Amiloride-blockable sodium currents in isolated taste receptor cells

Summary Isolated taste receptor cells from the frog tongue were investigated under whole-cell patch-clamp conditions. With the cytosolic potential head at –80 mV, more than 50% of the cells had a stationary inward Na current of 10 to 700 pA in Ringer's solution. This current was in some cells partially, in others completely, blockable by low concentrations of amiloride. With 110mm Na in the external and 10mm Na in the internal solution, the inhibition constant of amiloride was (at –80 mV) near 0.3 m. In some cells the amiloride-sensitive conductance was Na specific; in others it passed both Na and K. The Na/K selectivity (estimated from reversal potentials) varied between 1 and 100. The blockability bysmall concentrations of amiloride resembled that of channels found in some Na-absorbing epithelia, but the channels of taste cells showed a surprisingly large range of ionic specificities. Receptor cells, whichin situ express these channels in their apical membrane, may be competent to detect the taste quality salty. The same cells also express TTX-blockable voltage-gated Na channels.     

Ionic basis of methacholine-induced shrinkage of dissociated eccrine clear cells

Summary Apical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-m opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10–100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50–900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250mm, the maximal collective spike rate of a bud was in the range of 6–10 sec^–1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5–2 sec^–1. Buds of pure phasic response were also observed. Amiloride (30 m) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 m). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud. This is understandable since the stationary current of a bud arises from a multitude of taste cells, while each current spike is presumably generated by just one taste cell. We suggest that, in a Na-sensitive receptor cell, (i) the apical amiloride-blockable Na inward current serves as a generator current causing cell depolarization and firing of action potentials, and (ii) each current spike recorded from the taste pore arises mainly from a modulation of the apical Na inward current of this cell, because the action potential generated by the taste cell will transiently decrease or abolish the driving force for the apical Na inward current. The transients are indicators of receptor cell action potentials, which appear to be physiological responses of taste cellsin situ.     

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers

We determined the extent of Na⁺-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na⁺-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na⁺-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, -alanine, -aminoisobutyric acid (AIB), -methylaminoisobutyric acid (MeAIB), -amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2,7-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (I _sc) in voltage-clamped Caco-2 cell monolayers in Na⁺-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pH_i and inward I _sc. In conclusion, Caco-2 cells express a Na⁺-independent, H⁺-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.This study was supported by a Wellcome Trust Fellowship (to DTT). Charlotte Ward, Maureen Sinclair and Ken Elliott provided excellent technical assistance.     

Apical membrane Cl-butyrate exchange: Mechanism of short chain fatty acid stimulation of active chloride absorption in rat distal colon

The cellular model of short chain fatty acid stimulation of electroneutral Na-Cl absorption in large intestine proposes that SCFA, following its uptake across the apical membrane, recycles and is coupled to functional Na-H and Cl-short chain fatty acid exchanges. To establish the presence of a Cl-butyrate exchange (used as a model short chain fatty acid), studies of ³⁶Cl and ¹⁴C-butyrate uptake across apical membrane vesicles of rat distal colon were performed. An outward butyrate-gradient stimulated transient accumulation of ³⁶Cl uptake that was not inhibited by pH clamping with valinomycin (a K ionophore) and FCCP (a proton ionophore). Outward butyrate-gradient-stimulated ³⁶Cl uptake was inhibited by 4,4-diisothiocyanatostilbene2,2-disulfonic acid (DIDS) with a half-maximal inhibitory concentration (IC₅₀) of 68.4 m, and was saturated by both increasing extravesicular Cl concentration (K _m for Cl of 26.8 ±3.4 mm and a V _max of 12.4±0.6 nmol/mg protein·9 sec) and increasing intravesicular butyrate concentration (K _m for butyrate of 5.9 mm and a V _max for Cl of 5.9 nmol/mg protein · 9 sec). ³⁶Cl uptake was also stimulated by outward gradients of other short chain fatty acids (e.g., propionate, acetate and formate). In contrast, an outward Cl gradient failed to enhance ¹⁴C-butyrate uptake. Extravesicular Cl more than extravesicular butyrate enhanced ³⁶Cl efflux from apical membrane vesicles. These studies provide compelling evidence for the presence of an electroneutral, pH-activated, Cl-butyrate exchange which in concert with Na-H exchange is the mechanism by which butyrate stimulates electroneutral Na-Cl absorption.Abbreviations used AMV apical membrane vesicles - BLMV basolateral membrane vesicles - DIDS 4,4-diisothiocyanatostilbene 2,2-disulfonic acid - FCCP carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone - MES 1-[N-morpholino]ethanesulfonic acid - NMG N-memyl-d-glucamine - SCF Ashort chain fatty acid This study was supported in part by a Public Health Service research Grant (DK 14669) provided by the National Institute of Diabetes, Digestive and Kidney Diseases. Ms. Mary Guidone provided excellent secretarial assistance.     

