells↗
PubMed1999

A group of previously not recognized cytogenetic abnormalities in myeloid hematological malignancies.

We have identified a group of previously not reported chromosome abnormalities related to myeloid hematological malignancies. Cases 1 and 2 were observed to have an additional i(4)(p10) as the sole anomaly with similar clinical features of myeloid disorders; that is, acute nonlymphocytic leukemia (ANLL-M2) and myelodysplastic syndrome (MDS)-refractory anemia with an excess of blasts in transformation, respectively. Fluorescence in situ hybridization studies with the use of a 4p-specific microdissection probe further confirmed the presence of an i(4)(p10) in these patients. Case 3 was diagnosed with ANLL-M1 and had an additional i(8)(p10) as the only change, also confirmed by a whole-chromosome painting procedure. In cases 4-6, deletions of 18q at breakpoints q12, q23, and q21 were identified as the sole anomaly in a myeloproliferative disorder (MPD), MPD, and MDS, respectively. X-autosome translocations other than t(X;10)(p11;p11) and t(X;11)(q13;q23) have not been reported as recurrent or primary changes in hematological disorders. In the present study, a t(X;9)(q26;q22) and t(X;5)(q13;q33) as the sole anomaly were found in cases 7 and 8, respectively. Both cases had the same diagnosis of MDS. Considering that trisomies 4 (+4) and 8 (+8) are common anomalies in MDS and ANLL, our findings strongly indicate that amplification of genes on 4p and 8p, but not on 4q and 8q, may play a crucial role in the pathogenesis of MDS and ANLL. In addition, genes on 18q12-23 and on Xq13-26 may be involved in the pathogenesis of myeloid disorders.

Adult↗
PubMed1999

Parietal cells express high levels of Na-K-2Cl cotransporter on migrating into the gastric gland neck.

Na-K-2Cl cotransport and Cl/HCO3 exchange are prominent mechanisms for Cl- uptake in Cl--secreting epithelial cells. We used immunofluorescence microscopy to delineate the distributions of Na-K-2Cl cotransporter-1 (NKCC1) and anion exchanger-2 (AE2) proteins in rat gastric mucosa (zymogenic zone). Parietal cells (PCs) above the neck of the gastric gland contained abundant AE2 but little or no NKCC1, whereas those in the neck and base contained high NKCC1 but diminished AE2. Lower levels of NKCC1 were detected in surface mucous cells and in cells comprising the blind ends of all glands. Pulse labeling of proliferating cells with bromodeoxyuridine indicated that new PCs originate in the isthmus with scant NKCC1; the subset of PCs that migrate downward expresses NKCC1 abruptly on entering the neck, within 7 days of cell division. Our results suggest that downwardly migrating PCs replace one mechanism for Cl- entry (Cl/HCO3 exchange) with another (Na-K-2Cl cotransport).

Animals↗
PubMed1998

The alpha4 subunit of rat alpha4beta2 nicotinic receptors is phosphorylated in vivo.

The intracellular domains of the alpha4 and beta2 neuronal nicotinic subunits between transmembrane segments 3 and 4 contain a number of predicted phosphorylation sites but there is no direct evidence that any of these sites are actually phosphorylated in vivo. We expressed rat alpha4beta2 nicotinic receptors in Xenopus oocytes, labeled them by an overnight incubation in [32P]orthophosphate, and analyzed the immunoprecipitated receptors by autoradiography and Western blotting. Our results show that the oocytes contained three kinds of alpha4 subunits with apparent weights of 69, 79, and 89 kDa. The 89 kDa alpha4 subunit was the most heavily phosphorylated.

Amino Acid Sequence↗
PubMed1998

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), Reissner's 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 cell's 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.

Aging↗
PubMed1998

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.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1998

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).

Animals↗
PubMed1997

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 approximately 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 approximately 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 approximately 0.7 microM). 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.

Animals↗
PubMed1997

IL-6 secreted by astroglial cells regulates Na-K-Cl cotransport in brain microvessel endothelial cells.

We showed previously that cerebral microvessel endothelial cells (CMEC) exhibit a prominent Na-K-Cl cotransporter that functions to regulate intracellular volume and may also mediate vectorial ion transport across the blood-brain barrier (BBB). Astrocytes and their conditioned media induce BBB properties of the endothelium. Our previous studies demonstrated that exposure of CMEC to astroglial cells markedly increases cotransport activity, upregulates cotransporter protein expression, and also increases cotransporter protein phosphorylation. In the present study, we evaluated the possibility that astroglial effects on the Na-K-Cl cotransporter are mediated by astroglial cell-secreted interleukin-6 (IL-6). Cotransporter activity was assessed as bumetanide-sensitive K influx, and protein expression was evaluated by Western blot analysis. Exposing CMEC to IL-6 for 48 h caused a dose-dependent stimulation of Na-K-Cl cotransport activity. Both C6 glial cell-conditioned medium (C6CM)-induced and IL-6-induced increases in cotransport activity were neutralized by anti-IL-6 antibodies. A 48-h exposure of cells to IL-6 or C6CM also resulted in increased cotransporter protein expression. Furthermore, using an enzyme-linked immunosorbent assay for IL-6, we found significant amounts of IL-6 in C6CM. These data suggest that astroglia-secreted IL-6 may mediate the observed astroglial cell effects on CMEC Na-K-Cl cotransport and further support the hypothesis that astrocytes participate in maintenance of cerebral ionic homeostasis by regulating Na-K-Cl cotransporter function at the BBB.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

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 approximately 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.

Carrier Proteins↗
PubMed1996

Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl.

The effect of cytoplasmic Cl concentration ([Cl]i) on the activation state ([3H]benzmetanide binding rate) and phosphorylation state (32P incorporation) of the Na-K-Cl cotransporter was evaluated in secretory tubules isolated from the dogfish shark rectal gland. Reduction of [Cl]i at relatively constant cell volume (by removal of extracellular Cl or Na or by addition of bumetanide) increased cotransporter activation and phosphorylation. Raising extracellular K concentration ([K]o) from 4 to 80 mM, a maneuver that elevated [Cl]i above normal, reduced basal cotransport activity and rendered it entirely refractory to forskolin. High [K]o also blocked activation and phosphorylation in response to cell shrinkage, despite the fact that [Cl]i was already greatly elevated as a consequence of osmotic water loss. The phosphatase inhibitor calyculin A also promoted activation, but not in cells preexposed briefly to high [K]o. In summary, maneuvers than lower [Cl]i activate the cotransporter, whereas those that elevate [Cl]i (or prevent it from decreasing) block activation in response to secretory stimuli. Cell Cl appears to govern its own rate of entry via Na-K-Cl cotransport by impeding regulatory phosphorylation of the Na-K-Cl cotransport protein.

Animals↗
PubMed1995

[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 beta-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 approximately 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 approximately 170-kDa cotransport protein. Decreasing [Cl-]i (without concomitant cell shrinkage) by incubating cultures with apical nystatin and reduced apical [Cl-] ([Cl-]alpha) likewise increases both cotransport activity and cotransport protein phosphorylation. These effects become more pronounced with greater reductions in [Cl-]alpha; after 20 min of incubation with nystatin and 32 mM [Cl-]alpha, 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 approximately 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.

Adrenergic beta-Agonists↗