Altered pHi regulation and Na+/HCO3− transporter activity in choroid plexus of cilia-defective Tg737orpk mutant mouse

2007 ◽  
Vol 292 (4) ◽  
pp. C1409-C1416 ◽  
Author(s):  
Boglarka Banizs ◽  
Peter Komlosi ◽  
Mark O. Bevensee ◽  
Erik M. Schwiebert ◽  
Phillip D. Bell ◽  
...  
Keyword(s):  
Ion Transport ◽  
Choroid Plexus ◽  
Perinatal Period ◽  
Ependymal Cells ◽  
Intracellular Camp ◽  
Transport Activity ◽  
Wild Type ◽  
Choroid Plexus Epithelium ◽  
Fluid Movement ◽  
Camp Levels

Tg737 orpk mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737 orpk mutants is associated with defects in ion transport, we compared the steady-state pHi and Na+-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737 orpk mutant and wild-type mice. The data indicate that Tg737 orpk mutant choroid plexus epithelium have lower pHi and higher Na+-dependent HCO3− transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na+-dependent HCO3− transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737 orpk mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pHi and ion transport activity.

scholarly journals Mutational Activation of a Gαi Causes Uncontrolled Proliferation of Aerial Hyphae and Increased Sensitivity to Heat and Oxidative Stress in Neurospora crassa

Genetics ◽  
1999 ◽  
Vol 151 (1) ◽  
pp. 107-117
Author(s):  
Qi Yang ◽  
Katherine A Borkovich
Keyword(s):  
Signal Transduction ◽  
Neurospora Crassa ◽  
Signal Transduction Pathway ◽  
Sexual Cycle ◽  
Intracellular Camp ◽  
Wild Type ◽  
Independent Functions ◽  
Aerial Hyphae ◽  
Mutational Activation ◽  
Camp Levels

Abstract Heterotrimeric G proteins, consisting of α, β, and γ subunits, transduce environmental signals through coupling to plasma membrane-localized receptors. We previously reported that the filamentous fungus Neurospora crassa possesses a Gα protein, GNA-1, that is a member of the Gαi superfamily. Deletion of gna-1 leads to defects in apical extension, differentiation of asexual spores, sensitivity to hyperosmotic media, and female fertility. In addition, Δgna-1 strains have lower intracellular cAMP levels under conditions that promote morphological abnormalities. To further define the function of GNA-1 in signal transduction in N. crassa, we examined properties of strains with mutationally activated gna-1 alleles (R178C or Q204L) as the only source of GNA-1 protein. These mutations are predicted to inhibit the GTPase activity of GNA-1 and lead to constitutive signaling. In the sexual cycle, gna-1R178C and gna-1Q204L strains are female-fertile, but produce fewer and larger perithecia than wild type. During asexual development, gna-1R178C and gna-1Q204L strains elaborate abundant, long aerial hyphae, produce less conidia, and possess lower levels of carotenoid pigments in comparison to wild-type controls. Furthermore, gna-1R178C and gna-1Q204L strains are more sensitive to heat shock and exposure to hydrogen peroxide than wild-type strains, while Δgna-1 mutants are more resistant. In contrast to Δgna-1 mutants, gna-1R178C and gna-1Q204L strains have higher steady-state levels of cAMP than wild type. The results suggest that GNA-1 possesses several Gβγ-independent functions in N. crassa. We propose that GNA-1 mediates signal transduction pathway(s) that regulate aerial hyphae development and sensitivity to heat and oxidative stresses, possibly through modulation of cAMP levels.

scholarly journals TRPV4 activation affects transepithelial ion transport in the choroid plexus epithelium

2018 ◽  
Vol 32 (S1) ◽  
Author(s):  
Daniel Preston ◽  
Stefanie Simpson ◽  
Christian Schwerk ◽  
Horst Schroten ◽  
Bonnie Blazer‐Yost
Keyword(s):  
Ion Transport ◽  
Choroid Plexus ◽  
Choroid Plexus Epithelium

scholarly journals Nhe1 is a luminal Na+/H+ exchanger in mouse choroid plexus and is targeted to the basolateral membrane in Ncbe/Nbcn2-null mice

