Diarrhea-associated HIV-1 APIs potentiate muscarinic activation of Cl- secretion by T84 cells via prolongation of cytosolic Ca2+ signaling

Aspartyl protease inhibitors (APIs) effectively extend the length and quality of life in human immunodeficiency virus (HIV)-infected patients, but dose-limiting side effects such as lipodystrophy, insulin resistance, and diarrhea have limited their clinical utility. Here, we show that the API nelfinavir induces a secretory form of diarrhea in HIV-infected patients. In vitro studies demonstrate that nelfinavir potentiates muscarinic stimulation of Cl^- secretion by T84 human intestinal cell monolayers through amplification and prolongation of an apical membrane Ca²⁺-dependent Cl^- conductance. This stimulated ion secretion is associated with increased magnitude and duration of muscarinically induced intracellular Ca²⁺ transients via activation of a long-lived, store-operated Ca²⁺ entry pathway. The enhanced intracellular Ca²⁺ signal is associated with uncoupling of the Cl^- conductance from downregulatory intracellular mediators generated normally by muscarinic activation. These data show that APIs modulate Ca²⁺ signaling in secretory epithelial cells and identify a novel target for treatment of clinically important API side effects. nelfinavir; clotrimazole; barium     

Modulation of K+ channels by arachidonic acid in T84 cells. I.Inhibition of the Ca2+-dependent K+ channel

TheCl secretory response ofcolonic cells to Ca²⁺-mediatedagonists is transient despite a sustained elevation of intracellular Ca²⁺. We evaluated the effects ofsecond messengers proposed to limit Ca²⁺-mediatedCl secretion on thebasolateral membrane,Ca²⁺-dependentK⁺ channel(K_Ca) in colonic secretorycells, T84. Neither protein kinase C (PKC) nor inositoltetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affectedK_Ca in excised inside-out patches.In contrast, arachidonic acid (AA; 3 µM) potently inhibitedK_Ca, reducingNP_o, the productof number of channels and channel open probability, by 95%. Theapparent inhibition constant for this AA effect was 425 nM. AAinhibited K_Ca in the presence ofboth indomethacin and nordihydroguaiaretic acid, blockers of thecyclooxygenase and lipoxygenase pathways. In the presence of albumin,the effect of AA on K_Ca wasreversed. A similar effect of AA was observed onK_Ca during outside-out recording.We determined also the effect of thecis-unsaturated fatty acid linoleate,the trans-unsaturated fatty acidelaidate, and the saturated fatty acid myristate. At 3 µM, all ofthese fatty acids inhibited K_Ca,reducing NP_o by 72-86%. Finally, the effect of the cytosolic phospholipaseA₂ inhibitorarachidonyltrifluoromethyl ketone(AACOCF₃) on thecarbachol-induced short-circuit current(I_sc) responsewas determined. In the presence ofAACOCF₃, the peakcarbachol-inducedI_sc response wasincreased ~2.5-fold. Our results suggest that AA generation inducedby Ca²⁺-mediated agonists maycontribute to the dissociation observed between the rise inintracellular Ca²⁺ evoked by theseagonists and the associatedCl secretory response.

     

Apical ammonium inhibition of cAMP-stimulated secretion in T84 cells is bicarbonate dependent

Normal human colonic luminal (NH(4)(+)) concentration ([NH(4)(+)]) ranges from approximately 10 to 100 mM. However, the nature of the effects of NH(4)(+) on transport, as well as NH(4)(+) transport itself, in colonic epithelium is poorly understood. We elucidate here the effects of apical NH(4)(+) on cAMP-stimulated Cl(-) secretion in colonic T84 cells. In HEPES-buffered solutions, 10 mM apical NH(4)(+) had no significant effect on cAMP-stimulated current. In contrast, 10 mM apical NH(4)(+) reduced current within 5 min to 61 +/- 4% in the presence of 25 mM HCO(3)(-). Current inhibition was not simply due to an increase in extracellular K(+)-like cations, in that the current magnitude was 95 +/- 5% with 10 mM apical K(+) and 46 +/- 3% with 10 mM apical NH(4)(+) relative to that with 5 mM apical K(+). We previously demonstrated that inhibition of Cl(-) secretion by basolateral NH(4)(+) occurs in HCO(3)(-)-free conditions and exhibits anomalous mole fraction behavior. In contrast, apical NH(4)(+) inhibition of current in HCO(3)(-) buffer did not show anomalous mole fraction behavior and followed the absolute [NH(4)(+)] in K(+)-NH(4)(+) mixtures, where K(+) concentration + [NH(4)(+)] = 10 mM. The apical NH(4)(+) inhibitory effect was not prevented by 100 microM methazolamide, suggesting no role for apical carbonic anhydrase. However, apical NH(4)(+) inhibition of current was prevented by 10 min of pretreatment of the apical surface with 500 microM DIDS, 100 microM 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), or 25 microM niflumic acid, suggesting a role for NH(4)(+) action through an apical anion exchanger. mRNA and protein for the apical anion exchangers SLC26A3 [downregulated in adenoma (DRA)] and SLC26A6 [putative anion transporter (PAT1)] were detected in T84 cells by RT-PCR and Northern and Western blots. DRA and PAT1 appear to associate with CFTR in the apical membrane. We conclude that the HCO(3)(-) dependence of apical NH(4)(+) inhibition of secretion is due to the action of NH(4)(+) on an apical anion exchanger.     

