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1.
Adenylyl cyclase is involved in desensitization and recovery of ATP-stimulated Cl- secretion in MDCK cells 总被引:1,自引:0,他引:1
Woo Jae Suk; Inoue Chiyoko N.; Hanaoka Kazushige; Schwiebert Erik M.; Guggino Sandra E.; Guggino William B. 《American journal of physiology. Cell physiology》1998,274(2):C371
We investigated the process of and recovery from desensitizationof the P2 receptor-mediatedstimulation of Cl secretionin Madin-Darby canine kidney (MDCK) cell monolayers by assaying theresponse of short-circuit current(Isc). When thecells were exposed to repeated 3-min challenges of ATP or UTPinterspersed with 5-min washes, the response ofIsc desensitized rapidly followed by spontaneous recovery. The pattern of inhibition byvarious channel blockers or enzyme inhibitors revealed that both theinitial and recovered responses ofIsc have the same ionic and signaling mechanisms. The desensitization and recovery processes were confined to the membrane exposed to the repeated challenges. When added during the desensitized phase, 8-bromoadenosine 3',5'-cyclic monophosphate enhanced the ATP-stimulatedIsc response, whereas it did not during the initial or recovered phases. ATP-induced increases of intracellular adenosine 3',5'-cyclicmonophosphate showed similar desensitization and recovery in parallelwith the changes in the responses ofIsc. Thedesensitization process was attenuated by pretreatment with choleratoxin or pertussis toxin. Taken together, our results suggest that theadenylyl cyclase system plays a role in the desensitization andrecovery mechanism of the ATP-stimulatedCl secretion in MDCK cells. 相似文献
2.
Fröhlich O Klein JD Smith PM Sands JM Gunn RB 《American journal of physiology. Cell physiology》2004,286(6):C1264-C1270
Progress in understanding the cell biology of urea transporter proteins has been hampered by the lack of an appropriate cell culture system. The goal of this study was to create a polarized epithelial cell line that stably expresses the largest of the rat renal urea transporter UT-A isoforms, UT-A1. The gene for UT-A1 was cloned into pcDNA5/FRT and transfected into Madin-Darby canine kidney (MDCK) cells with an integrated Flp recombination target site. The cells from a single clone were grown to confluence on collagen-coated membranes until the resistance was >1,500 ·cm2. Transepithelial [14C]urea fluxes were measured at 37°C in a HCO3/CO2 buffer, pH 7.4, with 5 mM urea. The baseline fluxes were not different between unstimulated UT-A1-transfected MDCK cells and nontransfected or sham-transfected MDCK cells. However, only in the UT-A1-transfected cells was UT-A1 protein expressed (as measured by Western blot analysis) and urea transport stimulated by forskolin or arginine vasopressin. Forskolin and arginine vasopressin also increased the phosphorylation of UT-A1. Thionicotinamide, dimethylurea, and phloretin inhibited the forskolin-stimulated [14C]urea fluxes in the UT-A1-transfected MDCK cells. These characteristics mimic those seen in rat terminal inner medullary collecting ducts. This new polarized epithelial cell line stably expresses UT-A1 and reproduces several of the physiological responses observed in rat terminal inner medullary collecting ducts. urea transporter-A1; arginine vasopressin; collecting duct; Madin-Darby canine kidney cells 相似文献
3.
