Histamine-induced Cl- secretion in human nasal epithelium: responses of apical and basolateral membranes

1992 ◽  
Vol 263 (6) ◽  
pp. C1190-C1199 ◽  
Author(s):  
L. L. Clarke ◽  
A. M. Paradiso ◽  
R. C. Boucher
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Secretory Response ◽  
Bathing Solution ◽  
Equivalent Circuit Analysis ◽  
Cl Secretion ◽  
Influx Pathways ◽  
Cl Conductance

The mechanism by which receptors coupled to phospholipase C (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (10(-4) M, basolateral administration) stimulated a rapid increase in equivalent short-circuit current, an index of Cl- secretion, that returned to baseline within 5 min. Intracellular recordings with double-barreled Cl(-)-selective microelectrodes showed that the apical and basolateral membrane potentials rapidly hyperpolarized, the fractional resistance of the apical membrane increased, and the transepithelial resistance decreased in response to histamine. Intracellular Cl- activity remained constant. Equivalent circuit analysis revealed that the early portion (< 0.9 min) of the Cl- secretory response was driven by an activation of a hyperpolarizing basolateral conductance, likely K+, whereas the later (> 0.9 min) phase of Cl- secretion reflects activation of the apical membrane Cl- conductance. Histamine raised intracellular Ca2+ (Ca2+i) measured by fura-2 in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasma membrane influx pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (15 microM), coupled with reduced bathing solution Ca2+, blunted the rise in Ca2+i and the net transepithelial Cl- secretory response to histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance, and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca2+i may participate in the activation of both the basolateral and apical conductances.

Electrolyte transport by alkaline gland of little skate Raja erinacea

1985 ◽  
Vol 248 (3) ◽  
pp. R346-R352
Author(s):  
P. L. Smith
Keyword(s):  
Membrane Potential ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Transepithelial Resistance ◽  
Bathing Solution ◽  
Raja Erinacea ◽  
Cl Secretion ◽  
Transepithelial Potential

Transepithelial flux studies and conventional intracellular microelectrode measurements were employed to examine the mechanisms of ion transport by the alkaline gland of the male skate, Raja erinacea. These studies reveal that the transepithelial potential is 6.9 +/- 0.6 mV, lumen reference, and that the transepithelial resistance is 140 omega . cm2. The short-circuit current across this epithelium is entirely accounted for by net secretion of Cl, whereas transepithelial active transport of Na does not appear to be present in this tissue. Cl secretion and/or short-circuit current are reduced by serosal furosemide and abolished when the bathing solution Na is replaced with choline or when ouabain is added to the serosal bathing solution. Intracellular microelectrode studies reveal that the apical membrane potential is -43 mV, cell interior negative to the mucosal bathing solution. The transepithelial resistance in these tissues was 103 +/- 12 omega . cm2 and the apparent fractional resistance, i.e., the ratio of the change in apical membrane potential to the change in transepithelial potential produced by passing current across the epithelium was 0.39 +/- 0.09. Ion substitution experiments demonstrated that the apical membrane is dominated by a large Cl conductance while the basolateral membrane contains a barium-sensitive potassium conductance. These results suggest that the mechanism of Cl secretion by the alkaline gland is similar to the mechanism described for a variety of Cl secretory epithelia.

Effects of bradykinin on Na+ and Cl- transport in human nasal epithelium

1992 ◽  
Vol 262 (3) ◽  
pp. C644-C655 ◽  
Author(s):  
L. L. Clarke ◽  
A. M. Paradiso ◽  
S. J. Mason ◽  
R. C. Boucher
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Nasal Epithelium ◽  
Induced Response ◽  
Equivalent Circuit Analysis ◽  
Human Nasal Epithelium ◽  
Cl Conductance

Human nasal epithelium (HNE) is a Na+ absorptive epithelium but establishes a baseline Cl- secretory current in the presence of amiloride (10(-4) M, luminal). We compared the effects of an inflammatory mediator, bradykinin (BK), on ion transport in primary cultures of HNE using double-barreled Cl(-)-selective microelectrodes. In untreated HNE, BK (10(-5) M) transiently increased the equivalent short-circuit current (Ieq). Maximal Ieq occurred with hyperpolarization of the transepithelial potential difference (Vt), which was associated with hyperpolarization and decreased resistance of the basolateral membrane; a subsequent depolarization of Vt was observed that was associated with depolarization and decreased resistance of the apical membrane. Removal of bath Cl- did not affect the BK-induced Ieq response. In amiloride-treated HNE, the electrical pattern of the BK-induced response was identical, but the magnitude of the Ieq was reduced by 54% and the change in Ieq could be abolished by removal of bath Cl-. Equivalent-circuit analysis of the response in amiloride-treated tissues indicated activation of a hyperpolarizing conductance in the basolateral membrane, followed 20-30 s later by activation of an apical Cl- conductance. We conclude that BK stimulates both Na+ absorption in untreated HNE and Cl- secretion in amiloride-treated HNE by activating a basolateral (K+) conductance. Analysis of the entire Ieq response under both conditions also suggested that BK induces a delayed activation of apical membrane Na+ and Cl- conductances.