2009 ◽  
Vol 296 (6) ◽  
pp. C1291-C1300 ◽  
Author(s):  
Helle Hasager Damkier ◽  
Vikram Prasad ◽  
Christian Andreas Hübner ◽  
Jeppe Praetorius
Keyword(s):  
Choroid Plexus ◽  
Basolateral Membrane ◽  
Anion Transport ◽  
Wild Type ◽  
Membrane Domain ◽  
Choroid Plexus Epithelium ◽  
Major Fraction ◽  
Compensatory Response ◽  
Luminal Membrane ◽  
Transport Inhibitor

The choroid plexus epithelium (CPE) secretes the major fraction of the cerebrospinal fluid (CSF). The Na+-HCO3− transporter Ncbe/Nbcn2 in the basolateral membrane of CPE cells is important for Na+-dependent pHi increases and probably for CSF secretion. In the current study, the anion transport inhibitor DIDS had no effect on the residual pHi recovery in acidified CPE from Ncbe/Nbcn2 knockout mouse by 2′,7′- bis(2-carboxyethyl)-5( 6 )-carboxyfluorescein (BCECF)-fluorescence microscopy in the presence of CO2/HCO3− (Ncbe/Nbcn2-ko+DIDS 109% of control, P = 0.76, n = 5). Thus Ncbe/Nbcn2 mediates the DIDS-sensitive Na+-dependent pHi recovery in the CPE. The Na+/H+ exchanger-1 Nhe1 is proposed to mediate similar functions as Ncbe/Nbcn2 in CPE. Here, we immunolocalize the Nhe1 protein to the luminal membrane domain in mouse and human CPE. The Na+-dependent pHi recovery of Nhe1 wild-type (Nhe1-wt) mice in the absence of CO2/HCO3− was abolished in the Nhe1 knockout CPE (Nhe1-ko 0.37% of Nhe1-wt, P = 0.0007, n = 5). In Ncbe/Nbcn2-ko mice, Nhe1 was targeted to the basolateral membrane. Nevertheless, the luminal Na+-dependent pHi recovery was increased in Ncbe/Nbcn2-ko compared with wild-type littermates (Nhe1-ko 146% of Nhe1-wt, P = 0.007, n = 5). Whereas the luminal Nhe activity was inhibited by the Nhe blocker EIPA (10 μM) in the Ncbe/Nbcn2-wt, it was insensitive to the inhibitor in Ncbe/Nbcn2-ko (Ncbe/Nbcn2-ko+EIPA 100% of control, P = 0.98, n = 5). This indicates that a luminal EIPA-insensitive Nhe was induced in Ncbe/Nbcn2-ko CPE and that EIPA-sensitive Nhe activity was basolateral. The Nhe1 translocation in Ncbe/Nbcn2-ko CPE may reflect a compensatory response, which provides the cells with better means of regulating pHi or transporting Na+ after Ncbe/Nbcn2 disruption.

scholarly journals Phosphodiesterase 8A Regulates CFTR Activity in Airway Epithelial Cells

2021 ◽  
Vol 55 (6) ◽  
pp. 784-804
Keyword(s):  
Cystic Fibrosis ◽  
Large Family ◽  
Airway Epithelial Cells ◽  
Intracellular Camp ◽  
Wild Type ◽  
Ussing Chambers ◽  
Variable Expression ◽  
Gene And Protein Expression ◽  
Well Differentiated ◽  
Camp Levels

BACKGROUND/AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity. METHODS: We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]i) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers. RESULTS: PDE8 inhibition elevated [cAMP]i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators. CONCLUSION: These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.

scholarly journals Localization of metallothionein in the brain of rat and mouse.