A role for ERK1/2 in EGF- and ATP-dependent regulation of amiloride-sensitive sodium absorption 总被引:3,自引:0,他引:3
Falin R Veizis IE Cotton CU 《American journal of physiology. Cell physiology》2005,288(5):C1003-C1011
Receptor-mediated inhibition of amiloride-sensitive sodium absorption was observed in primary and immortalized murine renal collecting duct cell (mCT12) monolayers. The addition of epidermal growth factor (EGF) to the basolateral bathing solution of polarized monolayers reduced amiloride-sensitive short-circuit current (Isc) by 1525%, whereas the addition of ATP to the apical bathing solution decreased Isc by 4060%. Direct activation of PKC with phorbol 12-myristate 13-acetate (PMA) and mobilization of intracellular calcium with 2,5-di-tert-butyl-hydroquinone (DBHQ) reduced amiloride-sensitive Isc in mCT12 monolayers by 46 ± 4% (n = 8) and 22 ± 2% (n = 8), respectively. Exposure of mCT12 cells to EGF, ATP, PMA, and DBHQ caused an increase in phosphorylation of p42/p44 (extracellular signal-regulated kinase; ERK1/2). Pretreatment of mCT12 monolayers with an ERK kinase inhibitor (PD-98059; 30 µM) prevented phosphorylation of p42/p44 and significantly reduced EGF, ATP, and PMA-induced inhibition of amiloride-sensitive Isc. In contrast, pretreatment of monolayers with a PKC inhibitor (bisindolylmaleimide I; GF109203x; 1 µM) almost completely blocked the PMA-induced decrease in Isc, but did not alter the EGF- or ATP-induced inhibition of Isc. The DBHQ-mediated decrease in Isc was due to inhibition of basolateral Na+-K+-ATPase, but EGF-, ATP-, and PMA-induced inhibition was most likely due to reduced apical sodium entry (epithelial Na+ channel activity). The results of these studies demonstrate that acute inhibition of amiloride-sensitive sodium transport by extracelluar ATP and EGF involves ERK1/2 activation and suggests a role for MAP kinase signaling as a negative regulator of electrogenic sodium absorption in epithelia. mitogen-activated protein kinase; epithelial ion transport; epithelial sodium channel 相似文献
4.
Asymmetric distribution of muscarinic acetylcholine receptors in Madin-Darby canine kidney cells 总被引:1,自引:0,他引:1
Nadler Laurie S.; Kumar Geetha; Hinds Thomas R.; Migeon Jacques C.; Nathanson Neil M. 《American journal of physiology. Cell physiology》1999,277(6):C1220
We have characterized the muscarinic AChreceptors (mAChRs) expressed in Madin- Darby canine kidney (MDCK)strain II epithelial cells. Binding studies with themembrane-impermeable antagonist N-[3H]methylscopolaminedemonstrated that mAChRs are ~2.5 times more abundant on thebasolateral than on the apical surface. Apical, but not basolateral,mAChRs inhibited forskolin-stimulated adenylyl cyclase activity inresponse to the agonist carbachol. Neither apical nor basolateralmAChRs exhibited detectable carbachol-stimulated phospholipase Cactivity. Carbachol application to the apical or the basolateralmembrane resulted in a threefold increase in intracellularCa2+ concentration, which wascompletely inhibited by pertussis toxin on the apical side andpartially inhibited on the basolateral side. RT-PCR analysis showedthat MDCK cells express the M4 and M5 receptor mRNAs. These datasuggest that M4 receptors reside on the apical and basolateral membranes of polarized MDCK strain IIcells and that the M5 receptor mayreside in the basolateral membrane of a subset of cells. 相似文献
5.
RNA interference targeted to multiple P2X receptor subtypes attenuates zinc-induced calcium entry 总被引:2,自引:0,他引:2
Liang L Zsembery A Schwiebert EM 《American journal of physiology. Cell physiology》2005,289(2):C388-C396
A postulated therapeutic avenue in cystic fibrosis (CF) is activation of Ca2+-dependent Cl channels via stimulation of Ca2+ entry from extracellular solutions independent of CFTR functional status. We have shown that extracellular zinc and ATP induce a sustained increase in cytosolic Ca2+ in human airway epithelial cells that translates into stimulation of sustained secretory Cl transport in non-CF and CF human and mouse airway epithelial cells, cell monolayers, and nasal mucosa. On the basis of these studies, the Ca2+ entry channels most likely involved were P2X purinergic receptor channels. In the present study, molecular and biochemical data show coexpression of P2X4, P2X5, and P2X6 subtypes in non-CF (16HBE14o) and CF (IB3-1) human bronchial epithelial cells. Other P2X receptor Ca2+ entry channel subtypes are expressed rarely or not at all in airway epithelia, epithelial cell models from other CF-relevant tissues, or vascular endothelia. Novel transient lipid transfection-mediated delivery of small interference RNA fragments specific to P2X4 and P2X6 (but not P2X5) into IB3-1 CF human airway epithelial cells inhibited extracellular zinc- and ATP-induced Ca2+ entry markedly in fura-2 Ca2+ measurements and "knocked down" protein by >65%. These data suggest that multiple P2X receptor Ca2+ entry channel subtypes are expressed in airway epithelia. P2X4 and P2X6 may coassemble on the airway surface as targets for possible therapeutics for CF independent of CFTR genotype. purinergic receptors; zinc receptors; airway epithelia; cystic fibrosis; therapy 相似文献
6.
