Side specific effect of 5-hydroxytryptamine on NaCl transport in the apical and basolateral membrane of rat tracheal epithelia

5-Hydroxytryptamine (5-HT) can be released from mast cells and platelets through an IgE-dependent mechanism and may play a role in the pathogenesis of allergic bronchoconstriction. However, the effect of 5-HT on ion transport by the airway epithelium is still controversial. The objective of this study was to determine whether 5-hydroxytryptamine (5-HT) regulates NaCl transport by different mechanisms in the apical and basolateral membrane of tracheal epithelia. We studied the rat tracheal epithelium under short-circuit conditions in vitro. Short-circuit current (I(sc)) was measured in rat tracheal epithelial monolayers cultured on porous filters. 5-HT inhibited Na(+) absorption [measured via Na(+) short-circuit current (I(Na)(sc))] in the apical membrane and stimulated Cl(-) secretion [measured via Cl(-) short-circuit current (I(Cl)(sc))] in the basolateral membrane. Functional localization using selective 5-HT agonists and antagonists suggest that I(Cl)(sc)is stimulated by the basolateral membrane-resident 5-HT receptors, whereas I(Na)(sc) is inhibited by the apical membrane-resident 5-HT2 receptors. The basolateral addition of 5-HT increases intracellular cAMP content, but its apical addition does not. The addition of BAPTA/AM blocked the decrease of I(Na)(sc)which was induced by the apical addition of 5-HT, and 5-HT increased intracellular Ca concentrations. These results indicate that 5-HT differentially affects I(Na)(sc)and I(Cl)(sc)across rat tracheal monolayers through interactions with distinct receptors in the apical and the basolateral membrane. These effects may result in an increase of water movement towards the airway lumen.     

The mitochondrial barriers segregate agonist-induced calcium-dependent functions in human airway epithelia

In airway epithelia, purinergic receptor (P2Y2-R) stimulation of intracellular calcium (Ca2+i)-regulated ion transport is restricted to the membrane domain ipsilateral to receptor activation, implying compartmentalization of Ca2+i signaling. Because mitochondria can spatially restrict cellular Ca2+i signals, immunocytochemical, electron microscopic, and fluorescent studies of mitochondria localization were performed in human airway epithelia. Although concentrated at the apical domain, mitochondria were found distributed at both the apical and the basolateral poles and in close association with the endoplasmic reticulum. The role of mitochondria in locally restricting P2Y2-R-induced Ca2+i signals was investigated by measuring changes in mitochondrial Ca2+ (Ca2+m) in human airway epithelial monolayers. P2Y2-R activation induced Ca2+m accumulation in mitochondria confined to the domain ipsilateral to P2Y2-R stimulation, which was blocked by mitochondrial uncoupling with 1 microM CCCP and 2.5 microg/ml oligomycin. The role of mitochondria in restricting the cellular cross-talk between basolateral P2Y2-R-dependent Ca2+i mobilization and apical membrane Ca2+-activated Cl- secretion was investigated in studies simultaneously measuring Ca2+i and Cl- secretion in cystic fibrosis human airway epithelial monolayers. Activation of basolateral P2Y2-Rs produced similar increases in Ca2+i in monolayers without and with pretreatment with uncouplers, whereas Ca2+i-activated Cl- secretion was only efficiently triggered in mitochondria-uncoupled conditions. We conclude that (a) mitochondria function as a Ca2+i-buffering system in airway epithelia, compartmentalizing Ca2+i-dependent functions to the membrane ipsilateral to receptor stimulation; and (b) the mitochondria provide structural barriers that protect the airway epithelia against nonspecific activation of Ca2+i-modulated functions associated with Ca2+i signals emanating from the apical or the basolateral membrane domains.     

