Search

Mutations in Tg737 cause a wide spectrum of phenotypes, including random left-right axis specification, polycystic kidney disease, liver and pancreatic defects, hydrocephalus, and skeletal patterning abnormalities. To further assess the biological function of Tg737 and its role in the mutant pathology, we identified the cell population expressing Tg737 and determined the subcellular localization of its protein product called Polaris. Tg737 expression is associated with cells possessing either motile or immotile cilia and sperm. Similarly, Polaris concentrated just below the apical membrane in the region of the basal bodies and within the cilia or flagellar axoneme. The data suggest that Polaris functions in a ciliogenic pathway or in cilia maintenance, a role supported by the loss of cilia on the ependymal cell layer in ventricles of Tg737(orpk) brains and by the lack of node cilia in Tg737(Delta2-3betaGal) mutants.

Porphyromonas gingivalis is considered among the etiological agents of human adult periodontitis. Although in vitro studies have shown that P. gingivalis has the ability to invade epithelial cell lines, its effect on the epithelial barrier junctions is not known. Immunofluorescence analysis of human gingival epithelial cells confirmed the presence of tight-junction (occludin), adherens junction (E-cadherin), and cell-extracellular matrix junction (beta1-integrin) transmembrane proteins. These transmembrane proteins are expressed in Madin-Darby canine kidney (MDCK) cells. In addition, MDCK cells polarize and therefore serve as a useful in vitro model for studies on the epithelial cell barrier. Using the MDCK cell system, we examined the effect of P. gingivalis on epithelial barrier function. Exposure of the basolateral surfaces of MDCK cells to P. gingivalis (>10(9) bacteria/ml) resulted in a decrease in transepithelial resistance. Immunofluorescence microscopy demonstrated decreases in the amounts of immunoreactive occludin, E-cadherin, and beta1-integrin at specific times which were related to a disruption of cell-cell junctions in MDCK cells exposed to basolateral P. gingivalis. Disruption of cell-cell junctions was also observed upon apical exposure to bacteria; however, the effects took longer than those seen upon basolateral exposure. Cell viability was not affected by either basolateral or apical exposure to P. gingivalis. Western blot analysis demonstrated hydrolysis of occludin, E-cadherin, and beta1-integrin in lysates derived from MDCK cells exposed to P. gingivalis. Immunoprecipitated occludin and E-cadherin molecules from MDCK cell lysates were also degraded by P. gingivalis, suggesting a bacterial protease(s) capable of cleaving these epithelial junction transmembrane proteins. Collectively, these data suggest that P. gingivalis is able to invade the deeper structures of connective tissues via a paracellular pathway by degrading epithelial cell-cell junction complexes, thus allowing the spread of the bacterium. These results also indicate the importance of a critical threshold concentration of P. gingivalis to initiate epithelial barrier destruction.

We investigated the effects of various phenothiazines on the Na+-H+ exchanger in brush-border membrane vesicles isolated from the proximal small intestine of the rabbit. The Na+-H+ exchanger activity was assayed by measuring the dimethylamiloride-sensitive Na+ uptake into these vesicles in the presence of an outward-directed H+ gradient ([pH]o greater than [pH]i). All phenothiazines that were tested inhibited the Na+-H+ exchanger and chlorpromazine sulfoxide (CPZ-SO) was the most potent inhibitor among them. CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake in a dose-dependent manner and IC50 for this process was 90 microM. Even though CPZ-SO drastically reduced the initial rates of Na+ uptake, the equilibrium uptake of Na+ into the vesicles was not affected, indicating that the integrity of membrane vesicles was not altered by the drug. Furthermore, the inhibition of the exchanger activity by phenothiazines was not due to nonspecific dissipation of the H+ gradient, because CPZ-SO inhibited the dimethylamiloride-sensitive Na+ uptake even in the absence of a H+ gradient. The inhibition of the exchanger by CPZ-SO was freely and completely reversible. Kinetic analyses of the Na+-H+ exchanger carried out in the presence and absence of CPZ-SO revealed that the nature of the inhibition depended on the composition of the buffers used in the assay of the exchanger because the inhibition was of a mixed type in the mannitol medium, whereas it was of a competitive type in the KCl medium.