Polarized trafficking of the aquaporin-3 water channel is mediated by an NH2-terminal sorting signal 总被引:1,自引:0,他引:1
Epithelial renal collecting duct cells express multiple types of aquaporin (AQP) water channels in a polarized fashion. AQP2 is specifically targeted to the apical cell domain, whereas AQP3 and AQP4 are expressed on the basolateral membrane. It is crucial that these AQP variants are sorted to their proper polarized membrane domains, because correct AQP sorting enables efficient water transport. However, the molecular mechanisms involved in the polarized targeting and membrane trafficking of AQPs remain largely unknown. In the present study, we have examined the polarized trafficking and surface expression of AQP3 in Madin-Darby canine kidney type II (MDCKII) cells in an effort to identify the molecular determinants of polarized targeting specificity. When expressed in MDCKII cells, the majority of the exogenous wild-type AQP3 was found to be targeted to the basolateral membrane, consistent with its localization pattern in vivo. A potential sorting signal consisting of tyrosine- and dileucine-based motifs was subsequently identified in the AQP3 NH2 terminus. When mutations were introduced into this signaling region, the basolateral targeting of the resulting mutant AQP3 was disrupted and the mutant protein remained in the cytoplasm. AQP2-AQP3 chimeras were then generated in which the entire NH2 terminus of AQP2 was replaced with the AQP3 NH2 terminus. This chimeric protein was observed to be mislocalized constitutively in the basolateral membrane, and mutations in the AQP3 NH2-terminal sorting signal abolished this effect. On the basis of these results, we conclude that an NH2-terminal sorting signal mediates the basolateral targeting of AQP3. epithelial cells; protein sorting; Madin-Darby canine kidney cells; basolateral 相似文献
7.
Collagen gel overlay induces apoptosis of polarized cells in cultures: disoriented cell death 总被引:1,自引:0,他引:1
Tang Ming-Jer; Hu Jeng-Jay; Lin Hsi-Hui; Chiu Wen-Tai; Jiang Si-Tse 《American journal of physiology. Cell physiology》1998,275(4):C921
In this study, we attempted to investigate the response ofpolarized cells to inappropriate interaction with the extracellular matrix. Cell lines of epithelial [Madin-Darby canine kidney(MDCK) and LLC-PK1],endothelial [bovine aortic endothelial cells (BAEC)], andmesenchymal (ESK-4 and NIH/3T3) origins were employed. With collagengel overlay, MDCK cells underwent membrane remodeling and graduallydeveloped lumen formation within 24 h. Apoptosis could also be observedfollowing cell remodeling. The ratio of apoptosis was enhanced from12.1 ± 2.4% within 24 h to 58.4 ± 9.8% atday 3, and finally the monolayer wasdisintegrated. Collagen gel overlay-induced apoptosis was not a resultof physical stress, since agarose gel overlay did not induce anymorphological alterations. All epithelial and endothelial cellsexamined developed apoptosis in response to collagen overlay. Incontrast, collagen overlay did not affect growth of fibroblasts at all,although their growth under agarose gel was slightly hindered due tophysical stress. Collagen overlay-induced apoptosis seems to be aunique phenomenon for polarized cells and thus is defined as"disoriented cell death." Furthermore,anti-2-integrin antibody couldabolish collagen overlay-induced morphological changes and apoptosis inMDCK cells, indicating that signals through2-integrin on the apicalmembrane are required for disoriented cell death. Finally, Bcl-2overexpression prolonged survival of MDCK cells in response to collagenoverlay, but these cells eventually developed apoptosis due todownregulation of Bcl-2 protein. These findings indicate thatinappropriate cell-matrix interaction results in apoptosis, which mayaccount for cell death mechanisms during developmental processes orunder pathological conditions. 相似文献
8.