Mechanism of Cl secretion in canine trachea: Changes in intracellular chloride activity with secretion

Summary Cl-sensitive microelectrodes were employed to investigate the mechanism of Cl secretion by canine tracheal epithelium. In control tissues with a mean calculated short-circuit current (I _sc) of 18.1 A/cm², the intracellular Cl activity (a _Cl ⁱ ) was 47.2mm. This value is 30.1mm (or 27.0 mV) above the electrochemical equilibrium for Cl across the apical membrane. Epinephrine, which stimulates Cl secretion, increased the calculatedI _sc to 160 A/cm² and decreaseda _Cl ⁱ to 32.2mm, a value only 11.2mm (or 10.9 mV) above equilibrium for the apical membrane. These results indicate a secretagogue induced decrease in the impedance to Cl exit from the cell via the apical membrane. From these and prior measurements we calculate that epinephrine-induced Cl efflux from the cell can occur by simple diffusion across the apical membrane. Further implications of these calculations are also discussed.     

The fine structure of the polian vesicles of holothurians

Summary The Polian vesicles are tubules of variable length consisting of four layers: an external peritoneal epithelium, a connective tissue layer, a muscular layer, and an inner epithelium. The two simple epithelia produce a polysaccharide in their Golgi complexes. Their cellular junctions consist of an extensive zonula adhaerens, and an apical septate desmosome. Their surfaces possess microvilli and cilia. A basement membrane is lacking. The muscular layer is composed of paramyosin fibers. The connective tissue layer contains abundant collagen fibers that have a period of 640 Å and are embedded in an acid mucopolysaccharide matrix. Amoebocytes containing waste products and an acid mucopolysaccharide enter the Polian vesicles from the coelom, pass through them, and empty into the lumen along with material secreted by the inner epithelium. While establishing close contact with cells of the muscular layer and inner epithelium the amoebocytes seem to transfer part of their contents to these cells.Polian vesicles appear to be a very primitive excretory organ.Research performed under C.N.R. contract.     

The coelomic elements of sea urchins (Strongylocentrotus)

Summary Coelomocytes of Strongylocentrotus purpuratus and Strongylocentrotus franciscanus include phagocytic leukocytes, flagellated vibratile cells, and two types of spherule-bearing cells; one of which contains echinochrome, a red naphthaquinone pigment. Vibratile cells are PAS positive and contain a strongly acidic mucopolysaccharide. Substances with similar staining and chemical properties appear in the coelomic fluid under certain stress conditions. It is supposed that these substances enter the fluid by diffusion from the vibratile cells. Tests indicate that both spherule-cell types, like the spherule cells of holothurians (sea cucumbers), may undergo a cycle during which they pass from a basophilic to an acidophilic state. Chemical properties of the spherule cells approximate those of vertebrate mast cells. The variability of staining and chemical reactions of spherule cells explains why published accounts of their properties have differed so widely. It is suggested that the properties of mucoid substances in invertebrate spherule-bearing cells may be as variable as is true of vertebrate mast cells.This investigation was supported by Public Health Service Research Grant No. 5358 (to E. A. Steinhaus) from the National Institute of Allergy and Infectious Diseases.     

Morphological factors influencing transepithelial permeability: A model for the resistance of theZonula Occludens

Summary Epithelial cells are joined at their apical surfaces byzonulae occludentes. Claude and Goodenough (1973) demonstrated a correlation between the structure of thezonula occludens as seen in freeze-fracture preparations and the passive electrical permeability of several simple epithelia. In epithelia with high transepithelial resistance, thezonula occludens consisted of many strands. In epithelia with low transepithelial resistance thezonula occludens was much reduced, sometimes consisting of only one strand.Evidence is reviewed here that indicates that in a number of simple epithelia the structure of thezonula occludens is largely responsible for the magnitude of transepithelial conductance. An equation is derived relating transepithelial junctional resistance to the number of junctional strands:R=R _min p ^–n whereR is the transepithelial resistance of thezonula occludens,R _min is the minimum resistance of the junction (as when there areno strands in the zonula occludens),p is the probability a given strand is open andn is the number of strands in the junction. Using published experimental values ofR andn for different epithelia, the calculated value ofp was found to be as high as 0.4, which suggests that the strands in thezonula occludens are remarkably labile.Other morphological parameters relevant to transepithelial permeability are also considered, such as the width and depth of the intercellular spaces, and the size of the epithelial cells themselves.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

Endocytosis in the ureteric duct epithelium of the hagfish (Myxine glutinosa L.)

