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

Summary 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.     

Localization of Na+-pump sites in frog skin

The localization of Na+-pump sites (Na+-K+-ATPase) in the frog skin epithelium was determined by a freeze-dry radioautographic method for identifying [3H]ouabain-binding sites. Ventral pelvic skins of Rana catesbeiana were mounted in Ussing chambers and exposed to 10(-6) M [3H]ouabain for 120 min, washed in ouabain-free Ringer's solution for 60 min, and then processed for radioautography. Ouabain-binding sites were localized on the inward facing (serosal) membranes of all the living cells. Quantitative analysis of grain distribution showed that the overwhelming majority of Na+-pump sites were localized deep to the outer living cell layer, i.e., in the stratum spinosum and stratum germinativum. Binding of ouabain was correlated with inhibition of Na+ transport. Specificity of ouabain binding to Na+-K+-ATPase was verified by demonstrating its sensitivity to the concentration of ligands (K+, ATP) that affect binding of ouabain to the enzyme. Additional studies supported the conclusion that the distribution of bound ouabain reflects the distribution of those pumps involved in the active transepithelial transport of Na+. After a 30-min exposure to [3H]ouabain, Na+ transport declined to a level that was significantly less than that in untreated paired controls, and analysis of grain distribution showed that over 90% of the ouabain-binding sites were localized to the inner cell layers. Furthermore, in skins where Na+ transport had been completely inhibited by exposure to 10(-5) M ouabain, the grain distribution was identical to that in skins exposed to 10(-6) M. The results support a model which depicts all the living cell layers functioning as a syncytium with regard to the active transepithelial transport of Na+.     

Localization of kinesin in cultured cells

Kinesin was isolated from bovine brain and used to elicit polyclonal antibodies in rabbits. The specificities of the resulting antibodies were evaluated by immunoblotting. Antibodies purified from these sera by their affinity for brain kinesin react with a polypeptide of approximately 120 kD in extracts from bovine brain, PtK1 cells, and mouse neuroblastoma cells. They bind to a pair of polypeptides of approximately 120 kD present in crude kinesin prepared from Xenopus eggs and with a single polypeptide of approximately 115 kD in extracts from Drosophila embryos. Antibodies raised against kinesin prepared from fruit fly embryos (by W. M. Saxton, Indiana University, Bloomington, IN) and from neural tissues of the squid (by M. P. Sheetz, Washington University, St. Louis, MO) cross react with the mammalian, the fly, and the frog polypeptides. Kinesin antigen was localized in cultured cells by indirect immunofluorescence. PtK1 cells in interphase showed dim background staining of cytoplasmic membranous components and bright staining of a small, fibrous, juxtanuclear structure. Double staining with antibodies to microtubules showed that the fibrous object was usually located near the centrosome. On the basis of shape, size, and location, we identify the kinesin-positive structure as a primary cilium. PtK1 cells in mitosis are stained at their poles during all stages of division. The structure stained is approximately spherical, but wisps of faint fluorescence also extend into the body of the spindle. Antibodies to squid or fruit fly kinesin produce identical patterns in PtK1 cells. Controls with preimmune and preabsorbed sera show that the centrosome staining is not due simply to the common tendency of rabbit antisera to stain this structure. Similar centrosome and spindle pole staining was visible when antibodies to bovine brain or squid kinesin were applied to the A6 cell line (kidney epithelial cells from Xenopus laevis). Some possible functions of kinesin localized at the spindle poles are discussed.     

Action of light on frog pigment cells in culture

Solar radiation induces numerous biologic effects in skin but the mechanism underlying these responses is poorly understood. To study the etiology of these phenomena, we investigated the effect of light on cultured Xenopus laevis melanophores. Visible light stimulated a marked increase in intracellular cAMP levels within the first minute of irradiation. This light-induced elevation in cAMP was blocked by melatonin and was not seen in fibroblasts irradiated in a similar manner. These data show that the photoresponse of pigment cells from amphibian skin can be mediated by a cAMP-dependent mechanisms and suggest that a unique member of the rhodopsin family is involved in this process.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Regeneration of the skin pigment system during larval development of the clawed frog

We demonstrate regeneration capability of the skin pigment system of clawed frog larvae after local damage to melanophores without skin rupture. The contribution to recovery of pigmentation of the injured area of de novo differentiation of melanophores is compared to contribution of mitotic division of undamaged melanophores localized on the boundaries of the injured area. The regeneration process is observed during various stages of pigment system development of larvae. We establish that, compared to ontogenetic dynamics, pigmentation development in animals is more intense during the regeneration.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Localization of crystallins in Muellerian cells in the grass frog retina

Cell localization of 23 kDa- and 35 kDa-crystallins in the retina of adult common frogs Rana temporaria L. was studied using indirect immunofluorescence. Intense specific fluorescence of both crystallins was observed all over the retina, in both periphery and central area. It was localized in elongated radially oriented cells, whose bodies were located in the inner nuclear layer. These cells gave many fluorescing processes in the same layer and main processes in the outer nuclear and ganglion layers, one in each. The processes formed a strong network of fibers around the photoreceptor and ganglion cells. Intense fluorescence was also observed in the layer of nerve fibers and adjoining inner limiting membrane. The distribution and morphology of crystalline-containing cells mostly coincides with what is known for the Muller cells of vertebrate eye. The identity of the cells we described and Muller cells was also confirmed using the antiserum to glial fibrillary acidic protein.     

Distribution of actin microfilaments in frog skin epithelial granular cells

Summary— Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less babel along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na⁺ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.     