Turner John T.; Redman Robert S.; Camden Jean M.; Landon Linda A.; Quissell David O. 《American journal of physiology. Cell physiology》1998,275(2):C367
Because of thelack of salivary gland cell lines suitable for Ussing chamber studies,a recently established rat parotid acinar cell line, Par-C10, was grownon permeable supports and evaluated for development of transcellularresistance, polarization, and changes in short-circuit current(Isc) inresponse to relevant receptor agonists. Par-C10 cultures reachedconfluence in 3-4 days and developed transcellular resistancevalues of 2,000 · cm2.Morphological examination revealed that Par-C10 cells grew as polarizedmonolayers exhibiting tripartite junctional complexes and the acinarcell-specific characteristic of secretory canaliculi. Par-C10Isc was increasedin response to muscarinic cholinergic and - and -adrenergicagonists on the basolateral aspect of the cultures and to ATP and UTP(through P2Y2 nucleotidereceptors) applied apically. Ion replacement and inhibitor studiesindicated that anion secretion was the primary factor inagonist-stimulated Isc. RT-PCR,which confirmed the presence ofP2Y2 nucleotide receptor mRNA inPar-C10 cells, also revealed the presence of mRNA for the cysticfibrosis transmembrane conductance regulator and ClC-2 Cl channel proteins. These findingsestablish Par-C10 cells as the first cell line of salivary gland originuseful in transcellular ion secretion studies in Ussing chambers. 相似文献
9.
Clathrin assembly protein, AP180, was originally identified as a brain-specific protein localized to the presynaptic junction. AP180 acts to limit vesicle size and maintain a pool of releasable synaptic vesicles during rapid recycling. In this study, we show that polarized epithelial Madin-Darby canine kidney (MDCK) cells express two AP180-related proteins: the ubiquitously expressed 62-kDa clathrin assembly lymphoid myeloid leukemia (CALM, AP180-2) protein and a novel high-molecular-weight homolog that we have named AP180-3. Sequence analysis of AP180-3 expressed in MDCK cells shows high homology to AP180 from rat brain. AP180-3 contains conserved motifs found in brain-specific AP180, including the epsin NH2-terminal homology (ENTH) domain, the binding site for the -subunit of AP-2, and DLL repeats. Our studies show that AP180-3 from MDCK cells forms complexes with AP-2 and clathrin and that membrane recruitment of these complexes is modulated by phosphorylation. We demonstrate by immunohistochemistry that AP180-3 is localized to cytoplasmic vesicles in MDCK cells and is also present in tubule epithelial cells from mouse kidney. We observed by immunodetection that a high-molecular-weight AP180-related protein is expressed in numerous cells in addition to MDCK cells. clathrin assembly lympoid myeloid leukemia; kidney epithelial cells; epsin NH2-terminal homology domain; DLL repeats; clathrin; AP-2 相似文献
10.
Clarke Lane L.; Harline Matthew C.; Otero Miguel A.; Glover Geraldine G.; Garrad Richard C.; Krugh Brent; Walker Nancy M.; Gonzalez Fernando A.; Turner John T.; Weisman Gary A. 《American journal of physiology. Cell physiology》1999,276(4):C777
Desensitization ofP2Y2 receptor-activated anionsecretion may limit the usefulness of extracellular nucleotides insecretagogue therapy of epithelial diseases, e.g., cystic fibrosis(CF). To investigate the desensitization process for endogenousP2Y2 receptors, freshly excised orcultured murine gallbladder epithelia (MGEP) were mounted in Ussingchambers to measure short-circuit current (Isc), an indexof electrogenic anion secretion. Luminal treatment with nucleotidereceptor agonists increased theIsc with apotency profile of ATP = UTP > 2-methylthioATP >>,-methylene-ATP. RT-PCR revealed the expression ofP2Y2 receptor mRNA in the MGEPcells. The desensitization of anion secretion required a 10-minpreincubation with the P2Y2receptor agonist UTP and increased in aconcentration-dependent manner(IC50 106 M). Approximately 40%of the anion secretory response was unaffected by maximal desensitizingconcentrations of UTP. Recovery from UTP-induced desensitization wasrapid (<10 min) at preincubation concentrations less than theEC50 (1.9 × 106 M) but requiredprogressively longer time periods at greater concentrations.UTP-induced total inositol phosphate production and intracellularCa2+ mobilization desensitizedwith a concentration dependence similar to that of anion secretion. Incontrast, maximal anion secretion induced byCa2+ ionophore ionomycin wasunaffected by preincubation with a desensitizing concentration of UTP.It was concluded that 1)desensitization of transepithelial anion secretion stimulated by theP2Y2 receptor agonist UTP is timeand concentration dependent; 2)recovery from desensitization is prolonged (>90 min) at UTPconcentrations >105 M;and 3) UTP-induced desensitizationoccurs before the operation of the anion secretory mechanism. 相似文献
11.
