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Mouse proximal tubular cells (BUMPT), when cultured in the absence of growth factors, activate a default apoptotic pathway. Although Wnt signaling antagonizes the effect of proapoptotic triggers, its role in regulating the default pathway of apoptosis is less well defined. The present study examines the hypothesis that lithium ([Li.sup.+]) and (2'Z,3'E)-6-bromoindirubin-3'-oxime (BIO), two glycogen synthase kinase-3[beta] (GSK3[beta]) inhibitors, promote survival of growth factor-deprived renal epithelial cells by activating the Wnt pathway. These studies demonstrate that [Li.sup.+] and BIO activate Wnt signaling as indicated by the following changes: phosphorylation (inhibition) of GSK3[beta]; decreased phosphorylation of [beta]-catenin (a GSK3[beta] substrate); nuclear translocation of [beta]-catenin; specific transcriptional activation of Tcf/catenin-responsive pTopflash constructs; and an increase in the expression of cyclin D1 (indicative of a promitogenic cell response). In addition, [Li.sup.+] or BIO significantly increases the phosphorylation (activation) of Akt, an anti-apoptotic protein, and inhibits apoptosis (decreases both annexin-V staining and caspase-3 activation), during serum deprivation. Inhibition of phosphatidylinositol 3-kinase (responsible for Akt activation) either by wortmanin or LY-294002 prevented [Li.sup.+]-or BIO-induced Akt phosphorylation and reduces cell survival without altering the phosphorylation state of GSK3[beta]. [Li.sup.+] or BIO also increases the expression of insulin-like growth factor-II (IGF-II), a potent proliferative signaling protein. [Li.sup.+] or BIO-free conditioned medium harvested from [Li.sup.+] or BIO-exposed cells also induced Akt phosphorylafion, mimicking the protective effect of the two GSK3[beta] inhibitors on serum-starved cells. Furthermore, the effect of conditioned medium on Akt phosphorylation could be inhibited by either LY-294002 or IGF-binding protein. BIO, a specific GSK3[beta] inhibitor, replicated the protective effect of [Li.sup.+] on cell viability, suggesting that GSK3[beta] activation is important for initiating the apoptotic pathway. Taken together, these data suggest that [Li.sup.+] or BIO promotes renal epithelial cell survival by inhibiting apoptosis through GSK3[beta]-dependent activation of the Wnt pathway and subsequent release of IGF-II. Extracellular IGF-II serves as an autocrine survival factor that is responsible, in part, for activating the anti-apoptotic phosphatidylinositol-3-kinase-Akt pathway during serum deprivation. serum deprivation; renal epithelial cells; insulin-like growth factors; [beta]-catenin; glycogen synthase kinase-3[beta]; apoptosis

The function of intestinal keratins is unknown, although keratin 8 (K8)-null mice develop colitis, hyperplasia, diarrhea, and mistarget jejunal apical markers. We quantified the diarrhea in K8-null stool and examined its physiologic basis. Isolated crypt-units from K8-null and wild-type mice have similar viability. K8-null distal colon has normal tight junction permeability and paracellular transport but shows decreased short circuit current and net Na absorption associated with net CI secretion, blunted intracellular CI/HC[O.sub.3]-dependent pH regulation, hyperproliferation and enlarged goblet cells, partial loss of the membrane-proximal markers H,K-ATPase-[beta] and F-actin, increased and redistributed basolateral anion exchanger AE1/2 protein, and redistributed Na-transporter ENaC-[gamma]. Diarrhea and protein mistargeting are observed 1-2 d after birth while hyperproliferation/inflammation occurs later. The AE1/2 changes and altered intracellular pH regulation likely account, at least in part, for the ion transport defects and hyperproliferation. Therefore, colonic keratins have a novel function in regulating electrolyte transport, likely by targeting ion transporters to their cellular compartments.

