Jaekyung C. Song

PKC-ε regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-ε. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-ε, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

Levamisole inhibits intestinal Cl- secretion via basolateral K+ channel blockade.

BACKGROUND & AIMS Phenylimidazothiazoles have recently been shown to activate wild-type and mutant cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels in transfected cells and were proposed as therapy for cystic fibrosis. The aim of this study was to investigate the effects of phenylimidazothiazoles on regulated transepithelial Cl- transport in intact epithelia. METHODS T84 intestinal epithelial cells grown on permeable supports and stripped human colonic mucosal sheets were studied by conventional current-voltage clamping. Selective permeabilization of apical or basolateral membranes with the monovalent ionophore nystatin was used to isolate basolateral K+ and apical Cl- channel activity, respectively. 86Rb+ uptake was assessed for Na/K/2Cl cotransporter and Na+,K(+)-adenosine triphosphatase activity. RESULTS In T84 monolayers and human colon, levamisole and its brominated derivative bromotetramisole failed to activate transepithelial secretion. In fact, these compounds dose-dependently inhibited secretory responses to the cyclic adenosine monophosphate agonist forskolin and the Ca2+ agonist carbachol. In permeabilized T84 monolayers, phenylimidazothiazoles weakly activated apical Cl- currents (consistent with their reported action on CFTR) and did not affect bumetanide-sensitive or bumetanide-insensitive 86+Rb+ uptake. Instead, they profoundly inhibited the basolateral Ba(2+)-sensitive and Ba(2+)-insensitive K+ currents. CONCLUSIONS Phenylimidazothiazoles block K+ channels required for Cl(-)-secretory responses elicited by diverse pathways in model epithelia and native colon, an effect that outweighs their ability to activate apical Cl- channels.

Ammonia blockade of intestinal epithelial K+ conductance

Ammonia profoundly inhibits cAMP-dependent Cl- secretion in model T84 human intestinal crypt epithelia. Because colonic lumen concentrations of ammonia are high (10-70 mM), ammonia may be a novel regulator of secretory diarrheal responsiveness. We defined the target of ammonia action by structure-function analysis with a series of primary amines (ammonia, methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, and octylamine) that vary principally in size and lipid solubilities. The amine concentrations required for 50% inhibition of Cl- secretion in intact monolayers and 50% inhibition of outward K+ current ( I K) in apically permeabilized monolayers vs. the logs of the respective amine partition coefficients give two plots that are strikingly similar in character. Half-maximal inhibition of short-circuit current ( I sc) by ammonia was seen at 6 mM and for I K at 4 mM; half-maximal inhibition for octylamine was 0.24 mM and 0.19 mM for I sc and I K, respectively. The preferentially water-soluble hydrophilic amines (ammonia, methylamine, ethylamine) increase in blocking ability with decreasing size and lipophilicity. Conversely, the preferentially lipid-soluble hydrophobic (propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine) amines increase in blocking ability with increasing size and lipophilicity. Ammonia does not affect isolated apical Cl- conductance; amine-induced changes in cytosolic and endosomal pH do not correlate with secretory inhibition. We propose that ammonia in its protonated ammonium form ([Formula: see text]) inhibits cAMP-dependent Cl- secretion in T84 monolayers by blocking basolateral K+ channels.Ammonia profoundly inhibits cAMP-dependent Cl- secretion in model T84 human intestinal crypt epithelia. Because colonic lumen concentrations of ammonia are high (10-70 mM), ammonia may be a novel regulator of secretory diarrheal responsiveness. We defined the target of ammonia action by structure-function analysis with a series of primary amines (ammonia, methylamine, ethylamine, propylamine, butylamine, pentylamine, hexylamine, heptylamine, and octylamine) that vary principally in size and lipid solubilities. The amine concentrations required for 50% inhibition of Cl- secretion in intact monolayers and 50% inhibition of outward K+ current (IK) in apically permeabilized monolayers vs. the logs of the respective amine partition coefficients give two plots that are strikingly similar in character. Half-maximal inhibition of short-circuit current (Isc) by ammonia was seen at 6 mM and for IK at 4 mM; half-maximal inhibition for octylamine was 0.24 mM and 0.19 mM for Isc and IK, respectively. The preferentially water-soluble hydrophilic amines (ammonia, methylamine, ethylamine) increase in blocking ability with decreasing size and lipophilicity. Conversely, the preferentially lipid-soluble hydrophobic (propylamine, butylamine, pentylamine, hexylamine, heptylamine, octylamine) amines increase in blocking ability with increasing size and lipophilicity. Ammonia does not affect isolated apical Cl- conductance; amine-induced changes in cytosolic and endosomal pH do not correlate with secretory inhibition. We propose that ammonia in its protonated ammonium form (NH4+) inhibits cAMP-dependent Cl- secretion in T84 monolayers by blocking basolateral K+ channels.