Micromolarconcentrations of ATP stimulate biphasic change in transepithelialconductance across CaSki cultures on filters, an acute transientincrease (phase I response; triggered by P2Y2 receptor and mediated by calcium mobilization-dependent cell volume decrease) followed by a slower decrease in permeability (phase II response). Phase II response is mediated byaugmented calcium influx and protein kinase C-dependent increase intight junctional resistance. The objective of the study was todetermine the role of P2X4 receptor as a mediator ofphase II response. Human cervical epithelial cells expressP2X4 receptor mRNA (1.4-, 2.2-, and 4.4-kb isoforms byNorthern blot analysis) and P2X4 protein. Depletion ofvitamin A reversibly downregulated P2X4 receptor mRNA andprotein and ATP-induced calcium influx. Depletion of vitamin Aabrogated phase II response, and the effect could bepartially reversed only with retinoic acid receptor (RAR)-selectiveretinoids but not retinoid X receptor (RXR) agonists. Depletion ofvitamin A also abrogated protein kinase C increase in tight junctionalresistance, and the effect could not be reversed with retinoids.Depletion of vitamin A also abrogated phase I increase inpermeability and reversibly downregulated P2Y2 receptormRNA and ATP-induced calcium mobilization. However, in contrast tophase II response, both RAR and RXR agonists could fullyreverse those effects. These results suggest that phase IIresponse is mediated by a P2X4 receptor mechanism. 相似文献
12.
Polarity of A2b adenosine receptor expression determines characteristics of receptor desensitization 总被引:2,自引:0,他引:2
Sitaraman SV Si-Tahar M Merlin D Strohmeier GR Madara JL 《American journal of physiology. Cell physiology》2000,278(6):C1230-C1236
It is not knownif, in polarized cells, desensitization events can be influenced by thedomain on which the receptor resides. Desensitization was induced by5'-(N-ethylcarboxamido)adenosine (NECA) and wasquantitated by measurement of short-circuit current (Isc) in response to adenosine. NECA addedto either the apical or basolateral compartments rapidly desensitizedreceptors on these respective domains. Although apical NECA had noeffect on the basolateral receptor stimulation, basolateral NECAinduced a complete desensitization of the apical receptor. Wehypothesized that desensitization of apical receptor by basolateraldesensitization could relate to a trafficking step in which A2breceptor is first targeted basolaterally upon synthesis and transportedto the apical surface via vesicular transport/microtubules. Becausedesensitization is associated with downregulation of receptors, apicaladenosine receptor can thus be affected by basolateral desensitization. Both low temperature and nocodazole inhibited Iscinduced by apical and not basolateral adenosine. In conclusion:1) a single receptor subtype, here modeled by the A2b receptor,differentially desensitizes based on the membrane domain on which it isexpressed, 2) agonist exposure on one domain can result indesensitization of receptors on the opposite domain, 3)cross-domain desensitization can display strict polarity, and4) receptor trafficking may play a role in thecross-desensitization process. 相似文献
13.
Channel-forming peptide modulates transepithelial electrical conductance and solute permeability 总被引:3,自引:0,他引:3
Broughman JR Brandt RM Hastings C Iwamoto T Tomich JM Schultz BD 《American journal of physiology. Cell physiology》2004,286(6):C1312-C1323
NC-1059, a synthetic channel-forming peptide, transiently increases transepithelial electrical conductance (gTE) and ion transport (as indicated by short-circuit current) across Madin-Darby canine kidney (MDCK) cell monolayers in a time- and concentration-dependent manner when apically exposed. gTE increases from <2 to >40 mS/cm2 over the low to middle micromolar range. Dextran polymer (9.5 but not 77 kDa) permeates the monolayer following apical NC-1059 exposure, suggesting that modulation of the paracellular pathway accounts for changes in gTE. However, concomitant alterations in junctional protein localization (zonula occludens-1, occludin) and cellular morphology are not observed. Effects of NC-1059 on MDCK gTE occur in nominally Cl- and Na+-free apical media, indicating that permeation by these ions is not required for effects on gTE, although two-electrode voltage-clamp assays with Xenopus oocytes suggest that both Cl and Na+ permeate NC-1059 channels with a modest Cl permselectivity (PCl:PNa = 1.3). MDCK monolayers can be exposed to multiple NC-1059 treatments over days to weeks without diminution of response, alteration in the time course, or loss of responsiveness to physiological and pharmacological secretagogues. Together, these results suggest that NC-1059 represents a valuable tool to investigate tight junction regulation and may be a lead compound for therapeutic interventions. transepithelial resistance; cystic fibrosis; tight junction; epithelial barrier; amphipathic -helix 相似文献
14.
