Jeffrey B. Matthews

Na:K:2Cl Cotransporter (NKCC) of Intestinal Epithelial Cells SURFACE EXPRESSION IN RESPONSE TO cAMP

During intestinal chloride secretion, epithelial uptake of salts is accomplished largely by a bumetanide-sensitive Na:K:2Cl cotransporter designated here as NKCC. Using monoclonal antibodies directed against NKCC from the human crypt epithelial cell line, T84, we define its surface localization as a function of cotransporter activation. Immunoelectron microscopy, confocal localization, and selective surface biotinylation studies revealed that the 195-kDa NKCC protein is polarized to the basolateral domain. Following immunoprecipitation, several polypeptides coprecipitated with the 195-kDa cotransporter including two prominent proteins of molecular mass 160 and 130 kDa. Immunoblotting with three distinct anti-NKCC monoclonal antibodies in conjunction with deglycosylation experiments suggested that the 160- and 130-kDa bands represented novel proteins unrelated to the cotransporter. Stimulation of T84 monolayers with cAMP agonists, a condition which elicits chloride secretion and leads to microfilament-dependent NKCC activation, did not significantly increase the number of bumetanide-binding sites and only marginally increased surface expression of the 195-kDa cotransporter available for surface biotinylation. In contrast, cAMP agonist stimulation increased the surface expression of the coprecipitating 160- and 130-kDa proteins ∼6-fold. The increase in surface 160- and 130-kDa proteins was attenuated by phalloidin preloading the cells, a condition which also prevents activation of NKCC without influencing the activity of other membrane transporters participating in chloride secretion. These studies define the polarized distribution of the NKCC protein on intestinal epithelia, indicate that NKCC may be associated with two other previously unidentified membrane proteins and such association is influenced by the F-actin cytoskeleton.

Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells.

T84 cells, a human intestinal epithelial cell line, serve as a model of electrogenic Cl- secretion. We find that cAMP-elicited Cl- secretion in T84 cells is accompanied by a marked redistribution of F-actin in the basolateral portion of the cell. To prevent this F-actin redistribution and thereby assess its importance to Cl- secretion, we have defined simple conditions under which this model epithelium can be loaded with nitrobenzoxadiazole (NBD)-phallicidin. This reagent binds F-actin with high affinity thus stabilizing the F-actin cytoskeleton by preventing depolymerization, an event necessary for dynamic reordering of actin microfilaments. NBD-phallicidin loading is not cytotoxic as assessed by lactic dehydrogenase release, protein synthesis, transepithelial resistance, and the ability of the loaded cells to pump Na+ in an absorptive direction in response to the apical addition of a Na+ ionophore. However, cAMP-elicited redistribution of F-actin and the cAMP-elicited Cl- secretory response are both markedly impaired in NBD-phallicidin preloaded T84 cells. In contrast, the carbachol-elicited Cl- secretory response (Ca++ mediated) is not attenuated by NBD-phallicidin preloading nor is it accompanied by redistribution of F-actin. These findings suggest that the cAMP-elicited cytoskeletal redistribution we describe is an integral part of cAMP-elicited Cl- secretion in T84 cells.

Effects of substance P on human colonic mucosa in vitro.

Previous studies indicated that the peptide substance P (SP) causes Cl--dependent secretion in animal colonic mucosa. We investigated the effects of SP in human colonic mucosa mounted in Ussing chamber. Drugs for pharmacological characterization of SP-induced responses were applied 30 min before SP. Serosal, but not luminal, administration of SP (10-8 to 10-6 M) induced a rapid, monophasic concentration and Cl--dependent, bumetanide-sensitive short-circuit current ( I sc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG synthesis inhibitor indomethacin. SP caused TTX- and lodoxamide-sensitive histamine release from colonic mucosa. Two-photon microscopy revealed NK1(SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase inhibitor genistein concentration dependently blocked SP-induced I sc increase without impairing forskolin- and carbachol-mediated I sc increase. We conclude that SP stimulates Cl--dependent secretion in human colon by a pathway(s) involving mucosal nerves, mast cells, and the mast cell product histamine. Our results also indicate that tyrosine kinases may be involved in this SP-induced response.Previous studies indicated that the peptide substance P (SP) causes Cl--dependent secretion in animal colonic mucosa. We investigated the effects of SP in human colonic mucosa mounted in Ussing chamber. Drugs for pharmacological characterization of SP-induced responses were applied 30 min before SP. Serosal, but not luminal, administration of SP (10(-8) to 10(-6) M) induced a rapid, monophasic concentration and Cl--dependent, bumetanide-sensitive short-circuit current (Isc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG synthesis inhibitor indomethacin. SP caused TTX- and lodoxamide-sensitive histamine release from colonic mucosa. Two-photon microscopy revealed NK1 (SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase inhibitor genistein concentration dependently blocked SP-induced Isc increase without impairing forskolin- and carbachol-mediated Isc increase. We conclude that SP stimulates Cl--dependent secretion in human colon by a pathway(s) involving mucosal nerves, mast cells, and the mast cell product histamine. Our results also indicate that tyrosine kinases may be involved in this SP-induced response.

Na-K-2Cl cotransporter gene expression and function during enterocyte differentiation. Modulation of Cl- secretory capacity by butyrate.