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.

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

2001 ◽  
Vol 281 (2) ◽  
pp. C633-C648 ◽  
Author(s):  
Sasha Blaug ◽  
Kevin Hybiske ◽  
Jonathan Cohn ◽  
Gary L. Firestone ◽  
Terry E. Machen ◽  
...  
Keyword(s):  
Tight Junctions ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Fluid Secretion ◽  
Equivalent Circuit Analysis ◽  
Mean Values ◽  
Apical Side

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers on filters to analyze the apical membrane mechanisms that help mediate ion and fluid transport across the epithelium. RT-PCR showed the presence of cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial Na+ channel (ENaC) message, and immunomicroscopy showed apical membrane staining for both proteins. CFTR was also localized to the apical membrane of native human mammary duct epithelium. In control conditions, mean values of transepithelial potential (apical-side negative) and resistance ( R T) are −5.9 mV and 829 Ω · cm2, respectively. The apical membrane potential ( V A) is −40.7 mV, and the mean ratio of apical to basolateral membrane resistance ( R A/ R B) is 2.8. Apical amiloride hyperpolarized V A by 19.7 mV and tripled R A/ R B. A cAMP-elevating cocktail depolarized V A by 17.6 mV, decreased R A/ R B by 60%, increased short-circuit current by 6 μA/cm2, decreased R T by 155 Ω · cm2, and largely eliminated responses to amiloride. Whole cell patch-clamp measurements demonstrated amiloride-inhibited Na+ currents [linear current-voltage ( I-V) relation] and forskolin-stimulated Cl−currents (linear I-V relation). A capacitance probe method showed that in the control state, MEC monolayers either absorbed or secreted fluid (2–4 μl · cm−2 · h−1). Fluid secretion was stimulated either by activating CFTR (cAMP) or blocking ENaC (amiloride). These data plus equivalent circuit analysis showed that 1) fluid absorption across MEC is mediated by Na+ transport via apical membrane ENaC, and fluid secretion is mediated, in part, by Cl− transport via apical CFTR; 2) in both cases, appropriate counterions move through tight junctions to maintain electroneutrality; and 3) interactions among CFTR, ENaC, and tight junctions allow MEC to either absorb or secrete fluid and, in situ, may help control luminal [Na+] and [Cl−].

Sodium and chloride transport across the isolated porcine gallbladder

1989 ◽  
Vol 257 (1) ◽  
pp. C45-C51 ◽  
Author(s):  
S. M. O'Grady ◽  
P. J. Wolters
Keyword(s):  
Apical Membrane ◽  
Chloride Transport ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ringer Solution ◽  
Disulfonic Acid ◽  
Ussing Chambers ◽  
Cl Secretion ◽  
Conductive Pathway

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.

Activation of an apical Cl- conductance by Ca2+ ionophores in cystic fibrosis airway epithelia

1989 ◽  
Vol 256 (2) ◽  
pp. C226-C233 ◽  
Author(s):  
N. J. Willumsen ◽  
R. C. Boucher
Keyword(s):  
Cystic Fibrosis ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelia ◽  
Cl Secretion ◽  
Human Nasal Epithelium ◽  
Collagen Membranes ◽  
Cl Conductance ◽  
Dependent Mechanism

Cystic fibrosis (CF) airway epithelia express a defect in adenosine 3',5'-cyclic monophosphate (cAMP)-dependent regulation of apical membrane Cl- channels. Recent patch-clamp studies have raised the possibility that Ca2+ -dependent mechanisms for the activation of Cl- secretion may be preserved in CF airway epithelia. To determine 1) whether intact normal (N1) and CF airway epithelia exhibit a Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanism for activation of Cl- secretion and 2) whether Ca2+ -dependent mechanisms initiate Cl- secretion via activation of an apical membrane Cl- conductance (GCl-), nasal epithelia from N1 and CF subjects were cultured on collagen membranes, and responses to isoproterenol or Ca2- ionophores [A23187 10(-6) M; ionomycin (10(-5)M)] were measured with transepithelial and intracellular techniques. Isoproterenol induced activation of an apical membrane GCl- in N1 cultures but was ineffective in CF. In contrast, in both N1 and CF amiloride-pretreated cultures, A23187 induced an increase in the equivalent short-circuit current that was associated with an activation of an apical membrane Gc1- and was bumetanide inhibitable. A23187 addition during superfusion of the lumen with a low Cl- (3 mM) solution reduced intracellular Cl- activity of CF cells. A Ca2+ ionophore of different selectivity properties, ionomycin, was also an effective Cl- secretagogue in both N1 and CF cultures. We conclude that 1) the A23187 induced Cl- secretion via activation of an apical GCl- in N1 human nasal epithelium, and 2) in contrast to an isoproterenol-dependent path, a Ca2+ -dependent path for GCl- activation is preserved in CF epithelia.