Hillyard Stanley D.; Cantiello Horacio F.; Van Driessche Willy 《American journal of physiology. Cell physiology》1997,273(6):C1995
Skin from larval bullfrogs was mounted in an Ussing-type chamberin which the apical surface was bathed with a Ringer solution containing 115 mM K+ and thebasolateral surface was bathed with a Ringer solution containing 115 mMNa+. Ion transport was measured asthe short-circuit current(Isc) with alow-noise voltage clamp, and skin resistance(Rm) wasmeasured by applying a direct current voltage pulse. Membrane impedance was calculated by applying a voltage signal consisting of 53 sine wavesto the command stage of the voltage clamp. From the ratio of theFourier-transformed voltage and current signals, it was possible tocalculate the resistance and capacitance of the apical and basolateralmembranes of the epithelium(Ra andRb,Ca and Cb,respectively). With as the anion,Rm decreasedrapidly within 5 min following the addition of 150 U/ml nystatin to theapical solution, whereasIsc increasedfrom 0.66 to 52.03 µA/cm2 over a60-min period. These results indicate that nystatin becomes rapidlyincorporated into the apical membrane and that the increase inbasolateral K+ permeabilityrequires a more prolonged time course. Intermediate levels ofIsc were obtainedby adding 50, 100, and 150 U/ml nystatin to the apical solution. Thisproduced a progressive decrease in Ra andRb whileCa andCb remainedconstant. With Cl as theanion, Isc valuesincreased from 2.03 to 89.57 µA/cm2 following treatment with150 U/ml nystatin, whereas with gluconate as the anionIsc was onlyincreased from 0.63 to 11.64 µA/cm2. This suggests that theincrease in basolateral K+permeability produced by nystatin treatment, in the presence of morepermeable anions, is due to swelling of the epithelial cells of thetissue rather than the gradient for apicalK+ entry. Finally,Cb was notdifferent among skins exposed toCl,, or gluconate, despite the largedifferences inIsc, nor didinhibition of Iscby treatment with hyperosmotic dextrose cause significant changes inCb. These resultssupport the hypothesis that increases in cell volume activateK+ channels that are alreadypresent in the basolateral membrane of epithelial cells. 相似文献
15.
Toye AM Parker MD Daly CM Lu J Virkki LV Pelletier MF Boron WF 《American journal of physiology. Cell physiology》2006,291(4):C788-C801
The human electrogenic renal Na-HCO3 cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO3 from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na+ affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules. bicarbonate; intracellular pH; acidbase; SLC4A4; Na+-HCO3 cotransporter 1 相似文献
16.
Lin-Chien Huang Peter R. Thorne Srdjan M. Vlajkovic Gary D. Housley 《Purinergic signalling》2010,6(2):231-248
Purinergic signaling has broad physiological significance to the hearing organ, involving signal transduction via ionotropic
P2X receptors and metabotropic G-protein-coupled P2Y and P1 (adenosine), alongside conversion of nucleotides and nucleosides
by ecto-nucleotidases and ecto-nucleoside diphosphokinase. In addition, ATP release is modulated by acoustic overstimulation
or stress and involves feedback regulation. Many of these principal elements of the purinergic signaling complex have been
well characterized in the cochlea, while the characterization of P2Y receptor expression is emerging. The present study used
immunohistochemistry to evaluate the expression of five P2Y receptors, P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12, during development of the rat cochlea. Commencing in the late embryonic period, the P2Y receptors studied were found in
the cells lining the cochlear partition, associated with establishment of the electrochemical environment which provides the
driving force for sound transduction. In addition, early postnatal P2Y2 and P2Y4 protein expression in the greater epithelial ridge, part of the developing hearing organ, supports the view that initiation
and regulation of spontaneous activity in the hair cells prior to hearing onset is mediated by purinergic signaling. Sub-cellular
compartmentalization of P2Y receptor expression in sensory hair cells, and diversity of receptor expression in the spiral
ganglion neurons and their satellite cells, indicates roles for P2Y receptor-mediated Ca2+-signaling in sound transduction and auditory neuron excitability. Overall, the dynamics of P2Y receptor expression during
development of the cochlea complement the other elements of the purinergic signaling complex and reinforce the significance
of extracellular nucleotide and nucleoside signaling to hearing. 相似文献
17.
