Current-voltage relations of the basolateral membrane in tight amphibian epithelia: Use of nystatin to depolarize the apical membrane

1984 ◽  
Vol 77 (3) ◽  
pp. 213-222 ◽  
Author(s):  
Haim Garty
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Current Voltage ◽  
Amphibian Epithelia

Effects of oxytocin on cation content and electrophysiology of frog skin epithelium

1988 ◽  
Vol 255 (3) ◽  
pp. C357-C367 ◽  
Author(s):  
H. F. Schoen ◽  
A. Kaufman ◽  
D. Erlij
Keyword(s):  
Frog Skin ◽  
Rana Catesbeiana ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Membrane Conductance ◽  
Short Circuit Current ◽  
Flame Photometry ◽  
Intracellular Na Activity ◽  
Current Voltage

We measured effects of oxytocin on current-voltage (I-V) relations of frog (Rana catesbeiana) skins impaled with an intracellular microelectrode. In both Cl- and Cl(-)-free (SO4(2-) solutions, oxytocin caused an approximate doubling short-circuit current (Isc) and a depolarization of the cell membrane. Increase in apical membrane slope conductance, chord conductance, and permeability after oxytocin correlated with the increase in amiloride-sensitive Isc. Oxytocin also increased basolateral membrane conductance (gb). In Cl-, the shift in the voltage intercept of the apical membrane I-V relation (Ea) implied increased intracellular Na+ activity (a(Na)c) after oxytocin. In isolated frog skin epithelia, a similar increase in intracellular [Na+] after oxytocin was demonstrated by flame photometry. In SO4(2-), changes caused by oxytocin in both Ea and in flame photometrically determined cell [Na+] were minimal. The voltage intercept of the basolateral membrane I-V relations (Eb) was shifted by oxytocin in both Cl- and SO4(2-) solutions. Assuming that the basolateral membrane is selectively permeable to K+, changes in K+ obtained from Eb were in disagreement with those obtained by flame photometry. These results suggest that 1) the increase in a(Na)c caused by oxytocin is not essential to produce either the increase in gb or Isc and 2) ions other than K+ make an important contribution to basolateral membrane conductance.

Dibutyryl cAMP activates bumetanide-sensitive electrolyte transport in Malpighian tubules

1991 ◽  
Vol 261 (3) ◽  
pp. C521-C529 ◽  
Author(s):  
J. L. Hegarty ◽  
B. Zhang ◽  
T. L. Pannabecker ◽  
D. H. Petzel ◽  
M. D. Baustian ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Fluid Secretion ◽  
Ringer Solution ◽  
Malpighian Tubules ◽  
Membrane Voltage ◽  
Dibutyryl Camp ◽  
K Secretion ◽  
Transepithelial Voltage

The effects of dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and bumetanide (both 10(-4) M) on transepithelial Na+, K+, Cl-, and fluid secretion and on tubule electrophysiology were studied in isolated Malpighian tubules of the yellow fever mosquito Aedes aegypti. Peritubular DBcAMP significantly increased Na+, Cl-, and fluid secretion but decreased K+ secretion. In DBcAMP-stimulated tubules, bumetanide caused Na+, Cl-, and fluid secretion to return to pre-cAMP control rates and K+ secretion to decrease further. Peritubular bumetanide significantly increased Na+ secretion and decreased K+ secretion so that Cl- and fluid secretion did not change. In bumetanide-treated tubules, the secretagogue effects of DBcAMP are blocked. In isolated Malpighian tubules perfused with symmetrical Ringer solution, DBcAMP significantly hyperpolarized the transepithelial voltage (VT) and depolarized the basolateral membrane voltage (Vbl) with no effect on apical membrane voltage (Va). Total transepithelial resistance (RT) and the fractional resistance of the basolateral membrane (fRbl) significantly decreased. Bumetanide also hyperpolarized VT and depolarized Vbl, however without significantly affecting RT and fRbl. Together these results suggest that, in addition to stimulating electroconductive transport, DBcAMP also activates a nonconductive bumetanide-sensitive transport system in Aedes Malpighian tubules.