Loperamide, an opiate agonist, inhibited the Na+-H+ exchanger in brush-border membrane vesicles isolated from term human placenta, rabbit renal cortex and rabbit small intestine in a dose dependent manner. Because the placental Na+-H+ exchanger was the most sensitive to inhibition by loperamide (IC50 = 60 microM), characterization of the inhibition was done with the placental Na+-H+ exchanger. The inhibition of the placental Na+-H+ exchanger by loperamide was instantaneous and freely reversible. Kinetic analyses demonstrated that the inhibition was of a mixed type. Loperamide (70 microM) reduced the maximal velocity (Vmax) from 46.4 +/- 1.2 to 34.5 +/- 1.1 nmol/mg of protein/15 s and increased the apparent affinity constant (Kt) for Na+ from 12.3 +/- 1.0 to 16.5 +/- 1.5 mM. Loperamide interacted with the exchanger protein at more than one site. The effects of loperamide were not antagonized by naloxone, suggesting the noninvolvement of opiate receptors in this process. These results differentiate loperamide from other Na+-H+ exchanger inhibitors such as amiloride, cimetidine and clonidine, which interact with the exchanger at a single site in a strictly competitive manner.

The characteristics of lactate transport in brush-border membrane vesicles isolated from normal human full-term placentas were investigated. Lactate transport in these vesicles was Na+-independent, but was greatly stimulated when the extravesicular pH was made acidic. In the presence of an inwardly directed H+ gradient ([H+]o greater than [H+]i), transient uphill transport of lactate could be demonstrated. This H+ gradient-dependent stimulation was not a result of a H+ diffusion potential. Transport of lactate in the presence of the H+ gradient was not inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid or by furosemide, ruling out the participation of an anion exchanger in placental lactate transport. Many monocarboxylates strongly interacted with the lactate transport system, whereas, with the single exception of succinate, dicarboxylates did not. The monocarboxylates pyruvate and lactate, but not the dicarboxylate succinate, when present inside the vesicles, were able to exert a trans-stimulatory effect on the uptake of radiolabeled lactate. Kinetic analyses provided evidence for a single transport system with a Kt of 4.1 +/- 0.4 mM for lactate and a Vmax of 54.2 +/- 9.9 nmol/mg of protein/30 s. Pyruvate inhibited lactate transport competitively, by reducing the affinity of the system for lactate without altering the maximal velocity. It is concluded that human placental brush-border membranes possess a transport system specific for lactate and other monocarboxylates and that this transport system is Na+-independent and is energized by an inwardly directed H+ gradient. Lactate-H+ symport rather than lactate-OH- antiport appears to be the mechanism of the H+ gradient-dependent lactate transport in these membranes.

Syncytiotrophoblast brush-border membrane vesicles isolated from full-term human placentas were shown to transport Na+ against a concentration gradient in the presence of an outward proton gradient [( H+i] greater than [H+]o). This proton gradient-coupled Na+ uptake was markedly inhibited and the uphill transport abolished when the electrochemical proton gradient was dissipated by carbonylcyanide 4-(trifluoromethoxy) phenylhydrazone. The presence of nigericin also eliminated the concentrative uptake of Na+ in these vesicles. Dimethylamiloride and harmaline inhibited the proton gradient-induced Na+ uptake. The apparent inhibition constant for this process was 0.32 microM for dimethylamiloride was freely reversible and the inhibitor reduced the Na+ uptake by directly interacting with the exchanger protein rather than by dissipating the H+ gradient. The dimethylamiloride-sensitive Na+ uptake was saturable with respect to Na+. The affinity constant for Na+ was 7.8 +/- 1.2 mM and the maximal velocity was 38.7 +/- 2.4 nmol X mg protein-1 X min-1. The dimethylamiloride-insensitive Na+ uptake was not saturable and probably represented simple diffusion. The diffusional component accounted for only 10% of the total uptake. Li+ strongly competed with Na+ for the uptake process and the apparent inhibition constant was 3.6 +/- 0.4 mM. Tetraethylammonium also caused significant inhibition of Na+ uptake, whereas K+, Rb+, Cs+, and choline had no effect. These data provide evidence for the existence of a Na+-H+ exchanger in human placental brush-border membrane, and the properties of this exchanger are similar to those of the Na+-H+ exchanger identified in the brush-border membrane of mammalian kidney and small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin is actively transported into brush-border membrane vesicles isolated from normal human term placentas and an inward-directed NaCl gradient provides the driving force for this process. Uptake is negligible if Na+is replaced by Li+, K+, Rb+, Cs+or choline. The presence of Cl-seems necessary for the maximal activity of this Na+-dependent uptake system. Intravesicular K+(20–40 mM) stimulates serotonin uptake, the stimulation being considerably greater at pH 7.5 than at pH 6.5. But, in the absence of K+, uptake at pH 6.5 was twice the uptake at pH 7.5. Unlabeled serotonin and dopamine inhibit the uptake of radiolabeled serotonin and the IC50values are 70 nMand 20 µM, respectively. Histamine and 5-hydroxytryptophan do not significantly interact with the system (IC50> 1 mM). Kinetic analysis reveals that serotonin uptake in these vesicles occurs via a single, saturable, high affinity system (Kt= 51 ± 2 nM; Vmax= 6.4 ± 0.1 pmol/mg of protein/15 s). The transporter is highly sensitive to inhibition by imipramine (IC50= 32 nM) and desipramine (IC50= 160 nM) but relatively insensitive to reserpine and hydralazine.

