A common African variant of human connexin 37 is associated with Caucasian primary ovarian insufficiency and has a deleterious effect in vitro.

Folliculogenesis requires communication between granulosa cells and oocytes, mediated by connexin-based gap junctions. Connexin 37 (Cx37)-deficient female mice are infertile. The present study assessed Cx37 deficiency in patients with primary ovarian insufficiency (POI). A candidate gene study was performed in patients and controls from the National Genotyping Center (Evry, France) including 58 Caucasian patients with idiopathic isolated POI and 142 Caucasian controls. Direct genomic sequencing of the coding regions of the GJA4 gene (encoding Cx37) was performed with the aim to identify a deleterious variant associated with POI and absent in ethnically matched controls. A single Cx37 variant absent in the control population was identified, namely a c.946G>A heterozygous substitution leading to a p.Gly316Ser variant that was present in two POI patients. This variant was absent in all Caucasian controls from various databases, and has been observed exclusively in African populations. This variant was identified to have a dominant negative effect in HeLa cells in vitro to alter connexon function (by 67.2±7.17%), as determined by Gap-fluorescence recovery after photobleaching. The alteration principally resulted from a decrease of cell surface connexons due to altered trafficking (by 47.73±8.59%). In marked contrast to this observation, a p.Pro258Ser variant frequent in all ethnic populations in databases had no functional effect in vitro. In conclusion, the present study reported on a Cx37 variant in two Caucasian POI patients, which was absent in control Caucasian populations, and which had a deleterious effect in vitro. It is therefore suggested that in the genetic context of the Caucasian population, this variant may contribute to POI.

The Shigella type III effector IpgD recodes Ca2+ signals during invasion of epithelial cells.

The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca2+ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca2+ increases are transient and oscillatory, defining the so-called Ca2+ code that links cell responses to specific Ca2+ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca2+ signals. Here, we show that by hydrolyzing phosphatidylinositol-(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol-(1,4,5)trisphosphate (InsP3) levels. By modifying InsP3 dynamics and diffusion, IpgD favors the elicitation of long-lasting local Ca2+ signals at Shigella invasion sites and converts Shigella-induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP3-dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca2+ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca2+-dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.

Cadmium disorganises the scaffolding of gap and tight junction proteins in the hepatic cell line WIF B9.

BACKGROUND INFORMATION: Hepatocytes, which perform the main functions of the liver, are particularly vulnerable to toxic agents such as cadmium, an environmental pollutant. To identify the molecular targets for cadmium in hepatocytes, we have studied the effects of CdCl2 on the hybrid cell line WIF-B9 that exhibits stable structural and functional hepatocytic polarity. RESULTS: We showed that the toxicity of CdCl2 (1 µM, 24 h) resulted in a reduction in direct intercellular communication (via gap junctions) and in an increase in paracellular permeability (decrease in the sealing of tight junctions). These effects were not related to changes in the expression of the key proteins involved, Cx32 and claudin 2, the first being constitutive of gap junctions and the second of tight junctions in this cell line. Using immunofluorescence experiments, we observed a change in the location of Cx32 and claudin 2: these two proteins were less often found in the tight junction network that closes the bile canaliculi (BC). In control cells, 'Proximity Ligation Assay' (PLA Duolink®) has confirmed in situ that molecules of claudin 2 and Cx32 are very close to each other at the BC (probably less than 16 nm). This was no longer the case after treatment with CdCl2 . Localisation of occludin and Cx32 relative to each other was not modified by CdCl2 , but CdCl2 increased the PLA signal between molecules of JAM-A and Cx32. Finally, examination of freeze-fracture replicas obtained from cultures treated with CdCl2 showed the disruption of the network of tight junctions and the depletion or the disintegration of the junctional plaques associated with tight junctions. CONCLUSIONS: This study demonstrates in situ the changes induced by cadmium on the organisation of cell-cell junctions and points out the importance of the association Cx32/claudin 2 for the maintenance of normal hepatocyte functions.

Rat hepatocytes express functional P2X receptors.

Extracellular ATP regulates many hepatic functions by stimulating purinergic receptors. Only the G protein-coupled P2Y receptors have been studied in hepatocytes. We investigated the functional expression of P2X receptors, the ATP-gated channels in rat hepatocytes. P2X4 and P2X7 transcripts and proteins were detected by RT-PCR and by both Western blotting and immunocytochemistry. High concentrations of ATP, and 2'-and 3'-O-(4-benzoylbenzoyl)-ATP the preferring agonist of P2X7, induced membrane blebbing and significant uptake of 4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triethylammonio)propyl]diiodide, both of which were inhibited by oxidised ATP, a blocker of P2X receptors. These results provide evidence that P2X4 and P2X7 receptors are expressed and functional on rat hepatocytes, possibly playing an important role in the purinergic signaling complex in these cells.