Krishnan, Selvi, Vazhaikkurichi M. Rajendran, and Henry J. Binder. Apical NHE isoforms differentially regulate butyrate-stimulated Na absorption in rat distal colon. Am J Physiol Cell Physiol 285: C1246-C1254, 2003. First published July 23, 2003; 10.1152/ajpcell.00598.2002.--Bicarbonate and butyrate stimulate electroneutral Na absorption via apical membrane Na-H exchange (NHE) in rat distal colon, cAMP downregulates NHE-3 isoform and inhibits HC[O.sub.3]-dependent, but not butyrate-dependent, Na absorption. This study sought to determine whether 1) the apical membrane NHE-2 and NHE-3 isoforms differentially mediated HC[O.sub.3]- and butyrate-dependent Na absorption, and 2) cAMP had different effects on NHE-2 and NHE-3 isoforms. The effect of specific inhibitors of NHE-2 and NHE-3 isoforms (50 [micro]M HOE 694 and 2 [micro]M $3226, respectively) on unidirectional [sup.22][Na.sup] transepithelial fluxes performed across isolated mucosa from rat distal colon under voltage-clamp conditions was examined. HC[O.sub.3] stimulation of Na absorption was inhibited by EIPA, a nonspecific inhibitor of all NHE isoforms, by $3226 and dibutyryl cAMP but not by HOE 694. In contrast, butyrate stimulation of Na absorption was not altered by dibutyryl cAMP and was not inhibited by HOE 694 in the absence of dibutyryl cAMP, but in the presence of dibutyryl cAMP was HOE694 sensitive. In contrast, $3226 inhibited butyrate-stimulated Na absorption in the absence of dibutyryl cAMP, but not in its presence. We conclude that 1) HC[O.sub.3]-stimulated Na absorption is mediated solely by NHE-3 isoform, whereas butyrate-stimulated Na absorption is mediated by either NHE-3 or NHE-2 isoform, and 2) dibutyryl cAMP selectively inhibits NHE-3 isoform but stimulates NHE-2 isoform. Dibutyryl cAMP does not inhibit butyrate-stimulated Na absorption as a result of its differential effects on NHE-2 and NHE-3 isoforms. butyrate-dependent Na absorption; HC[O.sub.3]-dependent Na absorption; specific NHE isoform inhibitors; NHE-2 isoform; NHE-3 isoform

Colonic [K.sup.+] secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular [Ca.sup.2+]. However, the precise mechanisms by which this rise in [Ca.sup.2+] leads to [K.sup.+] efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] [Ca.sup.2+]-activated [K.sup.+] channels. In dietary [K.sup.+]-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the [K.sup.+] channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of [Ca.sup.2+]-activated [K.sup.+] channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active [K.sup.+] secretion was studied on epithelial strips of rat proximal colon using unidirectional [sup.86][Rb.sup.+] fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net [sup.86][Rb.sup.+] secretion. The mucosal addition of CLT completely inhibited carbachol-induced net [sup.86][Rb.sup.+] secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary [K.sup.+], carbachol-induced [.sup.86][Rb.sup.+] secretion was abolished in dietary [K.sup.+]-depleted animals. These results suggest that the rSK4 channel mediates [K.sup.+] secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent [K.sup.+] loss during dietary [K.sup.+] depletion. intermediate conductance [Ca.sup.2+]-activated [K.sup.+] channel; intermediate-conductance channel; carbachol; clotrimazole; [sup.86][RB.sup.+] flux; [K.sup.+] depletion

Hydrogen sulfide (H2S), previously only considered a toxic environmental air pollutant, is now increasingly recognized as an important signaling molecule able to modulate several cellular pathways in many human tissues. As demonstrated in recent studies, H2S is produced endogenously in response to different cellular stimuli and plays different roles in controlling a number of physiological responses. The precise role of H2S in inflammation is still largely unknown. In particular, the role of H2S in the regulation of the inflammatory response in acute and chronic infections is being actively investigated because of its potential therapeutic use. To study the effect of H2S as an anti-inflammatory mediator during bacterial infections, we developed an ex vivo model of primary cells and cell lines infected with Mycoplasma. Our data demonstrate a dichotomic effect of H2S on the NF-kB and Nrf-2 molecular pathways, which were inhibited and stimulated, respectively. [ABSTRACT FROM AUTHOR]

Colonic K[sup +] secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular Ca[sup 2+]. However, the precise mechanisms by which this rise in Ca[sup 2+] leads to K[sup +] efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] Ca[sup 2+]-activated K[sup +] channels. In dietary K[sup +]-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the K[sup +] channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of Ca[sup 2+]-activated K[sup +] channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active K[sup +] secretion was studied on epithelial strips of rat proximal colon using unidirectional [sup 86]Rb[sup +] fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net [sup 86]Rb[sup +] secretion. The mucosal addition of CLT completely inhibited carbachol-induced net [sup 86]Rb[sup +] secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary K[sup +], carbachol-induced [sup 86]Rb[sup +] secretion was abolished in dietary K[sup +]-depleted animals. These results suggest that the rSK4 channel mediates K[sup +] secretion induced by muscarinic agonists in the rat proximal colon and that... [ABSTRACT FROM AUTHOR]

Hydrogen sulfide (H 2 S), previously only considered a toxic environmental air pollutant, is now increasingly recognized as an important signaling molecule able to modulate several cellular pathways in many human tissues. As demonstrated in recent studies, H 2 S is produced endogenously in response to different cellular stimuli and plays different roles in controlling a number of physiological responses. The precise role of H 2 S in inflammation is still largely unknown. In particular, the role of H 2 S in the regulation of the inflammatory response in acute and chronic infections is being actively investigated because of its potential therapeutic use. To study the effect of H 2 S as an anti-inflammatory mediator during bacterial infections, we developed an ex vivo model of primary cells and cell lines infected with Mycoplasma. Our data demonstrate a dichotomic effect of H 2 S on the NF-kB and Nrf-2 molecular pathways, which were inhibited and stimulated, respectively.