Biomedical subjects

V Dodane

Publications and source records attributed to V Dodane.

8 recordsLinked to original sources

PubMed1999

ATP-Induced Ca(2+) release in cochlear outer hair cells: localization of an inositol triphosphate-gated Ca(2+) store to the base of the sensory hair bundle.

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Adenosine Triphosphate↗

PubMed1999

Effect of chitosan on epithelial permeability and structure.

Numerous studies have shown that chitosan, a mucoadhesive polymer, is a potential enhancer for transmucosal drug delivery. To further understand the mechanisms involved in chitosan action on the mucosal barrier, the activity of chitosan on the function and structure of monolayers of intestinal epithelial cells was investigated. In Caco-2 cells, chitosan caused a reversible, time and dose-dependent decrease in transepithelial electrical resistance. The effect of chitosan on tight junctions was confirmed by an increased permeability coefficient for mannitol transport when cells were treated with 0.1-0.5% w/v chitosan solution for 60 min compared to control cells. Involvement of tight junctions was visualized by confocal scanning microscopy using occludin and ZO-1, tight junctional proteins. Following an incubation with 0.01 or 0.1% w/v chitosan, labeling of both proteins varied in localization and decreased in fluorescent intensity at the cell periphery. In addition, a focal condensation of actin was observed preferentially at areas of cell-to-cell contacts. However, after 24-h recovery, the cell structure resembled untreated control cells. Simultaneous addition of cycloheximide, a protein synthesis inhibitor, prevented full recovery. This implied that protein synthesis was required for the cells to return to baseline levels. Chitosan treatment appeared to slightly perturb the plasma membrane as assessed by an increased release of lactate dehydrogenase. However, addition of 0.5% chitosan for 60 min did not affect cell viability as shown by Trypan blue dye exclusion. These data suggest that chitosan increases cell permeability by affecting paracellular and intracellular pathways of epithelial cells, in a reversible manner.

Animals↗

PubMed1998

Presynaptic localization of G protein isoforms in the efferent nerve terminals of the mammalian cochlea.

Heterotrimeric guanine nucleotide binding proteins (G proteins) are known to be involved in receptor-mediated synaptic activity. In order to determine which G protein isoforms, if any, are involved in synaptic regulation in the organ of Corti, we performed an extensive immunocytochemical screening. We localized a Galpha(q/11) isoform to the efferent nerve terminals using antibodies specific against the alpha subunit of these proteins. The label was observed in the efferent boutons contacting either the outer hair cells or the afferent fibers at the inner spiral bundle. We compared the localization of this isoform to that of the presynaptic protein SNAP-25 in double labeling experiments. Galpha(q/11) immunoreactivity was present predominantly in the cytoplasm of the presynaptic boutons in a region of high density of synaptic vesicles, while SNAP-25 was localized predominantly in the plasma membrane of the boutons. No label for these proteins was found at the afferent synapses, including the presynaptic terminals on hair cells. These results suggest that an isoform of the Gq subfamily of the G proteins might be involved in presynaptic modulation of neurotransmitter release at the cochlear efferents.

Animals↗

PubMed1998

Immunoelectronmicroscopy of soluble and membrane proteins with a sensitive postembedding method.

The application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.

Animals↗

PubMed1997

A collagen substrate enhances the sealing capacity of tight junctions of A6 cell monolayers.

A6 cells, a kidney derived epithelial cell line, when cultured either on a collagen-coated substrate or on polycarbonate substrate without collagen form confluent monolayers that are similar in cell density and overall morphology. However, the transepithelial electrical resistance (TER) of monolayers grown on the collagen-coated substrate is ninefold higher than that of monolayers grown without collagen. A comparative freeze-fracture study showed that this large difference in TER is not related to the length or number of tight junction strands but to differences in the specific conductance of individual strands. This conductance was obtained considering the TER, the linear junctional density and the mean number of tight junction strands. We estimated the specific linear conductance of the tight junction strands to be 2.56 x 10(-7) S/cm for cells grown on collagen and 30.3 x 10(-7) S/cm for the cells grown without collagen. We also examined changes in distribution and phosphorylation states of the zonula occludens associated protein, ZO-1, during monolayer formation. Immunocytochemistry reveals that the distribution of ZO-1 follows a similar time course and pattern independent of the presence or absence of collagen. While the amount of ZO-1 expression is identical in cells grown on both substrates, this protein is phosphorylated to a greater extent during the initial stages of confluence in cells cultured on collagen. We suggest that the phosphorylation levels of ZO-1 in A6 cells at the early stages of monolayer formation may determine the final molecular structure and specific conductance of the tight junctions strands.