Potassium currents in cultured cells of the rat retinal pigment epithelium

Whole-cell currents were investigated in cultured rat retinal pigment epithelial (RPE) cells. Two voltage-dependent conductances were discriminated. First, at potentials more positive than −30 mV, a time-dependent outward current was activated. Inhibition by Ba²⁺ (10 mM) and 4-aminopyridine (10 mM) indicated that this current was carried by potassium ions. This current showed no inactivation during 5 sec depolarizations. Second, an inward current, sensitive to Ba²⁺ (10 mM) and 4-aminopyridine (10 mM), was activated at potentials more negative than — 70 mV. Under extra- and intracellular potassium-free conditions, both currents disappeared. In summary, cultured rat RPE cells expressed one potassium conductance similar to the delayed rectifier and one similar to the inward rectifier. The delayed rectifier expressed characteristics comparable with those known in mammalian species and different from those in non-mammalian species.     

The volume of mitochondria-rich cells of frog skin epithelium

Summary 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.     

Cytoskeletal events underlying dendrite formation by cultured pigment cells

In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte-conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24–48 hr, and quantifiable by computerized image analysis. Cyto-chalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine-phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM-treated cells. Microtubules visualized with a monoclonal anti-tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering β actin or β tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte-induced changes involving β actin and β tubulin polymerization appear to require both new protein synthesis and post-translational regulation. The observed similarities between melanocytes and other neural crest-derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth. © 1992 Wiley-Liss, Inc.     

Single-channel recordings from cultured human retinal pigment epithelial cells

We have applied patch-clamp techniques to on-cell and excised-membrane patches from human retinal pigment epithelial cells in tissue culture. Single-channel currents from at least four ion channel types were observed: three or more potassium-selective channels with single-channel slope conductances near 100, 45, and 25 pS as measured in on-cell patches with physiological saline in the pipette, and a relatively nonselective channel with subconductance states, which has a main-state conductance of approximately 300 pS at physiological ion concentrations. The permeability ratios, PK/PNa, measured in excised patches were 21 for the 100-pS channels, 3 for the 25-pS channels, and 0.8 for the 300-pS nonselective channel. The 45-pS channels appeared to be of at least two types, with PK/PNa's of approximately 41 for one type and 3 for the other. The potassium-selective channels were spontaneously active at all potentials examined. The average open time for these channels ranged from a few milliseconds to many tens of milliseconds. No consistent trend relating potassium-selective channel kinetics to membrane potential was apparent, which suggests that channel activity was not regulated by the membrane potential. In contrast to the potassium-selective channels, the activity of the nonselective channel was voltage dependent: the open probability of this channel declined to low values at large positive or negative membrane potentials and was maximal near zero. Single-channel conductances observed at several symmetrical KCl concentrations have been fitted with Michaelis-Menten curves in order to estimate maximum channel conductances and ion-binding constants for the different channel types. The channels we have recorded are probably responsible for the previously observed potassium permeability of the retinal pigment epithelium apical membrane.     

Repigmentation of vitiliginous skin by cultured cells

Epidermal cells from pigmented areas of a patient with vitiligo were cultured in MCDB-153 medium, which supports the clonal growth of undifferentiated keratinocytes and melanocytes. The cells were grown on collagen-coated substrata. After the cells reached semiconfluence, the composite of substratum and cells was emplaced onto dermabraded vitiliginous areas as a graft. Re-epithelialization of the grafted areas was complete after 2 weeks. Repigmentation was evident after 1 month and continued over the observation period of several months. There was complete and normal differentiation of the graft, including a normal distribution of melanocytes in the basal layer. Ultrastructural studies showed a normal distribution of melanosomes in the melanocytes and showed keratinocytes that were indistinguishable from the uninvolved skin.     

Intrinsic pigment cell stimulating activity in the skin of the leopard frog, Rana pipiens.

Consistent with the concept that specific pigment patterns of amphibians might result from the highly localized distribution of stimulators and inhibitors of pigment cell expression in the skin, the spot pattern of the leopard frog, Rana pipiens, was examined through the use of the Xenopus neural tube explant assay system (Fukuzawa and Ide, 1988). Media conditioned with pieces of skin from dorsal black spotted areas promoted melanization of neural crest cells at a significantly higher level than did media conditioned with dorsal interspot skin in the absence of extra tyrosine. All conditioned media contained exceedingly low concentrations of tyrosine. With the addition of supplemental tyrosine, the melanization capacity of conditioned media from the interspot areas was elevated to that of the spotted skin. Control media conditioned with ventral frog skin inhibited melanization, as usual, because of the presumed presence of melanization inhibiting factor (MIF). It is considered that dorsal skin contains a melanization stimulating factor (MSF) which is present in significantly higher levels in spotted skin than in interspot areas and that expression of the particular pigmentary pattern of this leopard frog is regulated by the relative distribution of MIF, MSF, and possibly other intrinsic substances present in the skin.     

BDNF increases the phagocytic activity in cultured iris pigment epithelial cells

To investigate the effect of brain derived neurotrophic factor (BDNF) on the phagocytic activity in iris pigment epithelial (IPE) cells, purified porcine photoreceptor outer segments (POS) were applied to cultured IPE cells for three hours. To measure phagocytic activities, bound and total POS were differentially stained using a double immunofluorescence staining method. BDNF increased the binding of POS in IPE cells in a dose-dependent manner. Ingestion of POS, however, was not affected throughout the concentrations used in this study. To investigate the signal transduction pathways of BDNF, a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and MAPK/ERK kinase (MEK) inhibitor, PD98059, were used for this study. LY294002 had no effect on the binding and ingestion of POS in BDNF-treated IPE cells. On the other hand, PD98059 completely inhibited the increase of POS binding in BDNF-treated cells and also decreased the ingestion of POS. These results indicate that increased POS binding activity by BDNF and the decreased ingestion of POS were mediated through the MAPK pathway.