Calcium-stimulated short-circuit currents in the canine proximal colonic epithelium: effects of DK-PGD2, a metabolite of prostaglandin D2

Prostaglandin D2 (PGD2) has marked inhibitory effects on the canine proximal colonic epithelium set up in Ussing chambers. These effects involved a receptor that is pharmacologically distinct from the classical DP, presumably the recently identified CRTH2/DP2 variety. The mechanism underlying these effects was studied using 13,14-dihydro-15-keto-PGD2 (DK-PGD2), a stable metabolite of the parent prostanoid. The metabolite quickly reversed short circuit currents (I(sc)) stimulated by diverse agonists. Greater inhibitory effects were seen with stimulants such as carbachol and cyclopiazonic acid (CPA) rather than with forskolin or protein kinase A activators. Since the same stimulants were differentially affected by removal and replacement of serosal Ca2+, we tested the possibility that the prostanoid inhibited basolateral Ca2+ entry. In the absence of serosal Ca2+, tissues primed with CPA demonstrated concentration-dependent increases in I(sc), to cumulative additions of Ca2+ or Sr2+, though the former was more potent. Cl- removal and pretreatment with bumetanide virtually abolished responses, suggesting that the increase in I(sc) reflected Ca2+ dependent Cl- secretion. Though responses were insensitive to the L-type channel antagonist, verapamil, a marked inhibition was seen in the presence of metal cations (Gd3+, Cd2+, and La3+). Pretreatment with DK-PGD2 inhibited responses to Ca2+ in CPA-primed tissues. Thus, basolateral Ca2+ entry via store-operated Ca2+ channels may be the locus for the inhibitory effects of PGD2 in this tissue. These results could indicate a potential transduction mechanism for the novel DP receptor variously called CRTH2 or DP2.     

Apical trypsin increases ion transport and resistance by a phospholipase C-dependent rise of Ca2+

We investigated the mechanisms by which serine proteases alter lung fluid clearance in rat lungs and vectorial ion transport in airway and alveolar epithelial cells. Inhibition of endogenous protease activity by intratracheal instillation of soybean trypsin inhibitor (SBTI) or alpha(1)-antitrypsin decreased amiloride-sensitive lung fluid clearance across rat fluid-filled lungs; instillation of trypsin partially restored this effect. Gelatin zymography demonstrated SBTI-inhibitable trypsin-like activity in rat lung lavage fluid. Apical trypsin and human neutrophil elastase, but not agonists of protease activated receptors, increased Na(+) and Cl(-) short-circuit currents (I(sc)) and transepithelial resistance (R(TE)) across human bronchial and nasal epithelial cells and rat alveolar type II cells, mounted in Ussing chambers, for at least 2 h. The increase in I(sc) was fully reversed by amiloride and glibenclamide. The increase in R(TE) was not prevented by ouabain, suggesting that trypsin decreased paracellular conductance. Apical trypsin also induced a transient increase in intracellular Ca(2+) in human airway cells; treatment of these cells with BAPTA-AM mitigated the trypsin-induced increases of intracellular Ca(2+) and of I(sc) and R(TE). Increasing intracellular Ca(2+) in airway cells with either ionomycin or thapsigargin reproduced the increase in I(sc), whereas inhibitors of phospholipase C (PLC) prevented the increases in both Ca(2+) and I(sc). These data indicate trypsin-like proteases and elastase, either present in lung cells or released by inflammatory cells into the alveolar space, play an important role in the clearance of alveolar fluid by increasing ion transport and paracellular resistance via a PLC-initiated rise of intracellular Ca(2+).     