Modulation of Cl- secretion by benzimidazolones. I. Direct activation of a Ca(2+)-dependent K+ channel

1996 ◽  
Vol 271 (5) ◽  
pp. L775-L784 ◽  
Author(s):  
D. C. Devor ◽  
A. K. Singh ◽  
R. A. Frizzell ◽  
R. J. Bridges
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Secretory Response ◽  
K Channel ◽  
T84 Cells ◽  
K Channels ◽  
Cl Secretion ◽  
Direct Activation

We evaluated the effects of the novel benzimidazolone, 1-ethyl-2-benzimidazolinone (1-EBIO), on Cl- secretion across T84 monolayers. 1-EBIO stimulated a sustained Cl- secretory response at a half-maximal effective concentration of 490 microM. Charybdotoxin (CTX) inhibited the 1-EBIO-induced short-circuit current (Isc) with an inhibitory constant (Ki) of 3.6 nM, whereas 293B, an inhibitor of adenosine 3',5'-cyclic monophosphate-activated K+ channels, had no effect on the current induced by 1-EBIO. In contrast, CTX failed to inhibit the 293B-sensitive forskolin-induced Isc. The above results suggested that 1-EBIO may be activating the basolateral membrane Ca(2+)-dependent K+ channel (KCa) in these cells. This was further confirmed using nystatin to permeabilize the apical membrane in the presence of a mucosa-to-serosa K+ gradient and determining the effects of 1-EBIO on the basolateral K+ current (IK). Under these conditions, 1-EBIO induced a large increase in IK that was blocked by CTX. In membrane vesicles prepared from T84 cells, 1-EBIO stimulated 86Rb+ uptake in a CTX-sensitive manner; the Ki for inhibition by CTX was 3.5 nM. Similar to our intact monolayer studies, this 86Rb+ uptake was not blocked by 293B. The effects of 1-EBIO on the KCa in T84 cells was determined in excised inside-out patches. 1-EBIO (100 microM) increased the product of the number of channels and the open channel probability from 0.09 +/- 0.03 to 1.17 +/- 0.27 (n = 8); this effect on KCa activity required a minimal level of free Ca2+. Similar to its effect on T84 cells, 1-EBIO stimulated a sustained Cl- secretory current in rat colonic epithelium, which was partially blocked by CTX. Finally, 1-EBIO stimulated a sustained Cl- secretory response in primary cultures of murine tracheal epithelium. We conclude that the benzimidazolone, 1-EBIO, stimulates Cl- secretion in secretory epithelia via the direct activation of a Kca. 1-EBIO is the first pharmacological opener of this important class of epithelial K+ channels to be identified.

Ba2+ inhibition of VIP- and A23187-stimulated Cl- secretion by T84 cell monolayers

1986 ◽  
Vol 250 (3) ◽  
pp. C486-C494 ◽  
Author(s):  
K. G. Mandel ◽  
J. A. McRoberts ◽  
G. Beuerlein ◽  
E. S. Foster ◽  
K. Dharmsathaphorn
Keyword(s):  
Vasoactive Intestinal Polypeptide ◽  
Apical Membrane ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Ussing Chambers ◽  
Cell Monolayers ◽  
Cl Secretion ◽  
Cotransport System ◽  
Permeable Supports

