Mitochondria-rich Research Articles

Localization of chloride conductance to mitochondria-rich cells in frog skin epithelium.

Cell volume determinations and electrophysiological measurements have been made in an attempt to determine if mitochondria-rich (MR) cells are localized pathways for conductive movements of Cl across frog skin epithelium. Determinations of cell volume with video microscope techniques during transepithelial passage of current showed that most MR cells swell when the tissue is voltage clamped to serosa-positive voltages. Voltage-induced cell swelling was eliminated when Cl was removed from the mucosal bath solution. Using a modified vibrating probe technique, it was possible to electrically localize a conductance specifically to some MR cells in some tissues. These data are evidence supporting the idea that MR cells are pathways for conductive movements of Cl through frog skin epithelium.

The volume of mitochondria-rich cells of frog skin epithelium.

The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog, Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.

Identification and quantification of mitochondria-rich cells in transporting epithelia.

Fluorescent dyes specific for mitochondria have become important tools in the study of transporting epithelia. These dyes permit the localization and quantification of mitochondria-rich (MR) cells in these epithelia. To determine the specificity of the dye, dimethylaminostyrylmethylpyridiniumiodine ( DASPMI ), we combined fluorescence microscopy of this dye with the ultrastructural localization of the mitochondrial enzyme, cytochrome oxidase. Labeled cells were traced from the fluorescence-microscopic to the electron-microscopic level by devising several novel technical procedures. This new methodology assures a critical assessment of the specificity of fluorescent mitochondrial dyes in heterogeneous epithelia. Confirmation of DASPMI specificity allows the unequivocal identification of MR chloride cells in two epithelia in the head region of Fundulus heteroclitus and validates linear regression analysis of chloride cell number and short-circuit current in this species. In addition, this method provides a permanent, flat whole mount of labeled cells for morphological studies with the light microscope and with the scanning and transmission electron microscopes.

Modification of mitochondria-rich cells in different ionic conditions: changes in cell morphology and cell number in the skin of Xenopus laevis.

Xenopus laevis were kept in salt water (1.25% NaCl), distilled water, or tapwater for a month. Compared to the animals kept in tap water, the number of mitochondria-rich (MR) cells in the NaCl-adapted animals was significantly reduced, while it was increased in those maintained in distilled water. In addition, the MR-cells of NaCl-adapted animals lost their slender flask shape and developed large deposits of glycogen. The alteration of this cell type in conditions of high or low salinity may reflect a role of MR-cells in adaptation to different ionic environments.

Isolation and separation of toad bladder epithelial cells

The epithelium of the urinary bladder of Bufo marinus is composed of 5 cell types, i.e., granular (Gr), mitochondria-rich (MR) and goblet (G) cells which face the urinary lumen, microfilament-rich (MFR) and undifferentiated cells (Un) located basally. The epithelium was dissociated by collagenase and EGTA treatment. Fractionation of dispersed cells by isopycnic centrifugation on dense serum albumin solutions yielded 4 fractions: (i) a very light fraction (p approximately equal to 1.025) enriched in MR and MFR cells; (ii) a light fraction (p approximately equal to 1.045) enriched in vacuolated Gr cells; (iii) a heavy fraction (p approximately equal to 1.065) composed essentially of aggregated Gr cells, and (iv) a pellet (p approximately equal to 1.085) enriched in G and undifferentiated cells. Recoveries were based on cell counts and DNA measurements. DNA content per cell was 13.2 pg +/- 0.9 (n = 37). From 1 g fresh tissue, 62 +/- 5 x 10(6) (n = 10) cells were recovered before isopycnic centrifugation of which about 70% excluded Trypan blue. After centrifugation, 90 to 95% of the cells excluded the vital dye and approximately 3(9) x 10(6) cells were recovered from the gradient. Cell metabolism in each fraction was estimated by oxygen consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption measurements in absence or presence of ouabain, acetazolamide, and dinitrophenol. The consumption was threefold higher in the very light and light fractions when compared to the heavy and pellet fractions. Ouabain sensitive oxygen consumption (QO2) represented 12 to 35% of the total O2 consumption depending on the cell fraction, and acetazolamide sensitive QO2 varied from -0.8% in the heavy fractions to 20% in the lighter fractions. DNP increased QO2 in all fractions by 20 to 50%. Finally, the cells were able to reaggregate and form junctional complexes upon addition of calcium to the medium.

Cellular changes in the toad urinary bladder in response to metabolic acidosis.