EGF receptor transactivation and MAP kinase mediate proteinase-activated receptor-2-induced chloride secretion in intestinal epithelial cells

We examined the stimulus-secretion pathways whereby proteinase-activated receptor 2 (PAR-2) stimulates Cl(-) secretion in intestinal epithelial cells. SCBN and T84 epithelial monolayers grown on Snapwell supports and mounted in modified Ussing chambers were activated by the PAR-2-activating peptides SLIGRL-NH(2) and 2-furoyl-LIGRLO-NH(2). Short-circuit current (I(sc)) was used as a measure of net electrogenic ion transport. Basolateral, but not apical, application of SLIGRL-NH(2) or 2-furoyl-LIGRLO-NH(2) caused a concentration-dependent change in I(sc) that was significantly reduced in Cl(-)-free buffer and by the intracellular Ca(2+) blockers thapsigargin and BAPTA-AM, but not by the Ca(2+) channel blocker verapamil. Inhibitors of PKA (H-89) and CFTR (glibenclamide) also significantly reduced PAR-2-stimulated Cl(-) transport. PAR-2 activation was associated with increases in cAMP and intracellular Ca(2+). Immunoblot analysis revealed increases in phosphorylation of epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase, Src, Pyk2, cRaf, and ERK1/2 in response to PAR-2 activation. Pretreatment with inhibitors of cyclooxygenases (indomethacin), tyrosine kinases (genistein), EGFR (PD-153035), MEK (PD-98059 or U-0126), and Src (PP1) inhibited SLIGRL-NH(2)-induced increases in I(sc). Inhibition of Src, but not matrix metalloproteinases, reduced EGFR phosphorylation. Reduced EGFR phosphorylation paralleled the reduction in PAR-2-stimulated I(sc). We conclude that activation of basolateral, but not apical, PAR-2 induces epithelial Cl(-) secretion via cAMP- and Ca(2+)-dependent mechanisms. The secretory effect involves EGFR transactivation by Src, leading to subsequent ERK1/2 activation and increased cyclooxygenase activity.     

Effects of carbon monoxide on ion transport across rat distal colon

The aim of the present study was to investigate whether carbon monoxide (CO) induces changes in ion transport across the distal colon of rats and to study the mechanisms involved. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (CORM-2), a CO donor, evoked a concentration-dependent increase in short-circuit current (I(sc)). A maximal response was achieved at a concentration of 2.5·10(-4) mol/l. Repeated application of CORM-2 resulted in a pronounced desensitization of the tissue. Anion substitution experiments suggest that a secretion of Cl(-) and HCO(3)(-) underlie the CORM-2-induced current. Glibenclamide, a blocker of the apical cystic fibrosis transmembrane regulator channel, inhibited the I(sc) induced by the CO donor. Similarly, bumetanide, a blocker of the basolateral Na(+)-K(+)-2Cl(-) cotransporter, combined with 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid sodium salt, an inhibitor of the basolateral Cl(-)/HCO(3)(-) exchanger, inhibited the CORM-2-induced I(sc). Membrane permeabilization experiments indicated an activation of basolateral K(+) and apical Cl(-) channels by CORM-2. A partial inhibition by the neurotoxin, tetrodotoxin, suggests the involvement of secretomotor neurons in this response. In imaging experiments at fura-2-loaded colonic crypts, CORM-2 induced an increase of the cytosolic Ca(2+) concentration. This increase depended on the influx of extracellular Ca(2+), but not on the release of Ca(2+) from intracellular stores. Both enzymes for CO production, heme oxygenase I and II, are expressed in the colon as observed immunohistochemically and by RT-PCR. Consequently, endogenous CO might be a physiological modulator of colonic ion transport.     

On the role of calcium in the mechanism of ADH action

With an increased influx of Ca2+ in the cytoplasm, the response of cells to ADH in the urinary bladder of the frog was lowered by addition of ionophore A23187 from the side of the basolateral cell membrane, but inhibited when it was added from the apical cell membrane. The removal of calcium by EGTA from the serosal surface was accompanied by a sharp increase of osmotic permeability not only to water, but also to inulin; while when calcium was removed from the mucosal surface of the urinary bladder, osmotic permeability was not changed. After being added to the Ringer solution from the outer surface of the apical cell membrane, the inhibitors of Ca2+ channels (verapamil, Ni2+, Mn2+, Co2+) decreased the effect of ADH. These data indicate that Ca2+ applied onto the outer surface of apical plasma membrane plays an important role in the action of ADH.     

