Biomedical subjects

B J Hrnjez

Publications and source records attributed to B J Hrnjez.

9 recordsLinked to original sources

PubMed1999

PKC-epsilon 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-epsilon. 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-epsilon, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

Acetophenones↗

PubMed1999

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 (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.

Amines↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Calcium↗

PubMed1997

Effects of F-actin stabilization or disassembly on epithelial Cl- secretion and Na-K-2Cl cotransport.

Previous studies showed that cAMP-dependent transepithelial Cl- secretion of the intestinal cell line T84 is reduced by the F-actin stabilizer phalloidin, an effect in part attributable to inhibition of basolateral Na-K-2Cl cotransport. However, secretory responses are preserved in cells treated with the microfilament disrupter cytochalasin D. We explored the effects of cytochalasin D and two novel compounds derived from marine sponges on the Cl- secretory apparatus of T84 cells. Jasplakinolide (which stabilizes F-actin inhibited cAMP-dependent secretion and Na-K-2Cl cotransport. Latrunculin A (which sequesters G-actin monomers) profoundly altered the distribution of F-actin and reduced basal transepithelial resistance with minimal effect on secretion. Cytochalasin D, but not latrunculin A, activated Na-K-2Cl cotransport. The results provide further evidence that vectorial ion transport is influenced by the cytoskeleton and support a model in which disassembly of F-actin by specific pharmacological means or in response to secretory agonists favors activation of Na-K-2Cl cotransport.

Actins↗

PubMed1997

Ammonia effect on calcium-activated chloride secretion in T84 intestinal epithelial monolayers.

We recently showed that ammonia profoundly inhibits cyclic nucleotide-regulated Cl- secretion in model human T84 intestinal epithelia but does not impair the secretory response to the Ca2+ agonist carbachol. Using transepithelial transport, fura 2 fluorescence, and radioisotopic efflux techniques, we further explored this dichotomy and arrived at a preliminary explanation for the inhibitory action of ammonia. The secretory response to the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin is unaffected by ammonia, which suggests that an increase in intracellular Ca2+ stimulates secretory pathways that are insensitive to ammonia. Surprisingly, Cl- secretion elicited by the Ca2+ ionophores ionomycin and A23187 is markedly blunted in monolayers pretreated with ammonia. However, ammonia posttreatment does not inhibit the secretory response to ionophore, which suggests that ammonia may interfere with the ability of these ionophores to increase intracellular [Ca2+]. This hypothesis is directly supported by fura 2 experiments. The inhibitory action of ammonia parallels the behavior of the K+ channel blocker Ba2+, and ammonia reduces the basolateral 86Rb+ efflux rate constant in forskolin- but not in carbachol-treated monolayers. Ammonia, which is present in high concentrations in the normal gastro-intestinal tract, may serve as a novel endogenous regulator of epithelial electrolyte transport by interfering with a Ba(2+)-sensitive basolateral K+ conductance distinct from the Ca(2+)-activated basolateral K+ conductance.

Ammonia↗

PubMed1996

Adenosine scavenging: a novel mechanism of chloride secretory control in intestinal epithelial cells.

BACKGROUND: Adenosine released by cells during ischemia typically serves as a feedback inhibitor of further organ work. However, in ischemic intestine, adenosine appears to act via stimulatory A2b receptors to increase work in the form of chloride ion (Cl-) secretion. This unusual response may contribute to luminal fluid sequestration in intestinal ischemia. In nonischemic cells feed-forward activation of Cl- secretion does not occur despite the fact that adenosine may be continuously generated during normal cell metabolism. Thus we postulated that intestinal epithelia normally control the disposition of adenosine to prevent inappropriate activation of secretion. METHODS: Model T84 intestinal epithelia were studied by means of electrophysiologic and isotopic techniques. RESULTS: Dipyridamole and nitrobenzylthioinosine (inhibitors of nucleoside transport) and iodotubercidin (an inhibitor of adenosine kinase) caused adenosine to accumulate extracellularly and induced a Cl- secretory response that was prevented by adenosine receptor blockade. Uptake of exogenous adenosine was restricted to the basolateral compartment and was blocked by nucleoside transport inhibitors. CONCLUSIONS: Adenosine released from nonischemic intestinal epithelial cells is scavenged by a basolaterally restricted adenosine transporter. This system maintains extracellular adenosine levels below the prosecretory threshold and thus limits adenosine-elicited activation of Cl- secretion (and hence diarrhea) under normal conditions).

Adenosine↗

PubMed1995

Activation of Cl secretion during chemical hypoxia by endogenous release of adenosine in intestinal epithelial monolayers.

Intestinal ischemia is characterized by rapid early inhibition of absorptive function and the appearance of net secretion, although why active secretion persists in the setting of a mucosal energy deficit is unknown. The cryptlike epithelial line T84, a well-characterized model of intestinal Cl- secretion, develops a prominent increase in short-circuit current (Isc, indicative of active Cl- transport) in response to "hypoxia" induced by metabolic inhibitors. The increased Isc is associated with the initial decrease in monolayer ATP content. The Isc is transient and disappears with progressive energy depletion, although graded degrees of ATP depletion induce a more sustained Isc response. Chromatographic analysis and secretory bioassays show that the Isc response to metabolic inhibitors is related to the endogenous release of adenosine into the extracellular space in quantities sufficient to interact locally with stimulatory adenosine receptors. Unlike its classical role as a metabolic feedback inhibitor, adenosine appears to function as an autocrine "feed-forward" activator of active intestinal Cl- secretion. These studies suggest a novel role for adenosine in the conversion of the gut from an absorptive to a secretory organ during ischemic stress, thus contributing to the initial diarrheal manifestation of intestinal ischemia.

Adenosine↗

PubMed1995

Ammonia inhibits cAMP-regulated intestinal Cl- transport. Asymmetric effects of apical and basolateral exposure and implications for epithelial barrier function.

The colon, unlike most organs, is normally exposed to high concentrations of ammonia, a weak base which exerts profound and diverse biological effects on mammalian cells. The impact of ammonia on intestinal cell function is largely unknown despite its concentration of 4-70 mM in the colonic lumen. The human intestinal epithelial cell line T84 was used to model electrogenic Cl- secretion, the transport event which hydrates mucosal surfaces and accounts for secretory diarrhea. Transepithelial transport and isotopic flux analysis indicated that physiologically-relevant concentrations of ammonia (as NH4Cl) markedly inhibit cyclic nucleotide-regulated Cl- secretion but not the response to the Ca2+ agonist carbachol. Inhibition by ammonia was 25-fold more potent with basolateral compared to apical exposure. Ion substitution indicated that the effect of NH4Cl was not due to altered cation composition or membrane potential. The site of action of ammonia is distal to cAMP generation and is not due simply to cytoplasmic alkalization. The results support a novel role for ammonia as an inhibitory modulator of intestinal epithelial Cl- secretion. Secretory responsiveness may be dampened in pathological conditions associated with increased mucosal permeability due to enhanced access of lumenal ammonia to the basolateral epithelial compartment.

Ammonia↗