K+ channel inhibition accelerates intestinal epithelial cell wound healing

Restitution is the process by which superficial interruptions in the gastrointestinal mucosa are repaired by the flattening and spreading of epithelial cells surrounding the damage. During this process, mucosal epithelial cells undergo extensive reshaping and cytoskeletal remodeling. K+ channels, located primarily on the basolateral surface of gut epithelial cells, are central to both actin polymerization, via their control of membrane potential, and cell volume regulation. We questioned whether K+ channels are involved in restitution using an in vitro model of intestinal epithelium, monolayers of the human colon carcinoma cell line T84. We report that pharmacologic K+ channel inhibition accelerates wound healing in T84 cell monolayers. Both Ca++‐dependent and constitutively active channels are involved, as indicated by the sensitivity to clotrimazole, charybdotoxin, and barium. The ability of clotrimazole to accelerate wound resealing was also observed in Caco‐2 cell sheets. Pharmacologic stimulation of K+ channel activity had no effect on the repair rate. Analysis of the resealing process by time lapse and confocal microscopy revealed that K+ channel inhibitors abolished the initial wound retraction, briefly accelerated the repair rate, and altered the shape of the cell sheet abutting the injury during the early phase of resealing. We hypothesize that K+ channel inactivation interrupts the coregulation of f‐actin polymerization and volume control that is initiated by the healing process.

Protein kinase C activation downregulates the expression and function of the basolateral Na+/K+/2Cl− cotransporter

The basolateral Na+/K+/2Cl− cotransporter (NKCC1) has been shown to be an independent regulatory site for electrogenic Cl− secretion. The proinflammatory phorbol ester, phorbol 12‐myristate 13‐acetate (PMA), which activates protein kinase C (PKC), inhibits basal and cyclic adenosine monophosphate (cAMP)‐stimulated NKCC1 activity in T84 intestinal epithelial cells and decreases the steady state levels of NKCC1 mRNA in a time‐ and dose‐dependent manner. The levels of NKCC1 protein also fall in accordance with the NKCC1 mRNA transcript and these levels are unaffected by 4α‐phorbol, which does not activate PKC. Inhibition of maximal (cAMP‐stimulated) NKCC1 functional activity by PMA was first detected by 1 h, whereas decreases in the steady state levels of NKCC1 mRNA were not detectable until 4 h. NKCC1 mRNA expression recovers toward control levels with extended treatment of cells with PMA suggesting that the PMA effects on NKCC1 expression are mediated through activation of PKC. Although NKCC1 mRNA and protein levels return to control values after extended PMA exposure, NKCC1 functional activity does not recover. Immunofluorescence imaging suggest that the absence of functional recovery is due to failure of newly synthesized NKKC1 protein to reach the cell surface. We conclude that NKCC1 has the capacity to be regulated at the level of de novo expression by PKC, although decreased NKCC1 expression alone cannot account for either early or late loss of NKCC1 function. J. Cell. Physiol. 181:489–498, 1999.

Opposing effects of PKCA andpkce on basolateral membrane dynamics in intestinal epithelia

Song, Jaekyung Cecilia, Patangi K. Rangachari, and Jeffrey B. Matthews. Opposing effects of PKC and PKC on basolateral membrane dynamics in intestinal epithelia. Am J Physiol Cell Physiol 283: C1548–C1556, 2002. First published July 24, 2002; 10.1152/ajpcell.00105.2002.—PKC is a critical effector of plasma membrane dynamics, yet the mechanism and isoform-specific role of PKC are poorly understood. We recently showed that the phorbol ester PMA (100 nM) induces prompt activation of the novel isoform PKC followed by late activation of the conventional isoform PKC in T84 intestinal epithelia. PMA also elicited biphasic effects on endocytosis, characterized by an initial stimulatory phase followed by an inhibitory phase. Activation of PKC was shown to be responsible for stimulation of basolateral endocytosis, but the role of PKC was not defined. Here, we used detailed time-course analysis as well as selective activators and inhibitors of PKC isoforms to infer the action of PKC on basolateral endocytosis. Inhibition of PKC by the selective conventional PKC inhibitor Go-6976 (5 M) completely blocked the late inhibitory phase and markedly prolonged the stimulatory phase of endocytosis measured by FITC-dextran uptake. The PKC -selective agonist carbachol (100 M) induced prolonged stimulation of endocytosis devoid of an inhibitory phase. Actin disassembly caused by PMA was completely blocked by Go-6850 but not by Go-6976, implicating PKC as the key isoform responsible for actin disruption. The Ca2 agonist thapsigargin (5 M) induced early activation of PKC when added simultaneously with PMA. This early activation of PKC blocked the ability of PMA to remodel basolateral F-actin and abolished the stimulatory phase of basolateral endocytosis. Activation of PKC stabilizes F-actin and thereby opposes the effect of PKC on membrane remodeling in T84 cells.