House dust mite extract activates apical Cl− channels through protease‐activated receptor 2 in human airway epithelia

Adequate fluid secretion from airway mucosa is essential for maintaining mucociliary clearance, and fluid hypersecretion is a prominent feature of inflammatory airway diseases such as allergic rhinitis. House dust mite extract (HDM) has been reported to activate protease‐activated receptors (PARs), which play various roles in airway epithelia. However, the role of HDM in regulating ion transporters and fluid secretion has not been investigated. We examined the effect of HDM on ion transport in human primary nasal epithelial cells. The Ca²⁺‐sensitive dye Fura2‐AM was used to determine intracellular Ca²⁺ concentration ([Ca²⁺]_i) by means of spectrofluorometry in human normal nasal epithelial cells (NHNE). Short‐circuit current (Isc) was measured using Ussing chambers. Fluid secretion from porcine airway mucosa was observed by optical measurement. HDM extract (10 µg/Ml) effectively cleaved the PAR‐2 peptide and induced an increase of [Ca²⁺]_i that was abolished by desensitization with trypsin, but not with thrombin. Apical application of HDM‐induced Isc sensitive to both a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and a Ca²⁺‐activated Cl^? channel (CaCC) inhibitor. HDM extract also stimulated fluid secretion from porcine airway mucosa. HDM extract activated PAR‐2 and apical Cl^? secretion via CaCC and CFTR, and HDM‐induced fluid secretion in porcine airway mucosa. Our results suggest a role for PAR‐2 in mucociliary clearance and fluid hypersecretion of airway mucosa in response to air‐borne allergens such as HDM. J. Cell. Biochem. 109: 1254–1263, 2010. © 2010 Wiley‐Liss, Inc.     

COX-dependent and -independent pathways in bradykinin-induced anion secretion in rat epididymis

Lysylbradykinin (LBK) added to the apical or basolateral side of cultured rat epididymal monolayers stimulated a rise in short-circuit current (Isc) due to anion secretion. The concentration-response relationships for the apical and basolateral applications have EC50 value of 0.001 microM. The responses to apical or basolateral application of LBK were blocked by WIN64338, a specific B2 receptor antagonist, but not by Des-Arg9,[Leu8]-BK, a specific B1 receptor antagonist, indicating that the LBK effects were mediated through B2 bradykinin receptors. Experiments to desensitize the B2 receptors by repeated stimulation have demonstrated that the responses to apical or basolateral LBK were due to discrete receptors on the apical or basolateral surface. In epithelia clamped in the Ussing chambers, addition of LBK to the apical or basolateral surface evoked release of PGE2 into the apical and basolateral bathing solutions over the first 10 min following hormone addition. LBK added to the basolateral side elicited a greater release than it was added to the apical side. Pretreatment of the epithelia with piroxicam (5 microM) abolished PGE2 release elicited by apical or basolateral LBK and abrogated the Isc induced by basolateral LBK. However, the rise in Isc induced by apical LBK was reduced by 31.3% only. The anion secretion response to apical LBK was not affected by MDL-12330A, an adenylate cyclase inhibitor, but greatly attenuated by thapsigargin, an inhibitor of intracellular Ca2+ release. However, the reverse effects were seen for basolateral LBK. It is concluded that distinct pathways are involved in the stimulation of anion secretion by apical or basolateral LBK. The response to basolateral LBK was COX-dependent, mediated by PGE2 and involves cAMP as second messenger. In contrast, the response to apical LBK is largely COX-independent, not mediated by PCE2 and involves Ca2+ as intracellular messenger.     