Luminal flow rate regulates proximal tubule H-HCO3 transporters

1992 ◽  
Vol 262 (1) ◽  
pp. F47-F54 ◽  
Author(s):  
P. A. Preisig
Keyword(s):  
Proximal Tubule ◽  
Flow Rate ◽  
Initial Rate ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Rate Dependence ◽  
Unstirred Layer ◽  
Disulfonic Acid ◽  
Luminal Flow

In vivo microperfusion was used to examine the mechanism of luminal flow rate dependence of proximal tubule acidification. Luminal flow rate was acutely changed between 5 and 40 nl/min, while luminal and peritubular capillary composition were held constant. With inhibition of basolateral membrane base transport by peritubular 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), cell pH (pHi) provides a sensitive index of apical membrane H secretory activity. At a luminal perfusate [HCO3] of 25 mM, progressive increases in luminal flow rate (5----15----25----40 nl/min) caused progressive increases in pHi. This effect was of a smaller magnitude with a luminal perfusate [HCO3] of 60 mM and was further decreased at a luminal perfusate [HCO3] of 100 mM. This pattern of diminished flow rate dependence at higher luminal [HCO3] is consistent with the presence of a luminal unstirred layer, whose composition can be modified by luminal flow rate. The activity of the apical membrane Na-H antiporter, assayed as the initial rate of pHi recovery from an acid load in the presence of peritubular DIDS, was faster at 40 compared with 5 nl/min. Basolateral membrane Na-3HCO3 symporter activity, assayed as the initial rate of pHi recovery from an alkali load in the absence of luminal and peritubular chloride, was faster at 40 compared with 5 nl/min. This effect was eliminated by luminal amiloride, suggesting an indirect effect of flow mediated by changes in pHi secondary to flow rate-dependent changes in apical membrane Na-H antiporter activity. In summary, increases in luminal flow rate directly increase apical membrane H secretion, possibly by modification of a luminal unstirred layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Water permeability of apical and basolateral cell membranes of rat inner medullary collecting duct

1990 ◽  
Vol 259 (6) ◽  
pp. F986-F999 ◽  
Author(s):  
B. Flamion ◽  
K. R. Spring
Keyword(s):  
Water Flow ◽  
Water Permeability ◽  
Cell Membranes ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Basolateral Membrane ◽  
Volume Regulatory Decrease ◽  
Water Permeation ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

To quantify the pathways for water permeation through the kidney medulla, knowledge of the water permeability (Posmol) of individual cell membranes in inner medullary collecting duct (IMCD) is required. Therefore IMCD segments from the inner two thirds of inner medulla of Sprague-Dawley rats were perfused in vitro using a setup devised for rapid bath and luminal fluid exchanges (half time, t1/2, of 55 and 41 ms). Differential interference contrast microscopy, coupled to video recording, was used to measure volume and approximate surface areas of single cells. Volume and volume-to-surface area ratio of IMCD cells were strongly correlated with their position along the inner medullary axis. Transmembrane water flow (Jv) was measured in response to a variety of osmotic gradients (delta II) presented on either basolateral or luminal side of the cells. The linear relation between Jv and delta II yielded the cell membrane Posmol, which was then corrected for membrane infoldings. Basolateral membrane Posmol was 126 +/- 3 microns/s. Apical membrane Posmol rose from a basal value of 26 +/- 3 microns/s to 99 +/- 5 microns/s in presence of antidiuretic hormone (ADH). Because of amplification of basolateral membrane, the ADH-stimulated apical membrane remained rate-limiting for transcellular osmotic water flow, and the IMCD cell did not swell significantly. Calculated transcellular Posmol, expressed in terms of smooth luminal surface, was 64 microns/s without ADH and 207 microns/s with ADH. IMCD cells in anisosmotic media displayed almost complete volume regulatory decrease but only partial volume regulatory increase.