Summary Solutions containing ferritin or thorotrast particles were microperfused through the ureteric duct of the hagfish. The markers were taken up by the epithelial cells by way of endocytosis and were transported in bulk in apical vesicles. Newly formed apical vesicles containing marker showed bristle coating on the cytoplasmic side of their limiting membrane. This coating appeared to be lost during the movement of vesicles deeper into the cytoplasm.The findings indicate that the epithelial cells in the ureteric duct have capablity for extensive bulk uptake of macromolecules from the luminal fluid. The mechanisms involved in absorption appear to be similar to those in proximal convoluted tubules of mammals.The apical dense tubules observed with some fixation techniques appear to represent collapsed endocytotic vesicles.The authors are indebted to Finn Walwig, Cand. real., Marine Biology Station, University of Oslo, Dröbak, Norway for kindly supplying the hagfishes used in this study. The technical assistance of Miss Signe Fjeldsenden and Miss Britt-Marie Pettersson is gratefully acknowledged.This work has been supported by grants from the Karolinska Institutet Medical School, Stockholm, Sweden (Therese och Johan Anderssons Minne).     

Functional expression of rat renal Na/Pi-cotransport (NaPi-2) in Sf9 cells by the baculovirus system

The recently cloned Na/P _i -cotransport system NaPi-2 is an apical membrane protein of rat proximal tubular cells and is involved in proximal phosphate reabsorption. To make the protein available for further functional/structural studies, this transport system has been expressed in Sf9 insect cells using a recombinant baculovirus. Sf9 cells infected with NaPi-2 (or 6His tagged NaPi-2) expressed functional Na/P _i -cotransport up to 20- to 50-fold over noninfected Sf9 cells. Transport of phosphate in infected cells was highly dependent on sodium, exhibited a K _m for P _i of 0.114 mm and an apparent K _m for Na of 63 mm (Hill coefficient of approximately 3) and was stimulated by high external pH. Infected cells expressed a polypeptide of 65 kDa representing a nonglycosylated form of the 85 kDa mature NaPi-2 transporter as present in proximal tubular brush-border membranes. By confocal microscopy expression of NaPi-2 protein was observed in the plasma membrane, yet submembranous accumulation of NaPi-2 protein could not be excluded. This demonstrates that the rat proximal tubular Na/P _i -cotransport system NaPi-2 can be successfully expressed in Sf9 cells with characteristics similar to that in isolated brush-border membranes. The 6His tagging will permit isolation of the NaPi-2 cotransporter in amounts sufficient for structural/functional studies.We would like to thank W. Scherle and M. Lötscher (Institute of Anatomy) for their generous help using the confocal microscope and Ch. Gasser for the art work. Financial support by the Swiss National Fonds [Grant No. 32-30785.91 (to H.M.) and 32-28664.90 (to J.B.)] and by Stiftung für wissenschaftliche Forschung an der Universitát Zürich is greatly acknowledged.     

A study of the biochemistry and cytochemical localization of β-glycerophosphatase activity in root tips of maize and pea

Summary A critical study has been made of the standard lead phosphate precipitation technique for the localization of-glycerophosphatase activity in young root tips. The effects of fixatives on enzyme activity and on the loss of activity during fixation and incubation have been determined. Cytochemical studies showed that the most prominent sites of-glycerophosphatase activity were at particulate sites in the cytoplasm and in the nucleus. Good correlation was obtained between frozen section and electron microscope studies although a number of problems were encountered. These particularly concerned the penetration of the staining medium, the loss of activity during incubation and the use of the acetic acid rinse. Recommendations are made for the reliable localization of the enzyme. The use of controls in this method were studied and their validity discussed.The study was supported in part by a National Science Foundation Grant GB 12371 to Dr. J.Cronshaw.     

The fine structure of the olfactory tract in the teleost Carassius carassius L.