Vibrio cholerae ACE stimulates Ca(2+)-dependent Cl(-)/HCO(3)(-) secretion in T84 cells in vitro

ACE, accessory cholera enterotoxin, the thirdenterotoxin in Vibrio cholerae, has been reported toincrease short-circuit current (I_sc) in rabbitileum and to cause fluid secretion in ligated rabbit ileal loops. Westudied the ACE-induced change in I_sc andpotential difference (PD) in T84 monolayers mounted in modified Ussingchambers, an in vitro model of a Cl secretory cell. ACEadded to the apical surface alone stimulated a rapid increase inI_sc and PD that was concentration dependent andimmediately reversed when the toxin was removed. Ion replacement studies established that the current was dependent on Cland HCO₃. ACE acted synergistically with theCa²⁺-dependent acetylcholine analog, carbachol, tostimulate secretion in T84 monolayers. In contrast, the secretoryresponse to cAMP or cGMP agonists was not enhanced by ACE. TheACE-stimulated secretion was dependent on extracellular andintracellular Ca²⁺ but was not associated with an increasein intracellular cyclic nucleotides. We conclude that the mechanism ofsecretion by ACE involves Ca²⁺ as a second messenger andthat this toxin stimulates a novel Ca²⁺-dependent synergy.

     

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion‐current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage‐clamp technique. All agonists elicited Cl^? currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl^? (F_Cl) currents, while AII activated an oscillatory response; a robust Ca²⁺ influx linked specifically to F_Cl activation elicited an inward current (I_iw,Ca) which was carried mainly by Cl^? ions, through channels with a sequence of permeability of SCN^? > I^? > Br^? > Cl^?. Like F_Cl, I_iw,Ca was not dependent on oocyte [Ca²⁺]_i; instead both were eliminated by preventing [Ca²⁺]_i increase in the follicular cells, and also by U73122 and 2‐APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F_Cl and I_iw,Ca were produced by the expected, PLC‐stimulated Ca²⁺‐release and Ca²⁺‐influx, respectively, and by the opening of I_Cl(Ca) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca²⁺‐influx appeared to be driven through store‐operated, calcium‐like channels. The AII response, which is also known to require PLC activation, did not activate I_iw,Ca and was strictly dependent on oocyte [Ca²⁺]_i increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap‐junction channels. J. Cell. Physiol. 227: 3457–3470, 2012. © 2011 Wiley Periodicals, Inc.     

Distinct Ca2+- and cAMP-dependent anion conductances in the apical membrane of polarized T84 cells

Monolayers of the human colonic epithelial cell line T84 exhibitelectrogenic Cl secretionin response to the Ca²⁺ agonistthapsigargin and to the cAMP agonist forskolin. To evaluate directlythe regulation of apical Clconductance by these two agonists, we have utilized amphotericin B topermeabilize selectively the basolateral membranes of T84 cellmonolayers. We find that apical anion conductance is stimulated by bothforskolin and thapsigargin but that these conductances aredifferentially sensitive to the anion channel blocker DIDS. DIDSinhibits thapsigargin-stimulated responses completely but forskolinresponses only partially. Furthermore, the apical membrane anionconductances elicited by these two agonists differ in anion selectivity(for thapsigargin, I > Cl; for forskolin,Cl > I). However, theDIDS-sensitive component of the forskolin-induced conductance responseexhibits anion selectivity similar to that induced by thapsigargin(I > Cl). Thusforskolin-induced apical anion conductance comprises at least twocomponents, one of which has features in common with that elicited bythapsigargin.