PBA increases CFTR expression but at high doses inhibits Cl−secretion in Calu-3 airway epithelial cells

1999 ◽  
Vol 277 (4) ◽  
pp. L700-L708 ◽  
Author(s):  
Johannes Loffing ◽  
Bryan D. Moyer ◽  
Donna Reynolds ◽  
Bruce A. Stanton
Keyword(s):  
Protein Expression ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Apical Plasma Membrane ◽  
Airway Epithelial ◽  
High Concentration ◽  
Cycle Enzyme ◽  
Cf Transmembrane Conductance Regulator

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl−secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl−secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na+-K+-ATPase. PBA reduced CFTR Cl−currents across the apical membrane but had no effect on Na+-K+-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl−secretion by inhibiting CFTR Cl−currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05–2 mM) had no effect on cAMP-stimulated Cl−secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl−secretion. However, concentrations >5 mM might reduce transepithelial Cl−secretion by serous cells in submucosal glands in individuals expressing wt CFTR.

scholarly journals Electrophysiological effects of basolateral [Na+] in Necturus gallbladder epithelium.

1992 ◽  
Vol 99 (2) ◽  
pp. 241-262 ◽  
Author(s):  
G A Altenberg ◽  
J S Stoddard ◽  
L Reuss
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Resistance ◽  
Membrane Voltage ◽  
Gallbladder Epithelium ◽  
K Channels ◽  
Necturus Gallbladder ◽  
Electrophysiological Effects ◽  
Paracellular Diffusion ◽  
Cl Conductance

In Necturus gallbladder epithelium, lowering serosal [Na+] ([Na+]s) reversibly hyperpolarized the basolateral cell membrane voltage (Vcs) and reduced the fractional resistance of the apical membrane (fRa). Previous results have suggested that there is no sizable basolateral Na+ conductance and that there are apical Ca(2+)-activated K+ channels. Here, we studied the mechanisms of the electrophysiological effects of lowering [Na+]s, in particular the possibility that an elevation in intracellular free [Ca2+] hyperpolarizes Vcs by increasing gK+. When [Na+]s was reduced from 100.5 to 10.5 mM (tetramethylammonium substitution), Vcs hyperpolarized from -68 +/- 2 to a peak value of -82 +/- 2 mV (P less than 0.001), and fRa decreased from 0.84 +/- 0.02 to 0.62 +/- 0.02 (P less than 0.001). Addition of 5 mM tetraethylammonium (TEA+) to the mucosal solution reduced both the hyperpolarization of Vcs and the change in fRa, whereas serosal addition of TEA+ had no effect. Ouabain (10(-4) M, serosal side) produced a small depolarization of Vcs and reduced the hyperpolarization upon lowering [Na+]s, without affecting the decrease in fRa. The effects of mucosal TEA+ and serosal ouabain were additive. Neither amiloride (10(-5) or 10(-3) M) nor tetrodotoxin (10(-6) M) had any effects on Vcs or fRa or on their responses to lowering [Na+]s, suggesting that basolateral Na+ channels do not contribute to the control membrane voltage or to the hyperpolarization upon lowering [Na+]s. The basolateral membrane depolarization upon elevating [K+]s was increased transiently during the hyperpolarization of Vcs upon lowering [Na+]s. Since cable analysis experiments show that basolateral membrane resistance increased, a decrease in basolateral Cl- conductance (gCl-) is the main cause of the increased K+ selectivity. Lowering [Na+]s increases intracellular free [Ca2+], which may be responsible for the increase in the apical membrane TEA(+)-sensitive gK+. We conclude that the decrease in fRa by lowering [Na+]s is mainly caused by an increase in intracellular free [Ca2+], which activates TEA(+)-sensitive maxi K+ channels at the apical membrane and decreases apical membrane resistance. The hyperpolarization of Vcs is due to increase in: (a) apical membrane gK+, (b) the contribution of the Na+ pump to Vcs, (c) basolateral membrane K+ selectivity (decreased gCl-), and (d) intraepithelial current flow brought about by a paracellular diffusion potential.