1992 ◽  
Vol 40 (2) ◽  
pp. 309-315 ◽  
Author(s):  
N Nishimura ◽  
H Nishimura ◽  
A Ghaffar ◽  
C Tohyama
Keyword(s):  
Endothelial Cells ◽  
Choroid Plexus ◽  
Glial Cells ◽  
Adult Mouse ◽  
Ependymal Cells ◽  
Choroid Plexus Epithelium ◽  
Pia Mater ◽  
Adult Mouse Brain ◽  
Adult Rats ◽  
The Brain

Metallothionein (MT) is a low molecular mass protein inducible by heavy metals such as cadmium (Cd), zinc, and copper, and having high affinity for these metals. In the present study, we investigated the immunohistological localization of MT in the brains of rats and mice. In adult rat brain, almost no MT immunostaining was observed, whereas in adult mouse brain strong MT immunostaining was found in the ependymal cells, some glial cells, arachnoid, and pia mater. No immunostaining was detected in neurons and endothelial cells. In younger rats (1-3 weeks old), strong MT immunostaining was observed in ependymal cells, choroid plexus epithelium, arachnoid, and pia mater. The overall MT concentration in adult mouse brain appeared higher than that of the brains of young and adult rats. When adult rats were administered Cd, MT was induced not only in some glial cells, ependymal cells, arachnoid, and pia mater but also in endothelial cells. Although Cd treatment resulted in an increase in the MT immunostaining in the specific cells described above, the MT induction was not great enough to significantly affect the overall MT level in the brain. The present result suggest a possible link of MT with cell growth of choroid plexus epithelium and ependymal cells, as well as a detoxifying role of MT in the blood-brain barrier and the cerebrospinal fluid-brain barrier.

scholarly journals Environmental Signals Controlling Production of Hemagglutinin/Protease in Vibrio cholerae

2001 ◽  
Vol 69 (10) ◽  
pp. 6549-6553 ◽  
Author(s):  
Jorge A. Benitez ◽  
Anisia J. Silva ◽  
Richard A. Finkelstein
Keyword(s):  
Vibrio Cholerae ◽  
Deletion Mutant ◽  
Receptor Protein ◽  
Intracellular Camp ◽  
Wild Type ◽  
Camp Receptor Protein ◽  
Environmental Signals ◽  
Camp Receptor ◽  
Camp Levels

ABSTRACT Vibrio cholerae hemagglutinin/protease (Hap) was induced upon nutrient limitation and strongly repressed by glucose. Hap was not produced in a mutant defective in the cyclic AMP (cAMP) receptor protein, suggesting that intracellular cAMP levels mediate Hap expression. No difference was found in Hap production between anrpoS deletion mutant and its isogenic wild-type precursor, indicating that the alternate ςs factor is not essential for Hap expression. Based on these and previous results, we discuss the role of Hap in the pathogenesis of cholera.

scholarly journals G Protein Signaling Mediates Developmental Processes and Pathogenesis of Alternaria alternata

2006 ◽  
Vol 19 (11) ◽  
pp. 1280-1288 ◽  
Author(s):  
Daisuke Yamagishi ◽  
Hiroshi Otani ◽  
Motoichiro Kodama
Keyword(s):  
G Protein ◽  
Alternaria Alternata ◽  
Signal Transduction Pathway ◽  
Intracellular Camp ◽  
Protein Signaling ◽  
Wild Type ◽  
Targeted Disruption ◽  
Α Subunit ◽  
Cellulose Membranes ◽  
Camp Levels