Cholestatic bile acids inhibit gap junction permeability in rat hepatocyte couplets and normal rat cholangiocytes.

BACKGROUND/AIMS: The aim of this work was to study the effects of different bile acids on the permeability of gap junction channels (PGJC). We also looked at the effects of some bile acids on the coordination of intercellular calcium oscillations. METHODS: The permeability of gap junctions was assessed by fluorescent dye transfer and calcium signalling on fluorescent microscopy. RESULTS: Cholestatic bile acids such as taurolithocholate, taurolithocholate-sulfate and taurochenodeoxycholate inhibit the permeability of gap junctions in a dose-dependent and reversible manner in hepatocytes. Experiments performed in other cell types suggest that this effect is specific for cells having bile salt transporters, independently of the type of connexin expressed in these cells. Thus, cholestatic bile acids inhibit PGJC in normal rat cholangiocytes which express Cx43, but not in HeLa cells transfected with Cx26 or 32, which are expressed in hepatocytes. Calcium oscillations induced by bile acids in rat hepatocyte couplets are not coordinated and, by inhibiting the PGJC, cholestatic bile acids prevent the coordination of calcium oscillations induced by noradrenaline in these cells. CONCLUSIONS: Cholestatic, but not choleretic bile acids inhibit the PGJC in cells able to accumulate bile acids. This inhibition might contribute to the cholestatic effect of these bile acids.

Hypothalamic vasopressin release and hepatocyte Ca2+ signaling during liver regeneration: an interplay stimulating liver growth and bile flow.

Liver regeneration after partial hepatectomy is a plastic process during which the mechanisms that coordinate liver mass restoration compensate one another through a complex regulatory network of cytokines, growth factors, and hormones. Vasopressin, an agonist that triggers highly organized Ca2+ signals in the liver, may be one of these factors, although little in vivo evidence is available in support of this hypothesis. We provide evidence that hypothalamic vasopressin secretion is stimulated early after partial hepatectomy. Although hepatocytes were fully responsive to vasopressin during the first hours of regeneration, they became desensitized and exhibited slow oscillating Ca2+ responses to vasopressin on the following days. On the first day, hepatocyte V1a receptor density decreased and its lobular gradient increased in hepatectomized rats. By antagonizing the V1a receptor in vivo, we demonstrated that vasopressin contributes to NF-kappaB and cyclin (D1 and A) activation, to hepatocyte progression in the cell cycle, and to liver mass restoration. Finally, vasopressin exerted a choleretic effect shortly after hepatectomy, both in the isolated perfused liver and in the intact rat. In conclusion, we provide compelling in vivo evidence that vasopressin contributes significantly to growth initiation and bile flow stimulation in the early stages of liver regeneration.

Hormone receptor gradients supporting directional Ca2+ signals: direct evidence in rat hepatocytes.

BACKGROUND/AIMS: In the liver, InsP(3)-dependent agonists such as vasopressin and noradrenaline induce tightly coordinated sequences of intracellular Ca(2+) increases, leading to apparent unidirectional Ca(2+) waves. In previous works, we have postulated that cell-to-cell differences in hormone receptor density create a cellular sensitivity gradient that determines which cell initiates the intercellular Ca(2+) wave and the direction of propagation of the Ca(2+) signal. The aim of this study was to test directly this hypothesis. METHODS: Lobular distribution of V1a vasopressin receptors and alpha1 adrenergic receptors were observed by autoradiography in rat liver sections. Cell-to-cell differences in the number of these receptors were evaluated on hepatocyte multiplets using specific fluorescent probes. RESULTS: The relative amount of fluorescence associated with the V1a receptor differed significantly between cells within multiplets. The 'cell-after-cell' Ca(2+) increase induced by vasopressin was correlated with the number of V1a receptors. These observations may be more general, as autoradiography revealed similar lobular distributions of V1a receptors and alpha1 adrenergic receptors; the amounts of both were greatest in hepatocytes surrounding central veins. CONCLUSIONS: These data confirm that a fine gradient along liver cell plates contributes to the molecular basis of the unidirectional hormone-induced Ca(2+) signalling observed in the liver lobule.

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.