We used the short-circuit current (Isc) technique to investigate the effects of the isoflavone genistein on the electrogenic Cl secretion of the mouse jejunum. Genistein stimulated a sustained increase in Isc that was dose dependent. Bumetanide inhibited 76 ± 5% of the genistein-stimulated Isc consistent with activation of Cl secretion. Genistein failed to stimulate Isc following maximal activation of the cAMP pathway by forskolin. In addition, forskolin had a reduced effect on Isc of the mouse jejunum in the presence of genistein. Glibenclamide, a blocker of CFTR, eliminated the genistein-stimulated increase of Isc and reduced the forskolin-activated Isc. Clotrimazole, a Ca2+-activated K+ channel blocker, failed to reduce the genistein-stimulated Isc. Vanadate, a blocker of tyrosine-dependent phosphatases, reduced the genistein-activated Isc. Tyrphostin A23, a tyrosine kinase inhibitor, reduced basal Isc, after which genistein failed to stimulate Isc. These data suggest that genistein activated a sustained Cl secretory response of the mouse jejunum and that the effect of genistein was via a tyrosine-dependent phosphorylation pathway. 1-ethyl-2-benzimidazolone; vanadate; tyrphostin A23; cantharidic acid; phosphatase 相似文献
18.
ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells 总被引:4,自引:0,他引:4
Nakamura E Uezono Y Narusawa K Shibuya I Oishi Y Tanaka M Yanagihara N Nakamura T Izumi F 《American journal of physiology. Cell physiology》2000,279(2):C510-C519
In human osteoblast-like MG-63cells, extracellular ATP increased [3H]thymidineincorporation and cell proliferation and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-induced[3H]thymidine incorporation. ATP-induced[3H]thymidine incorporation was mimicked by thenonhydrolyzable ATP analogs adenosine5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2purinoceptor antagonist suramin, suggesting involvement of P2purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptorantagonist reactive blue 2 did not affect [3H]thymidineincorporation, whereas the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced[3H]thymidine incorporation, suggesting that ATP-inducedDNA synthesis was mediated by P2X receptors. RT-PCR analysis revealedthat MG-63 cells expressed P2X4, P2X5,P2X6, and P2X7, but not P2X1,P2X2, and P2X3, receptors. In fura 2-loadedcells, not only ATP, but also UTP, increased intracellularCa2+ concentration, and inhibitors for severalCa2+-activated protein kinases had no effect on ATP-inducedDNA synthesis, suggesting that an increase in intracellularCa2+ concentration is not indispensable for ATP-induced DNAsynthesis. ATP increased mitogen-activated protein kinase activity in aCa2+-independent manner and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-inducedkinase activity. Furthermore, the mitogen-activated protein kinasekinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors byactivating a mitogen-activated protein kinase pathway. 相似文献
19.