Signal transduction of mucous secretion by bronchial gland cells

Bronchial glands, which consist of mucous and serous cells, are abundant in human airways, playing a major role in the airway secretion. Cl(-) secretion is accompanied by water transport to the lumen in the acinar cells of bronchial glands. Agonists that increase [Ca(2+)]i induce the Cl(-) secretion in bronchial glands. Ca(2+) release from a IP(3)-sensitive Ca(2+) pool at the apical portion stimulates and opens Ca(2+)-sensitive Cl(-) channels at the apical membrane, producing Cl(-) secretion in bronchial glands. K(+) channels at the basolateral membranes are Ca(2+)-sensitive and activated by Ca(2+) release from a cADPribose-sensitive Ca(2+) pool, maintaining the Cl(-) secretion in bronchial glands. Further, cADP ribose in concert with IP(3) induce [Ca(2+)]i oscillation, inducing Cl(-) secretion in bronchial glands. Some tyrosine kinases are involved in the Cl(-) secretion in bronchial glands. Mucous and serous cells in bronchial glands take part in mucin secretion and the secretion of defensive substances (glycoconjugates), respectively. [Ca(2+)]i oscillations are shown to play a central role in the exocytosis of secretory granules in serous cells of bronchial glands. Other signal transductions of mucin and glycoconjugates in airway gland cells remain to be studied, although agonists which increase [cAMP]i are also well known to induce mucin and glycoconjugate secretion from airway glands.     

Endogenous ATP release inhibits electrogenic Na+ absorption and stimulates Cl− secretion in MDCK cells

Our previous studies with a line of Madin-Darby canine kidney (MDCK) cells (FL-MDCK) transfected with FLAG-labeled alpha, beta, and gamma subunits of epithelial Na(+) channel (ENaC) showed that, although most of the short-circuit current (I (sc)) was amiloride sensitive (AS-I (sc)), there was also an amiloride-insensitive component (NS-I (sc)) due to Cl(-) secretion (Morris and Schafer, J Gen Physiol 120:71-85, 2002). In the present studies, we observed a progressive increase in NS-I (sc) and a corresponding decrease in AS-I (sc) during experiments. There was a significant negative correlation between AS-I (sc) and NS-I (sc) both in the presence and absence of treatment with cyclic adenosine monophosphate (cAMP). NS-I (sc) could be attributed to both cystic fibrosis transmembrane conductance regulator (CFTR) and a 4, 4'-diisothiocyano-2, 2'-disulfonic acid stilbene (DIDS)-sensitive Ca(2+)-activated Cl(-) channel (CaCC). Continuous perfusion of both sides of the Ussing chamber with fresh rather than recirculated bathing solutions, or addition of hexokinase (6 U/ml), prevented the time-dependent changes and increased AS-I (sc) by 40-60%, with a proportional decrease in NS-I (sc). Addition of 100 muM adenosine triphosphate (ATP) in the presence of luminal amiloride produced a transient four-fold increase in NS-I (sc) that was followed by a sustained increase of 50-60% above the basal level. ATP release from the monolayers, measured by bioluminescence, was found to occur across the apical but not the basolateral membrane, and the apical release was tripled by cAMP treatment. These data show that constitutive apical ATP release, which occurs under both basal and cAMP-stimulated conditions, underlies the time-dependent rise in Cl(-) secretion and the proportional fall in ENaC-mediated Na(+) absorption in FL-MDCK cells. Thus, endogenous ATP release can introduce a significant confounding variable in experiments with this and similar epithelial cells, and it may underlie at least some of the observed interaction between Cl(-) secretion and Na(+) absorption.     

PAR2 activation interrupts E-cadherin adhesion and compromises the airway epithelial barrier: protective effect of beta-agonists