Addition of either 10(-8) M vasoactive intestinal polypeptide (VIP) or 10(-6) M A23187 to T84 cell monolayers, grown on permeable supports and mounted in Ussing chambers, stimulated net Cl- secretion. The effect of 10(-6) M A23187 on Cl- flux was consistently smaller than that observed with 10(-8) M VIP. In both cases the increase in net Cl- secretion accounted for the entire change in the observed short-circuit current (Isc). Since Cl- enters the cells through a basolaterally localized Na+-K+-Cl(-)-cotransport system (J. Clin. Invest. 75: 462, 1985), the fate of K+, which is cotransported with Cl- during VIP, and A23187-mediated Cl- secretion was explored. Unidirectional and net transepithelial 42K+ flux rates were negligible compared with 36Cl- flux rates (less than 4% of Cl- flux), indicating that little K+ was secreted along with Cl-. K+ recycling across the basolateral membrane was suggested from experiments in which 86Rb+ efflux (as a tracer for K+) was measured across the apical and basolateral membranes of 86Rb+ -preloaded monolayers under voltage-clamped conditions. In the absence of secretagogues, 86Rb+ efflux was 10-fold higher across the basolateral membrane than across the apical membrane. 86Rb+ efflux across the basolateral membrane was accelerated two- to threefold by addition of either VIP or A23187. In each case accelerated efflux was inhibited by 5 mM Ba2+. Cl- secretion induced by VIP or A23187 was also inhibited by serosal addition of Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Lovastatin inhibits cAMP- and calcium-stimulated chloride secretion by T84 cells

1993 ◽  
Vol 265 (2) ◽  
pp. C422-C431 ◽  
Author(s):  
T. W. Ecay ◽  
J. D. Valentich
Keyword(s):  
Apical Membrane ◽  
Short Circuit ◽  
Membrane Vesicle ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Cell Types ◽  
T84 Cells ◽  
Cyclic Monophosphate ◽  
Protein Isoprenylation ◽  
Cl Conductance

Isoprenylated proteins function in the processes of signal transduction and membrane vesicle trafficking. To investigate the role of isoprenylated proteins in secretagogue-stimulated epithelial ion transport, we studied the effects of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-stimulated Cl- secretion by monolayers of T84 colonic epithelial cells. Lovastatin reduces protein isoprenylation in many cell types. In T84 cells, lovastatin reversibly inhibits forskolin-stimulated equivalent short-circuit current (I(sc)eq) by 50% after 2 days of treatment. The concentration of lovastatin resulting in half-maximal effects on forskolin-stimulated I(sc)eq is consistent with inhibition of protein isoprenylation, and lovastatin effects on forskolin-stimulated I(sc)eq are not associated with inhibition of cholesterol or glycoprotein biosynthesis. Lovastatin blocks N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate- and ionomycin-stimulated Isc, suggesting that it inhibits a process beyond the stimulation of cAMP and Ca2+ second-messenger systems. In monolayers in which the basolateral membrane has been permeabilized with nystatin, lovastatin inhibits cAMP activation of a diphenylamine-2-carboxylate-sensitive, apical membrane Cl- conductance. Our results are consistent with the hypothesis that an isoprenylated protein is involved in the regulation of a secretagogue-activated apical membrane Cl- conductance in T84 cells.

scholarly journals Zinc inhibits cAMP-stimulated Cl secretion via basolateral K-channel blockade in rat ileum

2005 ◽  
Vol 288 (5) ◽  
pp. G956-G963 ◽  
Author(s):  
Kazi Mirajul Hoque ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
K Channel ◽  
Isolated Cells ◽  
Rat Ileum ◽  
Anion Secretion ◽  
Cl Secretion ◽  
Ion Absorption ◽  
Isotonic Buffer

Zn, an essential micronutrient and second most abundant trace element in cell and tissues, reduces stool output when administered to children with acute diarrhea. The mechanism by which Zn improves diarrhea is not known but could result from stimulating Na absorption and/or inhibiting anion secretion. The aim of this study was to investigate the direct effect of Zn on intestinal epithelial ion absorption and secretion. Rat ileum was partially stripped of serosal and muscle layers, and the mucosa was mounted in lucite chambers. Potential difference and short-circuit current were measured by conventional current-voltage clamp method.86Rb efflux and uptake were assessed for serosal K channel and Na-K-2Cl cotransport activity, respectively. Efflux experiments were performed in isolated cells preloaded with86Rb in the presence of ouabain and bumetanide, whereas uptake experiments were performed in low-Cl isotonic buffer containing Ba and ouabain. Neither mucosal nor serosal Zn affected glucose-stimulated Na absorption. In contrast, forskolin-induced Cl secretion was markedly reduced by serosal but not mucosal addition of Zn. Zn also substantially reversed the increase in Cl secretion induced by 8-bromoadenosine 3′,5′-cyclic monophosphate (8-BrcAMP) with half-maximal inhibitory concentration of 0.43 mM. In contrast, serosal Zn did not alter Cl secretion stimulated by carbachol, a Ca-dependent agonist. Zn inhibited 8-BrcAMP-stimulated86Rb efflux but not carbachol-stimulated86Rb efflux. Zn had no effect on bumetanide-sensitive86Rb uptake, Na-K-ATPase, or CFTR. We conclude from these studies that Zn inhibits cAMP-induced Cl secretion by blocking basolateral membrane K channels.

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