Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes.

We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.

Video-enhanced microscopy of organelle movement in an intact epithelium.

Digitally enhanced video microscopy has provided improved optical resolution in the study of intracellular organelle/particle movement, particularly in extruded axoplasm and certain thin single cell systems. We report here, for the first time, particle movement in an intact, isolated epithelium, the killifish proximal convoluted tubule. Cytoplasmic particles exhibited predominantly unidirectional linear movement approaching several microns in length, sometimes with multiple turns. The velocities of 34 particles measured in 11 cells averaged 0.29 microns/sec (range, 0.007-3.1 microns/sec). Microtubules--the well-established basis for organelle movement in cells--were present but were sparsely represented in electron micrographs of these cells. Video-enhanced microscopic techniques can now be applied to the study of organelle/particle movement in an intact epithelium.

Sugar-stimulated ion absorption is not different in seahare and vertebrate intestine.

We have reexamined the notion that sugars stimulate ion absorption differently in invertebrate and vertebrate intestine. In the seahare intestine, mucosal sugar presumably increases the rate of transcellular Na+ and Cl- absorption, whereas only transcellular Na+ absorption is increased in the vertebrate small intestine. Our data indicate that the seahare intestine responds to mucosal D-galactose like the vertebrate small intestine: namely, the apical membrane electrical potential difference depolarizes, the ratio of the mucosal to serosal membrane resistances decreases, and the short-circuit current (Isc) increases. Because mucosal substitution of tetramethylammonium for Na+ abolished the increased Isc, this stimulation resulted from an increase in rheogenic Na+ absorption. Unidirectional transepithelial Cl- fluxes indicate that mucosal D-galactose had no effect on the net Cl- flux under short-circuit conditions. Further, ion substitution experiments indicate that the apical membrane is K+ conductive rather than Cl- conductive as previously reported. These electrophysiological as well as parallel histological findings indicate that studies previously reported on the seahare intestine were in fact conducted on the esophagus.

Seasonal variations in the fine structure of the Necturus maculosus urinary bladder epithelium: low transporters and high transporters.

Although the urinary bladder of Necturus maculosus provides an important model system for studying the mechanisms of active Na absorption, little critical attention has been paid to the fine structure of its epithelium. Moreover, two distinct groups of urinary bladders, low and high Na transporters, have been described based on short-circuit current or transepithelial potential difference. In the present study, over an 11-month period, stable electrical parameters (short-circuit current, transepithelial potential difference, and resistance) were recorded from 63 chamber-mounted bladders. Analysis of these parameters revealed a highly significant difference between two groups (low transporters and high transporters) occurring at different times of the year. Consistent with these data, in urine collected from the bladders, the Na concentration in low transporters was significantly higher than that in high transporters. A subpopulation of these bladders was subsequently fixed and examined at the light and/or electron microscopic level. Low-transporting bladders were characterized unequivocally by a thin, stratified squamous epithelium only 6-15 micron thick. High-transporting bladders were composed predominantly of columnar-shaped granular cells up to 70 micron in height, with ciliated, mitochondria-rich, and basal cells present in small numbers. There is thus a correlation between transport activity, as measured by electrophysiological techniques and urine sodium analysis, and the structure of the tissue. Moreover, these parameters exhibit significant seasonal variation, the underlying mechanisms of which remain obscure.

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.

Inhibition of active sodium transport by cytochalasin B in rat jejunum in vitro.

The effects of cytochalasin B on electrophysiological properties and sodium transport in rat jejunum in vitro are described. Stripped paired rat jejunal segments were maintained in Ussing chambers with Leibovitz's (L-15) tissue culture medium bubbled with 100% oxygen. L-15 medium contains galactose as the only sugar, and an assortment of amino acids and cofactors to nourish the tissue. Electrophysiological parameters of short-circuit current (Isc) and transepithelial potential difference could be maintained for up to 4 h in control tissues. Upon application of cytochalasin B (20 micrograms/ml), on the mucosal side, Isc and potential difference fell within 1 h from 1.93 +/- 0.12 to 1.09 +/- 0.14 (mean +/- S.E.) muequiv./cm2 per h and from 5 to 2.5 mV. Tissue resistance remained unchanged at approx. 110 omega X cm2 for up to 4 h. 22Na net flux was 4.1 +/- 0.9 muequiv./cm2 per h during the last control period and fell to zero within 1 h after cytochalasin B treatment. Transmission electron micrographs revealed no gross morphological changes at this dose. Absorptive junctional morphology was apparently not altered by cytochalasin B treatment, a finding which was consistent with the stable transepithelial electrical resistance observed during exposure to this drug. Active sodium transport processes coupled to hexose, amino acid, and chloride movements are all possible in L-15 medium. However, following exposure to 20 micrograms/ml cytochalasin B, all net sodium transport is completely inhibited. The data are consistent with the hypothesis of a common regulator for active sodium transport processes which is modulated through structural changes in cytoskeletal organization.