Animals↗

PubMed1996

Identification of isoforms of G proteins and PKC that colocalize with tight junctions.

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near zonula occludens complex. Antibodies against alpha-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, G alpha 12 and PKC zeta, were the only isoforms of these proteins at the cell borders. In subconfluent monolayers, G alpha 12 and PKC zeta were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that G alpha 12 and PKC zeta may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions.

Animals↗

PubMed1994

Modulation of tight junction morphology and permeability by an epithelial factor.

We report evidence of a factor secreted at the apical side of epithelial monolayers which modulates tight junction structure and permeability. This activity was detected within 4-7 days of conditioning of the apical medium by MDCK, A6 or Caco-2 epithelial cell lines cultured on permeable membranes in bipartite chambers. Apical conditioned medium (ACM), applied to the basolateral surface of a confluent monolayer, increased the transepithelial electrical resistance (TER), progressively reaching values 12-22% higher than the baseline within 5-10 min. After 40-60 min, the TER returned slowly to the basal value. This phenomenon was not observed either when using preheated ACM or the ACM filtrate obtained through a 30,000 MW cutoff membrane. The ACM maintained its activity even when applied to cell lines from different organs and species, as demonstrated when ACM from MDCK monolayers promoted an increase of 22% in the TER of Caco-2 cells. The increase of TER induced by the ACM treatment is accompanied by a change in the distribution of the number of tight junction strands, from an initial pattern, dominated mostly by junctions with one or two strands, to a new pattern after treatment dominated by junctions with two or three strands. Our results suggest the existence of a mechanism in epithelial cells that could signal leakage of apically secreted components to the basolateral side, thereby modulating the junction structure and permeability.

Animals↗

PubMed1991

Longitudinal study of solute excretion and glomerular ultrastructure in an experimental model of aging rats free of kidney disease.

Because experimental studies of kidney aging are frequently complicated by the presence of renal disease, we set out to define a model minimizing renal pathology and thus revealing basic aging phenomena. Male and female Wistar/Lou rats were conceived, born, and bred to 42 months in a specific pathogen-free husbandry. They had free access to water and to a protein diet containing 2% fish and 15% vegetable proteins. The mean survival ages of this colony were 39 months for females and 35 months for males. Body weight, 24-hour food and water intake, urinary volume, and solute excretion were measured every 6 months in a group of 12 males and 12 females. Throughout the study, the mean body weight remained close to 180 gm in females and 320 gm in males. Despite this size difference, absolute daily food intake was similar in the two sexes and almost constant over the studied period. Age-related changes in proteinuria and phosphate excretion were greater in males than in females. Decreased urine osmolality and increased urinary volume, on the other hand, were more pronounced in old females than in males. Renal loss of calcium was noticed in both sexes and glucosuria remained discrete. Kidneys examined at 12, 24, and 36 months in both sexes and also at 42 months in females were free of major pathology such as pronounced glomerulosclerosis, tubular nephrosis, tubular cast, or hydronephrosis. In the oldest animals a few foci of interstitial inflammation occasionally were seen. The sole significant morphologic change was a regular but moderate thickening of the glomerular basement membrane, which roughly doubled its size from 12 to 36 months. Morphometric studies failed to demonstrate an increase in mesangial matrix or mesangial cellularity. No changes in foot processes, slit diaphragms, or endothelial fenestrae were seen with increasing age. These observations indicate that basic age-related changes in kidney structure and function of rats fed ad libitum can be reduced to a few parameters provided that adequate strains, diet, and husbandry conditions are selected for experimentation.

Aging↗