Inhibition of Na transport by 2-chloroadenosine: dissociation from production of cyclic nucleotides

1990 ◽  
Vol 68 (10) ◽  
pp. 1357-1362
Author(s):  
Russell F. Husted ◽  
Gerard P. Clancy ◽  
Abigail Adams-Brotherton ◽  
John B. Stokes
Keyword(s):  
Adenosine Receptors ◽  
Cyclic Nucleotides ◽  
Cell Function ◽  
Apical Membrane ◽  
Second Messengers ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Good Evidence ◽  
Camp Content ◽  
Adenosine Transport

The adenosine analogue 2-chloroadenosine (2-CA) is often used to determine the biologic effects of adenosine because 2-CA is less susceptible to degradation than adenosine. We studied the effects of 2-CA on primary cultures of rat inner medullary collecting ducts because there is good evidence that adenosine can influence cell function through its effects on second messengers. 2-CA inhibited Na+ transport across the apical membrane and increased cAMP content of the cells. The major adenosine receptors in these cells appear to be the stimulatory (A2) type. Stimulation of cAMP by 2-CA was more potent when applied to the apical membrane than to the basolateral membrane, an effect opposite to that of vasopressin. These results imply that adenosine receptors are more numerous or more effective on the apical membrane than on the basolateral membrane. Inhibition of Na+ transport was probably not mediated by an adenosine receptor as evidenced by (i) a lack of effect of adenosine and other adenosine analogues on Na+ transport; (ii) a lack of effect of nonmetabolizable cyclic nucleotides on Na+ transport; and (iii) a clear discrepancy in the temporal course of 2-CA effects on a second messenger system (cAMP) and 2-CA inhibition of Na+ transport. Dipyridimole, an inhibitor of adenosine transport, also reduced Na+ transport. Taken together, the data suggest that 2-CA inhibits Na+ transport by interfering with adenosine transport or metabolism.Key words: cAMP, cGMP, 2-chloroadenosine, vasopressin, Na+ transport, dipyridimole, adenosine metabolism.

Activation of CFTR Cl- conductance in polarized T84 cells by luminal extracellular ATP

1995 ◽  
Vol 268 (2) ◽  
pp. C425-C433 ◽  
Author(s):  
M. J. Stutts ◽  
E. R. Lazarowski ◽  
A. M. Paradiso ◽  
R. C. Boucher
Keyword(s):  
Adenosine Receptor ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Extracellular Atp ◽  
T84 Cells ◽  
Apical Cell Membrane ◽  
Cl Secretion ◽  
Cl Conductance

Luminal extracellular ATP evoked a bumetanide-sensitive short-circuit current in cultured T84 cell epithelia (90.2 +/- 18.2 microA/cm2 at 100 microM ATP, apparent 50% effective concentration, 11.5 microM). ATP appeared to increase the Cl- conductance of the apical membrane but not the driving force for Cl- secretion determined by basolateral membrane K+ conductance. Specifically, the magnitude of Cl- secretion stimulated by ATP was independent of basal current, and forskolin pretreatment abolished subsequent stimulation of Cl- secretion by ATP. Whereas ATP stimulated modest production of adenosine 3',5'-cyclic monophosphate (cAMP) by T84 cells, ATP caused smaller increases in intracellular Ca2+ and inositol phosphate activities than the Ca(2+)-signaling Cl- secretagogue carbachol. An inhibitor of 5'-nucleotidase, alpha,beta-methyleneadenosine 5'-diphosphate, blocked most of the response to luminal ATP. The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline blocked both the luminal ATP-dependent generation of cAMP and Cl- secretion when administered to the luminal but not submucosal bath. These results demonstrate that the Cl- secretion stimulated by luminal ATP is mediated by a A2-adenosine receptor located on the apical cell membrane. Thus metabolism of extracellular ATP to adenosine regulates the activity of cystic fibrosis transmembrane conductor regulator (CFTR) in the apical membrane of polarized T84 cells.

scholarly journals Polarized Distribution of IQGAP Proteins in Gastric Parietal Cells and Their Roles in Regulated Epithelial Cell Secretion

2003 ◽  
Vol 14 (3) ◽  
pp. 1097-1108 ◽  
Author(s):  
Rihong Zhou ◽  
Zhen Guo ◽  
Charles Watson ◽  
Emily Chen ◽  
Rong Kong ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Actin Cytoskeleton ◽  
Acid Secretion ◽  
Rho Gtpase ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Parietal Cells ◽  
Actin Dynamics ◽  
Cell Secretion ◽  
Gastric Parietal Cells

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.

scholarly journals Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

1992 ◽  
Vol 103 (3) ◽  
pp. 677-687 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Transport Pathways ◽  
Intracellular Targeting ◽  
Polarized Delivery ◽  
Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

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