     

Wortmannin inhibition of forskolin-stimulated chloride secretion by T84 cells

The time- and dose-dependent effects of wortmannin on transepithelial electrical resistance (Rte) and forskolin-stimulated chloride secretion in T84 monolayer cultures were studied. In both instances, maximal effects developed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (Isc) technique, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibition of myosin light chain kinase (MLCK) and mitogen-activated protein kinases (MAPK). Previous work demonstrated that 500 nM wortmannin did not inhibit the cAMP activation of apical membrane chloride channels. We show here that 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter activity, but inhibits basolateral membrane Na/K-ATPase activity significantly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-stimulated Isc. Similarly, the p38- and MEK-specific MAPK inhibitors SB203580 and PD98059 did not reduce forskolin-stimulated Isc. In contrast, the non-specific MAPK inhibitor apigenin reduced forskolin-stimulated Isc and basolateral membrane Na/K-ATPase activity similar to wortmannin. In isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the functional expression of basolateral membrane Na/K-ATPase by controlling the abundance of enzyme molecules in the plasma membrane.     

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.     

Membrane association domains in Ca2+-dependent activator protein for secretion mediate plasma membrane and dense-core vesicle binding required for Ca2+-dependent exocytosis

Ca2+-dependent activator protein for secretion (CAPS) is a cytosolic protein essential for the Ca2+-dependent fusion of dense-core vesicles (DCVs) with the plasma membrane and the regulated secretion of a subset of neurotransmitters. The mechanism by which CAPS functions in exocytosis and the means by which it associates with target membranes are unknown. We identified two domains in CAPS with distinct membrane-binding properties that were each essential for CAPS activity in regulated exocytosis. The first of these, a centrally located pleckstrin homology domain, exhibited three properties: charge-based binding to acidic phospholipids, binding to plasma membrane but not DCV membrane, and stereoselective binding to phosphatidylinositol 4,5-bisphosphate. Mutagenesis studies revealed that the former two properties but not the latter were essential for CAPS function. The central pleckstrin homology domain may mediate transient CAPS interactions with the plasma membrane during Ca2+-triggered exocytosis. The second membrane association domain comprising distal C-terminal sequences mediated CAPS targeting to and association with neuroendocrine DCVs. The CAPS C-terminal domain was also essential for optimal activity in regulated exocytosis. The presence of two membrane association domains with distinct binding specificities may enable CAPS to bind both target membranes to facilitate DCV-plasma membrane fusion.     

Basolateral Ca2+-dependent K+-channels play a key role in Cl- secretion induced by taurodeoxycholate from colon mucosa

The diarrhea associated with malabsorption of bile salts such as the secondary hydrophobic taurodeoxycholate (TDC) may be partly explained by the TDC-induced increase in colon Cl(-) secretion. We, therefore, investigated the effects of TDC (0.5-8 mM) on electrical parameters and electrolyte transport of rat proximal colon mucosa mounted in Ussing chambers. Colonic secretion, measured as short circuit current (I(SC)), progressively increased on mucosal incubation with TDC ranging 0.5-2 mM; up to TDC 2 mM, a spontaneous recovery toward control values with no changes in epithelial resistance (Rt), and lactate dehydrogenase (LDH) release was observed. In contrast, for TDC > 2 mM, I(SC) increased further and the effect was progressive and associated with a significant decrease in the Rt and increased LDH release, implying a cytolytic effect. Mucosal preincubation with the Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fully prevented the precytolytic effect of TDC on I(SC). Serosal preincubation with furosemide, a Na(+)/K(+)/2Cl(-) cotransporter inhibitor, significantly reduced TDC-induced increase in I(SC). Inhibition of the basolateral Ca(2+)-dependent K(+) channel-rSK4-with serosal clotrimazole or incubation with mucosal Ca(2+)-free (EGTA) buffer completely prevented precytolytic TDC-induced increase in I(SC). In conclusion, Cl(-) secretion is activated in colon mucosa by TDC low concentrations; while at higher concentrations, a detergent cytotoxic effect intervenes. Activation of the Ca(2+)-dependent basolateral K(+) pathway, through TDC-induced apical Ca(2+) influx, provides the Na(+)/K(+)/2Cl(-) basolateral activation, thereby the driving force for the apical exit of Cl(-) ions. These findings further enhance the knowledge of the pathogenic mechanisms of diarrhea associated with bile salt malabsorption.