Production of ammonium by Helicobacter pylori mediates occludin processing and disruption of tight junctions in Caco-2 cells.

Tight junctions, paracellular permeability barriers that define epithelial cell polarity, play an essential role in transepithelial transport, cell-cell adhesion and lymphocyte transmigration. They are also important for the maintenance of innate immune defence and intestinal antigen uptake. Ammonium (NH4+) is elevated in the gastric aspirates of Helicobacter pylori-infected patients and has been implicated in the disruption of tight-junction functional integrity and the induction of gastric mucosal damage during H. pylori infection. The precise mechanism of the effect of ammonium and the molecular targets of ammonium in host tissue are not yet identified. To study the effects of ammonium on epithelial tight junctions, the human colon carcinoma cell line Caco-2 was cultured on permeable supports and the transepithelial resistance (TER) was measured at different time intervals following exposure to ammonium salts or H. pylori-derived ammonium. A biphasic response to treatment with ammonium was found. Acute exposure to ammonium salts or NH3/NH4+ derived from urea metabolism by wild-type H. pylori resulted in a 20-30 % decrease in TER. After 24 h, the NH4Cl-treated cells showed a partial recovery of TER. In contrast, the control culture, or cultures that were exposed to supernatants derived from urease-deficient H. pylori, showed no significant decrease in TER. Occludin-specific immunoblots revealed the expression of a low-molecular-weight form of occludin of 42 kDa upon NH3/NH4+ exposure. The results indicate that modulation of tight-junction function by H. pylori is ammonium-dependent and linked to the accumulation of a low-molecular-weight and detergent-soluble form of occludin.

Nitric oxide specifically reduces the permeability of Cx37-containing gap junctions to small molecules.

Gap junction intercellular communication (GJIC) plays a significant role in the vascular system. Regulation of GJIC is a dynamic process, with alterations in connexin (Cx) protein expression and post-translational modification as contributing mechanisms. We hypothesized that the endothelial autacoid nitric oxide (NO) would reduce dye coupling in human umbilical vein endothelial cells (HUVECs). In our subsequent experiments, we sought to isolate the specific Cx isoform(s) targeted by NO or NO-activated signaling pathways. Since HUVEC cells variably express three Cx (Cx37, Cx40, and Cx43), this latter aim required the use of transfected HeLa cells (HeLaCx37, HeLaCx43), which do not express Cx proteins in their wild type form. Dye coupling was measured by injecting fluorescent dye (e.g., Alexa Fluor 488) into a single cell and determining the number of stained adjacent cells. Application of the NO donor SNAP (2 microM, 20 min) reduced dye coupling in HUVEC by 30%. In HeLa cells, SNAP did not reduce dye transfer of cells expressing Cx43, but decreased the dye transfer from Cx37-expressing cells to Cx43-expressing cells by 76%. The effect of SNAP on dye coupling was not mediated via cGMP. In contrast to its effect on dye coupling, SNAP had no effect on electrical coupling, measured by a double patch clamp in whole cell mode. Our results demonstrate that NO inhibits the intercellular transfer of small molecules by a specific influence on Cx37, suggesting a potential role of NO in controlling certain aspects of vascular GJIC.

Role of basolateral membrane conductance in the regulation of transepithelial sodium transport across frog skin.

Circuit analyses of the principal cell compartment of frog skin ( Rana temporaria and R. esculenta) were made using microelectrode measurements under short-circuit conditions and with the aid of the Na(+) channel blocker amiloride. Under control conditions, intracellular potential ranged between -65 and -5 mV, and the conductances of the apical and basolateral membranes were related directly to the short-circuit current and inversely to the cellular potential. Blockade of apical Na(+) uptake by amiloride hyperpolarized the cells to nearly the same value, irrespective of the potential under transporting conditions. Under these conditions, basolateral membrane conductance increased greatly, which led to paradoxical reactions of the transepithelial Na(+) transport at lower concentrations of amiloride. The half-maximal inhibitory concentration of amiloride estimated from the response of the apical membrane conductance (99+/-10 nM) was about 5 times lower than the value derived from transepithelial current or conductance in the same tissues. The results are discussed in the context of the importance of the membrane potential for acute control of membrane conductance and transepithelial transport.

Xanthine derivatives without PDE effect stimulate voltage-activated chloride conductance of toad skin.

The effect of xanthine derivatives on the voltage-activated Cl(-) conductance (G(Cl)) of amphibian skin was analyzed. 3-Isobutyl-1-methylxanthine (IBMX) and the recently synthesized xanthine derivatives 3,7-dimethyl-1-propyl xanthine (X-32) and 3,7-dimethyl-1-isobutyl xanthine (X-33), which lack inhibitory effects on phosphodiesterases in CHO and Calu-3 cells, increased voltage-activated G(Cl) without effect on baseline conductance at inactivating voltage. Half-maximal stimulation of G(Cl) occurred at 108 +/- 9 microM for X-32 and X-33 after apical or basolateral application. The stimulation of G(Cl), which occurs only in the presence of Cl(-) in the mucosal solution, is caused by a shift of the voltage sensitivity to lower clamp potentials and an increase of the maximally activated level. Furosemide reversed both the shift of sensitivity and the increase in magnitude. These patterns are fundamentally different from those seen after application of membrane-permeant, nonmetabolized analogs of cAMP, and they indicate that the xanthines stimulate G(Cl) directly. This notion is strengthened by the lack of influence on intracellular cAMP content, which is consistent with the observations in CHO and Calu-3 cells. We propose that the xanthine derivatives increase the voltage sensitivity of a regulative component in the conductive Cl(-) pathway across amphibian skin.

The route of passive chloride movement across amphibian skin: localization and regulatory mechanisms.

Transepithelial Cl(-) conductance (G(Cl)) in amphibian skin can be activated in several species by serosa positive potentials. Mitochondria-rich cells (MRC) or tight junctions (TJ) between the epithelial cells are possible sites for this pathway. The properties and the techniques used to investigate this pathway are reviewed in the present paper. In situ techniques are preferable, since specific properties of the MRC are apparently not maintained in isolated cells. Volume measurements and electronprobe microanalysis of intracellular ions suggest the localization of voltage-activated G(Cl) to MRC. G(Cl) correlates poorly with the density of MRC. The vibrating voltage probe allows quantitative correlation of the local Cl(-) current through morphologically identified structures and the transepithelial Cl(-) current. Our analysis shows that 80% of the voltage-activated Cl(-) current is accounted for by current through MRC or their immediate vicinity. The activation patterns of this current and the inhibition by the alpha(1)-adrenergic agonist, epinephrine, conform to those of the transepithelial current. However, less than 20% of the MRC are active at a certain moment and the activity is spontaneously variable with time. The molecular nature of this pathway, physiological control mechanisms and their relation to the temporal activity of MRC remain to be studied.