A G protein α subunit gene (AGA1) has been cloned and characterized from a toxigenic and necrotrophic Alternaria alternata pathogen. Targeted disruption of AGA1 in the apple pathotype of A. alternata gave rise to mutants that differed in colony and conidial morphology as well as sporulation. The conidia of wild type and ΔAGA1 mutants showed equal germination on cellulose membranes. However, wild-type germ tubes formed readily from different points around the conidia, grew randomly, and were often branched, whereas those of the mutants formed only at one or both ends of the conidia and tended to grow in straight paths. Targeted disruption of AGA1 also resulted in reduction of pathogenicity on apple leaves, although the mutant produced host-specific AM-toxin, a fungal secondary metabolite associated with pathogenicity of the pathogen, at levels similar to the wild-type strain. Measurement of the intracellular cAMP levels of the mutant revealed that it was consistently higher than that of the wild type, indicating that AGA1 negatively regulates cAMP levels similar to mammalian Gαi systems. These results indicate that the signal transduction pathway represented by AGA1 appears to be involved in developmental pathways leading to sporulation and pathogenesis of A. alternata.

Organization of tight junctions in the choroid plexus epithelium

1978 ◽  
Vol 36 (2) ◽  
pp. 336-337
Author(s):  
B. Van Deurs ◽  
J. K. Koehler
Keyword(s):  
Choroid Plexus ◽  
Present Report ◽  
Freeze Fracture ◽  
Barrier Properties ◽  
Cell Junctions ◽  
Structural Basis ◽  
Apical Surface ◽  
Choroid Plexus Epithelium ◽  
Solid Nitrogen ◽  
Special Composition

The choroid plexus epithelium constitutes a blood-cerebrospinal fluid (CSF) barrier, and is involved in regulation of the special composition of the CSF. The epithelium is provided with an ouabain-sensitive Na/K-pump located at the apical surface, actively pumping ions into the CSF. The choroid plexus epithelium has been described as “leaky” with a low transepithelial resistance, and a passive transepithelial flux following a paracellular route (intercellular spaces and cell junctions) also takes place. The present report describes the structural basis for these “barrier” properties of the choroid plexus epithelium as revealed by freeze fracture.Choroid plexus from the lateral, third and fourth ventricles of rats were used. The tissue was fixed in glutaraldehyde and stored in 30% glycerol. Freezing was performed either in liquid nitrogen-cooled Freon 22, or directly in a mixture of liquid and solid nitrogen prepared in a special vacuum chamber. The latter method was always used, and considered necessary, when preparations of complementary (double) replicas were made.

Transmembrane Calcium Influx Associated with von Willebrand Factor Binding to GP Ib in the Initiation of Shear-Induced Platelet Aggregation

1993 ◽  
Vol 69 (05) ◽  
pp. 496-502 ◽  
Author(s):  
Yasuo Ikeda ◽  
Makoto Handa ◽  
Tetsuji Kamata ◽  
Koichi Kawano ◽  
Yohko Kawai ◽  
...  
Keyword(s):  
Shear Force ◽  
Calcium Influx ◽  
Fold Increase ◽  
Intracellular Camp ◽  
Recombinant Fragment ◽  
Transmembrane Flux ◽  
Irreversible Aggregation ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Camp Levels

SummaryWe found that the binding of multimeric vWF to GP Ib under a shear force of 108 dynes/cm2 resulted in the transmembrane flux of Ca2+ ions with a two-to three-fold increase in their intracellular concentration ([Ca2+]i). The blockage of this event, obtained by inhibiting the vWF-GP Ib interaction, suppressed aggregation. In contrast, the blockage of vWF binding to GP IIb-IIIa, as well as the prevention of activation caused by increased intracellular cAMP levels, inhibited aggregation but had no significant effect on [Ca2+]i increase. A monomeric recombinant fragment of vWF containing the GP Ib-binding domain of the molecule (residues 445-733) prevented all effects mediated by multimeric vWF but, by itself, failed to support the increase in [Ca2+]i and aggregation. These results suggest that the binding of multimeric vWF to GP Ib initiates platelets aggregation induced by high shear stress by mediating a transmembrane flux of Ca2+ ions, perhaps through a receptor-dependent calcium channel. The increase in [Ca2+]i may act as an intracellular message and cause the activation of GP IIb-IIIa; the latter receptor then binds vWF and mediates irreversible aggregation.

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