The airway epithelium is an important barrier between the environment and subepithelial tissues. The epithelium is also divided into functionally restricted apical and basolateral domains, and this restriction is dependent on the elements of the barrier. The protease-activated receptor-2 (PAR2) receptor is expressed in airway epithelium, and its activation initiates multiple effects including enhanced airway inflammation and reactivity. We hypothesized that activation of PAR2 would interrupt E-cadherin adhesion and compromise the airway epithelial barrier. The PAR2-activating peptide (PAR2-AP, SLIGRL) caused an immediate approximately 50% decrease in the transepithelial resistance of primary human airway epithelium that persisted for 6-10 min. The decrease in resistance was accompanied by an increase in mannitol flux across the epithelium and occurred in cystic fibrosis transmembrane conductance receptor (CFTR) epithelium pretreated with amiloride to block Na and Cl conductances, confirming that the decrease in resistance represented an increase in paracellular conductance. In parallel experiments, activation of PAR2 interrupted the adhesion of E-cadherin-expressing L cells and of primary airway epithelial cells to an immobilized E-cadherin extracellular domain, confirming the hypothesis that activation of PAR2 interrupts E-cadherin adhesion. Selective interruption of E-cadherin adhesion with antibody to E-cadherin decreased the transepithelial resistance of primary airway epithelium by >80%. Pretreatment of airway epithelium or the E-cadherin-expressing L cells with the long-acting beta-agonist salmeterol prevented PAR2 activation from interrupting E-cadherin adhesion and compromising the airway epithelial barrier. Activation of PAR2 interrupts E-cadherin adhesion and compromises the airway epithelial barrier.     

Fenofibrate inhibits intestinal Cl- secretion by blocking basolateral KCNQ1 K+ channels

Fibrates are peroxisome proliferator-activated receptor-alpha (PPARalpha) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current (I(sc)) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic Cl(-) secretion by isolated jejunum and colon without affecting electroneutral fluxes of (22)Na(+) or (86)Rb(+) (K(+)) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 microM fenofibrate inhibited cAMP-stimulated I(sc) within 5 min. In T84 cells, fenofibrate rapidly inhibited approximately 80% the Cl(-) secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol (Ca(2+)). Both fenofibrate and clofibrate inhibited cAMP-stimulated I(sc) with an IC(50) approximately 1 muM, whereas other PPARalpha activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral cAMP-stimulated K(+) channels (putatively KCNQ1/KCNE3) without affecting Ca(2+)-stimulated K(+) channel activity, whereas clofibrate inhibits both K(+) pathways. Fenofibrate had no effect on apical cAMP-stimulated Cl(-) channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 microM fenofibrate rapidly inhibits K(+) currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 beta-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated Cl(-) secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent.     

Role of capacitative Ca2+ entry in bronchial contraction and remodeling.

Asthma is characterized by airway inflammation, bronchial hyperresponsiveness, and airway obstruction by bronchospasm and bronchial wall thickening due to smooth muscle hypertrophy. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) may serve as a shared signal transduction element that causes bronchial constriction and bronchial wall thickening in asthma. In this study, we examined whether capacitative Ca2+ entry (CCE) induced by depletion of intracellular Ca2+ stores was involved in agonist-mediated bronchial constriction and bronchial smooth muscle cell (BSMC) proliferation. In isolated bronchial rings, acetylcholine (ACh) induced a transient contraction in the absence of extracellular Ca2+ because of Ca2+ release from intracellular Ca2+ stores. Restoration of extracellular Ca2+ in the presence of atropine, an M-receptor blocker, induced a further contraction that was apparently caused by a rise in [Ca2+]cyt due to CCE. In single BSMC, amplitudes of the store depletion-activated currents (I(SOC)) and CCE were both enhanced when the cells proliferate, whereas chelation of extracellular Ca2+ with EGTA significantly inhibited the cell growth in the presence of serum. Furthermore, the mRNA expression of TRPC1, a transient receptor potential channel gene, was much greater in proliferating BSMC than in growth-arrested cells. Blockade of the store-operated Ca2+ channels by Ni2+ decreased I(SOC) and CCE and markedly attenuated BSMC proliferation. These results suggest that upregulated TRPC1 expression, increased I(SOC), enhanced CCE, and elevated [Ca2+]cyt may play important roles in mediating bronchial constriction and BSMC proliferation.     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Mechanisms of cystic fibrosis transmembrane conductance regulator activation by S-nitrosoglutathione