The responses of canine lingual epithelium to D-glucose weremeasured in an Ussing chamber to determine the possible contributionof the osmotic changes of taste cells to the response of saccharides.With the mucosal solution containing 50 mM NaCl, 2 mM HEPES,pH 7.4 (solution A) and the serosal solution containing KrebsHenseleit(KH) buffer the addition of up to 0.5 M D-glucose in the mucosalsolution increased the short circuit current (Isc) in a sigmoidalmanner. The D-glucose-stimulated Isc was inhibited by 0.1 mMamiloride or 1 mM ouabain added to either the mucosal or theserosal solution, and partially inhibited by 5 mM BaCl2 addedto the serosal solution. The inhibition by these three compoundswas also observed in the presence of 0.5 M NaCl. Ouabain alsoinhibited transport when added to solution A. These experimentssuggest that in canine lingual epithelium the paracellular pathwaypermits molecules as large as ouabain (mol. wt 586) to diffusefrom the mucosal to the serosal solution and vice versa underall osmotic conditions. These results may explain the phenomenonof intravascular taste. Such is not the case in rat tongue whereouabain only inhibited transport when added to the serosal solution.Increasing the osmolality of the serosal KH buffer by additionof relatively membrane-impermeable saccharides such as sucroseor L-glucose did not significantly alter the Isc, whereas makingthe serosal KH solution hypo-osmotic resulted in a transientdecrease in Isc. These data suggest that the increase in Iscinduced by saccharides, such as D-glucose, is not simply anosmotic response of the epithelium but more likely the consequenceof saccharides binding weakly to receptors. That the responseto both salts by themselves and in the presence of saccharidesexhibits the same cation selectivity, and that both are inhibitedby amiloride, ouabain, BaCl2 and LaCl3 suggest that in caninelingual epithelia, in contrast to rat epithelium, the responsesto hyperosmotic concentrations of salts and saccharides mightoccur via the same transcellular pathways. 相似文献
20.
Devor DC Bridges RJ Pilewski JM 《American journal of physiology. Cell physiology》2000,279(2):C461-C479
Forskolin,UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen(Methoxsalen; 8-MOP), and genistein were evaluated for theireffects on ion transport across primary cultures of human bronchialepithelium (HBE) expressing wild-type (wt HBE) and F508(F-HBE) cystic fibrosis transmembrane conductance regulator. In wtHBE, the baseline short-circuit current (Isc)averaged 27.0 ± 0.6 µA/cm2 (n = 350). Amiloride reduced this Isc by 13.5 ± 0.5 µA/cm2 (n = 317). In F-HBE,baseline Isc was 33.8 ± 1.2 µA/cm2 (n = 200), and amiloride reducedthis by 29.6 ± 1.5 µA/cm2 (n = 116), demonstrating the characteristic hyperabsorption of Na+ associated with cystic fibrosis (CF). In wt HBE,subsequent to amiloride, forskolin induced a sustained,bumetanide-sensitive Isc(Isc = 8.4 ± 0.8 µA/cm2; n = 119). Addition ofacetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid further reduced Isc, suggesting forskolin also stimulatesHCO3 secretion. This was confirmed by ionsubstitution studies. The forskolin-induced Iscwas inhibited by 293B, Ba2+, clofilium, and quinine,whereas charybdotoxin was without effect. In F-HBE the forskolinIsc response was reduced to 1.2 ± 0.3 µA/cm2 (n = 30). In wt HBE, mucosal UTPinduced a transient increase in Isc ( Isc = 15.5 ± 1.1 µA/cm2;n = 44) followed by a sustained plateau, whereas inF-HBE the increase in Isc was reduced to5.8 ± 0.7 µA/cm2 (n = 13). In wtHBE, 1-EBIO, NS004, 8-MOP, and genistein increased Isc by 11.6 ± 0.9 (n = 20), 10.8 ± 1.7 (n = 18), 10.0 ± 1.6 (n = 5), and 7.9 ± 0.8 µA/cm2(n = 17), respectively. In F-HBE, 1-EBIO, NS004, and8-MOP failed to stimulate Cl secretion. However, additionof NS004 subsequent to forskolin induced a sustained Clsecretory response (2.1 ± 0.3 µA/cm2,n = 21). In F-HBE, genistein alone stimulatedCl secretion (2.5 ± 0.5 µA/cm2,n = 11). After incubation of F-HBE at 26°C for24 h, the responses to 1-EBIO, NS004, and genistein were allpotentiated. 1-EBIO and genistein increased Na+ absorptionacross F-HBE, whereas NS004 and 8-MOP had no effect. Finally,Ca2+-, but not cAMP-mediated agonists, stimulatedK+ secretion across both wt HBE and F-HBE in aglibenclamide-dependent fashion. Our results demonstrate thatpharmacological agents directed at both basolateral K+ andapical Cl conductances directly modulate Clsecretion across HBE, indicating they may be useful in ameliorating theion transport defect associated with CF. 相似文献