The urinary bladder of Bufo marinus excretes H+ and NH+4, and the H+ excretion is increased when the animal is placed in metabolic acidosis. The mitochondria-rich (MR) cells mediate the H+ excretion by the bladder. The purpose of this study was to determine if there is a change in MR cells of the bladder during metabolic acidosis. Bladders from normal toads and from toads that had been placed in metabolic acidosis were used. The bladders were mounted between plastic chambers and H+ excretion measured. The bladder was then fixed and prepared for scanning (SEM) and transmission (TEM) electron micrograph studies. SEM's at low magnification were used to count the various cell types and the TEM's were used to confirm the different cell types. Fields were randomly selected and a total of 2500 cells counted in each group. The bladders from toads in metabolic acidosis had a consistently higher ratio of MR cells to granular cell than did the normal bladders. These results indicate that during metabolic acidosis there is an increased number of MR cells in the bladder, and this increased the bladder's capacity to excrete H+.

Differential effects of vasopressin on the water, calcium and lysosomal enzyme contents of mitochondria-rich and lysosome-rich (granular) epithelial cells isolated from bullfrog urinary bladder

Treatment of bullfrog urinary bladder with arginine vasopressin (AVP) elicited a dose-dependent increase in the basal movement of water and sodium across isolated tissues. Epithelial cells from the mucosal surface and incubated with 10 ml) AVP/ml for 30 min retained a greater amount of intracellular water and calcium than cells not treated with hormone. The epithelial cells were further separated into two major fractions by density gradient centrifugation; cells damaged during these manipulations were separated from viable cells and discarded. Morphologcal examination of the two respective fractions indicated that they largely consisted of mitochondria-rich (MR) and granular (G) cell types which line the lumen of bullfrog bladder. The calcium content of MR cells averaged 25% greater than that of G-type cells. G cells had a markedly higher content of the characteristic lysosomal hydrolases, acid phosphatase and cathepsin B1, than that found in MR cells. Incubation of G cells with AVP elicited significant increments in water and calcium contents and extracellular release of lysosomal enzymes as compared to untreated cells. Among MR cells treated with AVP, cell calcium declines slightly but no significant increase in water content or extracellular hydrolase activity was detected in comparison with paired control cells. The physiological significance of acid proteinase release from G cells treated with AVP was evaluated in experiments with intact bladder. Proteinase inhibitors which suppress the activity of cathepsin Bl selectively antagonized the action of hormone on water permeation. The data suggest that alterations in the calcium and lysosomal hydrolase activity associated with G cells exposed to AVP may contribute to the hormone-induced water flow observed across the intact epithelium.

Binding of aldosterone by mitochondria-rich cells of the toad urinary bladder

ALDOSTERONE causes a large increase in the active transport of sodium by the urinary bladder1 of the toad. This response, analogous to the effects of the steroid on the kidney, has been the object of considerable biochemical study2. Aldosterone also enhances the stimulation by neurohypophyseal hormones of sodium and hydro-osmotic flux3,4. Because the response in sodium transport seems quantitatively related to the increment of cyclic AMP generated by vasopressin5, augmentation of the sodium response is presumably related to the larger increment of intracellular nucleotide induced by a given dose of vaso-pressin in steroid-treated bladders6. Using a technique for separating the two major cell types of the bladder mucosal epithelium—the granular (G) and the mitochondria-rich (MR) cells—we found that a response to neurohypophyseal hormones, as reflected by hormone-induced increases in the intracellular cyclic AMP content, is apparently limited to the MR cell7. This apparent synergism of the neurohypophyseal and steroid hormones on transport suggests that their loci of action are in close proximity. We therefore examined the binding of 3H-aldosterone by the two major cell types.

Aldosterone effect in the epithelium of the frog skin—A new story about an old enzyme

It is established that aldosterone stimulates active epithelial sodium transport. Evidence is accumulating that this effect on transport is mediated through an intermediate step of protein synthesis. So far, morphobioelectric results of our laboratory indicate that only one type of cell, namely the mitochondriarich (MR) cell in all epithelia studied is involved. This cell responds in a characteristic manner to the hormone, with respect to induction and protein synthesis. Carbonic anhydrase activity is not only selectively located in the mitochondria-rich cells but its activity pattern parallels our earlier morphological observations. As there is evidence that sodium selection of the outer membrane in frog skin is strongly pH dependent[1], we tentatively propose that epithelial transport regulation by aldosterone is mediated through carbonic anhydrase which is induced, synthesized and secreted by the mitochondria-rich cells. Carbonic anhydrase may control the pH in the extracellular compartment next to the outer membrane and thus regulate the sodium permeability of the latter.