We investigated the mechanisms by which S-nitrosoglutathione (GSNO) alters cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride (Cl(-)) secretion across Calu-3 cells, an extensively used model of human airway gland serous cells. Confluent monolayers of Calu-3 cells, grown under an air-liquid interface, were mounted in Ussing chambers for the measurements of chloride short circuit current (I(sc)) and trans-epithelial resistance (R(t)). Addition of GSNO into the apical compartment of these chambers resulted in significant and sustained increase of I(sc) with an IC(50) of 3.2 +/- 1 mum (mean +/- 1 S.E.; n = 6). Addition of either glibenclamide or pre-treatment of Calu-3 cells with the soluble guanylate cyclase inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one totally prevented the GSNO-induced increase of I(sc). Conversely, BAY 41-2272, a sGC stimulator, increased I(sc) in a dose-response fashion. The GSNO increase of I(sc) was reversed by addition of two phosphatases (PP2A1, PP2A2) into the apical compartment of Ussing chambers containing Calu-3 monolayers. Oxy-myoglobin (oxy-Mb, 300 mum) added into the apical compartment of Ussing chambers either prior or after GSNO either completely prevented or immediately reversed the increase of I(sc). However, smaller concentrations of oxy-Mb (1-10 mum), sufficient to scavenge NO in the medium (as assessed by direct measurement of NO in the Ussing chamber using an ISO-NO meter) decreased I(sc) partially. Oxy-Mb did not reverse the increase of I(sc) following addition of GSNO and cysteine (50 mum). These findings indicate that GSNO stimulates Cl secretion via both cGMP-dependent and cGMP-independent mechanisms.     

Energized mitochondria increase the dynamic range over which inositol 1,4,5-trisphosphate activates store-operated calcium influx

In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.     

Purinergic-induced ion current in monolayers of C7-MDCK cells: role of basolateral and apical ion transporters

This study examines purinergic modulation of short-circuit current (I(SC)) in monolayers of C7- and C11-MDCK cells resembling principal and intercalated cells from collecting ducts. In C7 monolayers, basolateral and apical application of ATP led to similar elevation of I(SC), consisting of a transient phase with maximal I(SC) increment of approximately 10 microA/cm2 terminating in 2-3 min, and a sustained phase with maximal I(SC) less than 2 microA/cm2 and terminating in 10 min. ATP-induced I(SC) was insensitive to the presence of Na+, Cl- and inhibitors of K+ (Ba2+, charibdotoxin (ChTX), clotrimazole (CLT), apamin) and Na + (amiloride) channels in the mucosal solution. Inhibitors of Cl- channels, DIDS and NPPB, added to apical membranes at a concentration of 100 microM, did not affect ATP-induced I(SC), whereas at 500 microM, NPPB inhibited it by 70-80%. Substitution of Cl- with NO3- in serosal medium decreased ATP-induced I(SC) by 2-3-fold and elevation of [K+]o from 6 to 60 mM changed its direction. Basolateral NPPB inhibited I(SC) by 10-fold with ED50 of approximately 30 microM, whereas ChTX (50 nM) and CLT (2 microM) diminished this parameter by 30-50%. Suppression of Na+, K+, Cl- cotransport with bumetanide did not affect the transient phase of ATP-induced I(SC) and slightly diminished its sustained phase. ATP increased ouabainand bumetanide-resistant K+ (86Rb) influx across the basolateral membrane by 7-8-fold, which was partially inhibited with ChTX and CLT. ATP-treated C11 cells exhibited negligible I(SC), and their presence did not affect I(SC) triggered by ATP in C7 cells. Thus, our results show that transcellular ion current in ATP-treated C7 cells is mainly caused by the coupled function of apical and basolateral anion transporters providing transient Cl- secretion. These transporters possess different sensitivities to anion channel blocker NPBB and are under the control of basolateral K+ channels(s) inhibited by ChTX and CLT.