The basolateral Na-K-2Cl cotransporter (NKCC1) is a key component of the intestinal crypt cell secretory apparatus. Its fate during the transition to absorptive enterocyte and the potential impact of its altered expression on secretory output have not been addressed. In this report, NKCC1 mRNA was found to be expressed in rat jejunal crypt but not villus cells. Butyrate treatment of intestinal epithelial HT29 cells induced a differentiation pattern that recapitulated the rat intestinal crypt-villus axis, with NKCC1 mRNA levels decreasing in a time- and dose-dependent fashion in parallel with upregulation of apical brush-border markers. Butyrate but not acetate or proprionate decreased basal and cAMP-stimulated bumetanide-sensitive K+ (86Rb) uptake in both HT29 cells and the Cl--secreting T84 line. Butyrate markedly decreased transepithelial Cl- secretion in confluent T84 monolayers without effect on cAMP-regulated apical Cl- efflux. We conclude that NKCC1 regulation during enterocyte differentiation occurs at the level of gene expression, and that selective downregulation of NKCC1 gene expression and function by butyrate leads to a profound decrease in transepithelial Cl- secretion. These data emphasize the importance of NKCC1 in determining epithelial secretory capacity and suggest the possibility of modulation of the enterocytic transport phenotype as therapy for diarrheal disorders.

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.

Is CT angiography sufficient for prediction of resectability of periampullary neoplasms

The optimal preoperative evaluation of periampullary neoplasms remains controversial. The aim of this study was to analyze the accuracy of helical computed tomography (CT) and CT angiography with three-dimensional reconstruction in predicting resectability. Between March 1996 and May 1999, a total of 100 patients with periampullary neoplasms were prospectively staged by helical CT and CT angiography with three-dimensional reconstruction. Vascular involvement was graded from 0 to 4, with grade 0 representing no vascular involvement and grade 4 total encasement of either the superior mesenteric vein or artery. Patients with grade 4 lesions were considered unresectable. Sixty-eight patients underwent surgical exploration with intent to perform a pancreaticoduodenectomy. Forty-four lesions were grade 0, five were grade 1, eight were grade 2, and 11 were grade 3. Resectability for grades 0 to 3 was 96%, l00%, 50%, and 9%, respectively, for an overall resectability rate of 76%. Resectability in patients with vascular encroachment (grade 2) is usually determined by the extent of local disease rather than the presence of extrapancreatic disease. Resection is rarely possible in patients with evidence of vascular encasement (grade 3). Additional imaging modalities such as diagnostic laparoscopy are superfluous in patients with no evidence of local vascular involvement on CT angiography (grades 0 and 1) because of the high resectability rate and infrequency of unsuspected distant metastatic deposits.

Molecular Regulation of Na+-K+-2C1- Cotransporter (NKCC1) and Epithelial Chloride Secretion

Defective regulation of epithelial secretion underlies the clinical manifestations of a number of important human diseases ranging from cystic fibrosis to secretory diarrhea. Although much attention has focused on the role of apical membrane chloride (Cl-) channels in this process, emerging evidence highlights the importance of a basolateral Na+-K+-2Cl- cotransporter (NKCC1) as an independent regulatory site that may determine the overall rate of epithelial secretion. The cotransporter NKCC1 is expressed not only in epithelial cells but also in virtually all mammalian cells, where it plays a more generalized role in cell volume homeostasis, cell ionic composition, and, possibly, the control of cell growth. Emerging molecular evidence indicates that NKCC1 function is regulated in the short and long term at the level of protein phosphorylation, membrane targeting, and gene expression. An improved understanding of NKCC1 may lead to new therapeutic approaches to secretory diarrhea as well as diverse clinical conditions in which cell ion composition is disturbed.

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.

Monochloramine directly modulates Ca2+-activated K+ channels in rabbit colonic muscularis mucosae

BACKGROUND & AIMS Mesenteric ischemia, infection, and inflammatory bowel disease may eventuate in severe colitis, complicated by toxic megacolon with impending intestinal perforation. Monochloramine (NH(2)Cl) is a membrane-permeant oxidant generated during colitis by the large amount of ambient luminal NH(3) in the colon. Reactive oxygen metabolites can modulate smooth muscle ion channels and thereby affect colonic motility, which is markedly impaired in colitis. METHODS Effects of NH(2)Cl on ionic currents in the innermost smooth muscle layer of the colon, the tunica muscularis mucosae, were examined using the patch clamp technique. Membrane potential in whole tissue strips was measured using high-resistance microelectrodes. RESULTS Whole cell voltage clamp experiments showed that NH(2)Cl (3-30 micromol/L) enhanced outward currents in a dose-dependent manner, increasing currents more than 8-fold at a test potential of +30 mV. Tail current analysis showed that the currents enhanced by NH(2)Cl were K(+) currents. Inhibition by tetraethylammonium and iberiotoxin suggested that these currents represented activation of large-conductance, Ca(2+)-activated K(+) channels. The membrane-impermeant oxidant taurine monochloramine, however, had no effect on whole cell currents. Single-channel studies in inside-out patches showed that NH(2)Cl increased open probability of a 257-pS channel in symmetrical (140 mmol/L) K(+). In the presence of NH(2)Cl, the steady-state voltage dependence of activation was shifted by -22 mV to the left with no change in the single-channel amplitude. The sulfhydryl alkylating agent N-ethylmaleimide prevented NH(2)Cl-induced channel activation. NH(2)Cl also hyperpolarized intact muscle strips, an effect blocked by iberiotoxin. CONCLUSIONS NH(2)Cl, at concentrations expected to be found during colitis, may contribute to smooth muscle dysfunction by a direct oxidant effect on maxi K(+) channels.

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.