The kinin receptor is expressed in the Malpighian tubule stellate cells in the mosquito Aedes aegypti (L.): a new model needed to explain ion transport?

It is known that insect kinins increase diuresis and fluid secretion in the Aedes aegypti Malpighian tubule, causing a rapid drop of the transepithelial resistance and increasing chloride conductance from the hemolymph towards the tubule lumen. The tubule is composed of both principal and stellate cells. The main route for increased chloride influx upon kinin treatment is proposed to be paracellular, with septate junctions acquiring increased chloride selectivity and conductance. Therefore, kinin treatment renders the Ae. aegypti tubule a “leaky epithelium”, and under this model the kinin receptor is postulated to be expressed in principal cells. However, in another dipteran, the fruit fly Drosophila melanogaster, the main route for chloride transport is transcellular through stellate cells. In both the fruit fly and the mosquito Anopheles stephensi the kinin receptor has been immunolocalized in stellate cells, where it regulates transepithelial chloride permeability. Here we show that in Ae. aegypti, similarly, the stellate cells express the kinin receptor. This was confirmed through immunohistochemistry with two specific anti-kinin receptor antibodies and confocal analysis. The receptor is detected as a 75 kDa band in western blot. These results indicate that the currently accepted model for chloride transport must be re-evaluated in Ae. aegypti and suggest the kinin regulatory signals controlling intercellular junctions originate in the stellate cells.     

Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules

Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli–Sertoli cell junction and Sertoli–spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.     

Exotic aqueous behavior of synthetic lipids: formation of vesicular nanotubes

The work reported herein deals with the synthesis and the aggregation behavior studies of synthetic lipids bearing a non-ionic polar head made up of a tris(hydroxymethyl) aminomethane (tris) moiety linked with an aminoglycerol interface. The hydrophobic chains with variable lengths were grafted onto the hydroxyl functions of the aminoglycerol residue through ester or carbamate bonds. Tiny chemical modifications within this family of non-ionic surfactants brought about major variations in their aggregation behavior. They formed vesicles, tubules, and also small stable end-capped tubules - called vesicular nanotubes -, when the polar head bore two heptadecyl chains linked through a carbamate bond. Various techniques (nanosizer measurements, freeze fracture electron microscopy (FFEM), transmission electron microscopy (TEM), carboxyfluorescein (CF)) encapsulation were used to specify the structure of these assemblies. Notably, the vesicular nanotubes exhibited a small size, a fair polydispersity, great stability in an aqueous solution (up to 1 year) and a good efficiency to entrap and slowly release a probe such as carboxyfluoresceine: all these properties are perfectly suitable for their use as potential drug carriers.     

The role of intracellular cAMP in renal gluconeogenesis in view of differential action of various cAMP analogues

Effects of various cAMP analogues on gluconeogenesis in isolated rabbit kidney tubules have been investigated. In contrast to N(6),2'-O-dibutyryladenosine-3',5'-cyclic monophosphate (db-cAMP) and cAMP, which accelerate renal gluconeogenesis, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP) and 8-(4-chlorophenylthio)-cAMP (pCPT-cAMP) inhibit glucose production. Stimulatory action of cAMP and db-cAMP may be evoked by butyrate and purinergic agonists generated during their extracellular and intracellular metabolism resulting in an increase in flux through fructose-1,6-bisphosphatase and in consequence acceleration of the rate of glucose formation. On the contrary, Br-cAMP is poorly metabolized in renal tubules and induces a fall of flux through glyceraldehyde-3-phosphate dehydrogenase. The contribution of putative extracellular cAMP receptors to the inhibitory Br-cAMP action is doubtful in view of a decline of glucose formation in renal tubules grown in the primary culture supplemented with forskolin. The presented data indicate that in contrast to hepatocytes, in kidney-cortex tubules an increased intracellular cAMP level results in an inhibition of glucose production.     

Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2

The multidrug transporter MDR-1 P-glycoprotein (Pgp) has been recently pointed out as an important mechanism underlying chemical interaction between drugs and many commonly ingested substances, including grapefruit juice (GFJ). Modulation of intestinal Pgp dependent transport by GFJ may lead to changes in bioavailability of drugs that are substrates of Pgp itself, by affecting their presystemic clearance. Since other cellular sites expressing Pgp and devoted to drug disposition, like kidney proximal tubules, could be involved in these pharmacokinetic interactions, we investigated the effect of GFJ on the expression and activity of Pgp in the human immortalized tubular cell line HK-2. Two flavonoid compounds related to GFJ, kaempferol and naringenin, were also tested for their effects on HK-2 Pgp. HK-2 cells cultured for 4 days in the presence of GFJ, showed a dose-dependent decrease in Pgp immunoblottable amount as well as a decrease in MDR-1 mRNA level, as shown by western blot analysis and RT-PCR, respectively. Both kaempferol and naringenin were also able to significantly decrease Pgp immunoblottable amount. To test whether the downregulation of HK-2 Pgp due to GFJ exposition could influence the cell sensitivity to drugs that are transported by Pgp itself, HK-2 cells precultured with GFJ were exposed to scalar concentrations of Cyclosporin A or Vinblastine and cell viability examined 36 hours later. The cytotoxicity of both drugs was increased. The calcein-AM test in untreated cells showed that GFJ, kaempferol or naringenin inhibited Pgp activity. Downregulation of Pgp as well inhibition of its function by GFJ or its related components in tubular cells could have a role in changing disposition kinetics of some important therapeutic agents.     

Expression analysis of the novel gene collagen triple helix repeat containing-1 (Cthrc1)

We recently identified collagen triple helix repeat containing-1 (Cthrc1) as a novel gene induced in adventitial fibroblasts after arterial injury. Cthrc1 is a 30 kDa secreted protein that has the ability to inhibit collagen matrix synthesis. Cthrc1 is also glycosylated and retains a signal sequence consistent with the presence of Cthrc1 in the extracellular space. In injured arteries and skin wounds, we have found Cthrc1 expression to be associated with myofibroblasts and sites of collagen matrix deposition. Furthermore, we demonstrated that Cthrc1 inhibits collagen matrix deposition in vitro. Using in situ hybridization and immunohistochemistry, we characterized the expression domains of Cthrc1 during murine embryonic development and in postnatal tissues. In mouse embryos, Cthrc1 was expressed in the visceral endoderm, notochord, neural tube, developing kidney, and heart. Abundant expression of Cthrc1 was observed in the developing skeleton, i.e., in cartilage primordia, in growth plate cartilage with exclusion of the hypertrophic zone, in the bone matrix and periostium. Bones from adults showed expression of Cthrc1 only in the bone matrix and periostium while the articular cartilage lacked expression. Cthrc1 is typically expressed at epithelial-mesenchymal interfaces that include the epidermis and dermis, basal corneal epithelium, airway epithelium, esophagus epithelium, choroid plexus epithelium, and meninges. In the adult kidney, collecting ducts and distal tubuli expressed Cthrc1. Collectively, the sites of Cthrc1 expression overlap considerably with those reported for TGF-beta family members and interstitial collagens. The present study provides useful information towards the understanding of potential Cthrc1 functions.     

Chlamydia trachomatis causes centrosomal defects resulting in chromosomal segregation abnormalities

Chlamydiae traffic along microtubules to the microtubule organizing center (MTOC) to establish an intracellular niche within the host cell. Trafficking to the MTOC is dynein dependent although the activating and cargo-linking function of the dynactin complex is supplanted by unknown chlamydial protein(s). We demonstrate that once localized to the MTOC, the chlamydial inclusion maintains a tight association with cellular centrosomes. This association is sustained through mitosis and leads to a significant increase in supernumerary centrosomes, abnormal spindle poles, and chromosomal segregation defects. Chlamydial infection thus can lead to chromosome instability in cells that recover from infection.