The Na+-H+ exchanger of the human placental brush-border membrane was inhibited by pretreatment of the membrane vesicles with a histidyl-group-specific reagent, diethyl pyrocarbonate and with a carboxy-group-specific reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. In both cases the inhibition was irreversible and non-competitive in nature. But, if the membrane vesicles were treated with these reagents in the presence of amiloride, cimetidine or clonidine, there was no inhibition. Since amiloride, cimetidine and clonidine all interact with the active site of the exchanger in a mutually exclusive manner, the findings provide evidence for the presence of essential histidyl and carboxy groups at or near the active site of the human placental Na+-H+ exchanger. This conclusion was further substantiated by the findings that Rose Bengal-catalysed photo-oxidation of histidine residues as well as covalent modification of carboxy residues with NN′-dicyclohexylcarbodi-imide irreversibly inhibited the Na+-H+ exchanger and that amiloride protected the exchanger from inhibition caused by NN′-dicyclohexylcarbodi-imide.

The Na+-H+ exchanger of human placental brush-border membrane was strongly inhibited by cimetidine, a histamine type II receptor antagonist. The inhibition was freely reversible and the apparent inhibition constant for the process was 38 microM. The nature of inhibition was found to be competitive with respect to Na+. Cimetidine inhibition was specific as other related compounds had no inhibitory effect. Treatment of the membrane vesicles with diethylpyrocarbonate inactivated the exchanger, but the presence of cimetidine during the treatment provided complete protection from this inhibition. These results show that histidine residues are located at the external Na+-binding site of the exchanger and cimetidine interacts with this site. The Na+-H+ exchanger of rabbit renal brush-border membrane was also susceptible to inhibition by the drug. However, transport of proline and glucose in placental brush-border membrane vesicles was not affected by cimetidine. The placental Na+-H+ exchanger was also inhibited by amiloride and the inhibition was freely reversible and competitive with respect to Na+. These results show that the characteristics of the inhibition of Na+-H+ exchanger by cimetidine and amiloride are very similar.

Normal cell polarity protects epithelial cells against Pseudomonas aeruginosa invasion and cytotoxicity. Using epithelial cell clones with selective defects in sorting of membrane constituents, and using hepatocyte growth factor pretreatment, we found that polarized susceptibility to P. aeruginosa can be altered without disrupting tight junctions. The results also showed that cellular susceptibility factors for invasion and cytotoxicity are not the same, although both are localized to the basolateral cell surface in polarized epithelial cells.

Hepatocyte growth factor (HGF) and E-cadherin are important for epithelial morphogenetic events. We examined the effects of HGF on E-cadherin localization and interaction with beta-catenin in polarized Madin-Darby canine kidney (MDCK) cell monolayers grown on filters. Surface biotinylation experiments showed that HGF increases apically accessible E-cadherin. Confocal immunofluorescence microscopy of HGF-treated cells showed localization of E-cadherin at membrane domains contacting the apical compartment and an increase in accessibility of apically applied antibodies to lateral E-cadherin below the tight junction. Coimmunoprecipitation of beta-catenin/E-cadherin complexes showed that the amount of E-cadherin associated with beta-catenin increased during the first 24 h of HGF treatment with a return to baseline values after 48 and 72 h. Metabolic labeling showed that HGF increased the synthetic rate of beta-catenin and the amount of newly synthesized E-cadherin associated with immunoprecipitated beta-catenin, with the peak effect occurring after 12 h of treatment and returning to baseline after 24 h. HGF treatment inhibited transcytosis of immunoglobulin A by the polymeric immunoglobulin receptor. We conclude that HGF treatment of polarized MDCK cells grown on filters decreases cell polarity and alters E-cadherin/beta-catenin interaction and synthesis.

An inward-directed H+ gradient markedly stimulated lactate uptake in rabbit intestinal brush-border membrane vesicles, and uphill transport against a concentration gradient could be demonstrated under these conditions. Uptake of lactate was many-fold greater in the presence of a H+ gradient than in the presence of a Na+ gradient. Moreover, there was no evidence for uphill transport of lactate in the presence of a Na+ gradient. The H+-gradient-dependent stimulation of lactate uptake was not due to the effect of a H+-diffusion potential. The uptake process in the presence of a H+ gradient was saturable [Kt (concn. giving half-maximal transport) for lactate 12.7 +/- 4.5 mM] and was inhibited by many monocarboxylates. It is concluded that a H+ gradient, not a Na+ gradient, is the driving force for active transport of lactate in rabbit intestinal brush-border membrane vesicles.