Summary Electronmicrographic montages of the olfactory tract at two levels in each of two fish (Carassius carassius L.) were constructed and fibre diameters measured using a Zeiss TGZ 3 particle size analyzer. Medial and lateral tract divisions, rhinocele and dorsal tela were identified. Ciliated ependymal cells line the rhinocele. Meninges form the outer covering of both tract divisions and the tela roofing the central canal.The lateral tract consists of 10–14 fasciculi in which myelinated nerve fibres are prominent. These fibres range in diameter between 0.2 and 1.8 (mean 0.7 ) consistent with conduction velocities averaging 0.6 m/sec recorded in the carp lateral olfactory tract.The medial division of the olfactory tract contains two larger fasciculi within which are numerous fine unmyelinated nerve fibres (mean diameter 0.17 ) arranged in bundles partly enveloped by glial cell processes. Myelinated nerve fibres are unevenly distributed within both fasciculi and have mean diameters of 0.6 .An interesting observation is the consistent presence of synapses within the largest bundle of the medial tract at all levels.Supported by Grant 5 Ro5 TW00154-03 from the National Institutes of Health, United States Public Health Service.The authors are indebted to the Fisheries and Wildlife Department who generously provided the fish from Snob's Creek Fish Hatchery, and gratefully acknowledge the technical assistance of Mr. T. Armitage, Mr. J. Simmons and Miss D. Harrison.     

Patch-clamp study of isolated taste receptor cells of the frog

Summary Taste discs were dissected from the tongue ofR. ridibunda and their cells dissociated by a collagenase/low Ca/mechanical agitation protocol. The resulting cell suspension contained globular epithelial cells and, in smaller number, taste receptor cells. These were identified by staining properties and by their preserved apical process, the tip of which often remained attached to an epithelial (associated) cell. When the patch pipette contained 110mm KCl and the cells were superfused with NaCl Ringer's during whole-cell recording, the mean zero-current potential of 22 taste receptor cells was –65.2 mV and the slope resistance 150 to 750 M. Pulse-depolarization from a holding voltage of –80 mV activated a transient TTX-blockable inward Na current. Activation became noticeable at –25 mV and was half-maximal at –8 mV. Steady-state inactivation was half-maximal at –67 mV and complete at –50 mV. Peak Na current averaged –0.5 nA/cell. The Ca-ionophore A23187 shifted the activation and inactivation curve to more negative voltages. Similar shifts occurred when the pipette Ca was raised. External Ni (5mm) shifted the activation curve towards positive voltages by 10 mV. Pulse depolarization also activated outward K currents. Activation was slower than that of Na current and inactivation slower still. External TEA (7.5mm) and 4-aminopyridine (1mm) did not block, but 5mm Ba blocked the K currents. K-tail currents were seen on termination of depolarizing voltage pulses. A23187 shifted theI _K(V)-curve to more negative voltages. Action potentials were recorded when passing pulses of depolarizing outward current. Of the frog gustatory stimulants, 10mm Ca caused a reversible 5-to 10-mV depolarization in the current-clamp mode. Quinine (0.1mm, bitter) produced a reversible depolarization accompanied by a full block of Na current and, with slower time-course, a partial block of K currents. Cyclic AMP (5mm in the external solution or 0.5 m in the pipette) caused reversible depolarization (to –40 to –20 mV) due to partial blockage of K currents, but only if ATP was added to the pipette solution. Similar responses were elicited by stimulating the adenylate cyclase with forskolin. Blockage of cAMP-phosphodiesterase enhanced the response to cAMP. These results suggest that cAMP may be one of the cytosolic messengers in taste receptor cells. Replacement of ATP by AMP-PNP in the pipette abolished the depolarizing response to cAMP. Inclusion of ATP--S in the pipette caused slow depolarization to –40 to –20 mV, due to partial blockage of K currents. Subsequently, cAMP was without effect. The remaining K currents were blockable by Ba. These results suggest that cAMP initiates phosphorylation of one set of K channels to a nonconducting conformation.     

Lysozyme acts as a chemorepellent and secretagogue in Paramecium by activating a novel receptor-operated Ca++ conductance

Using combined intracellular recordings and behavioral bioassays, it was found that lysozyme has two different effects in Paramecium, depending upon the concentrations used. At low concentrations (0.5 nm to 1.0 m) it acts as an effective chemorepellent that causes reliable electrophysiological changes. Lysozyme-induced somatic depolarizations, isolated by blocking K⁺ channels with Cs-TEA, showed concentration dependencies that were well correlated with chemorepulsion. Ion dependency experiments showed that these were Ca⁺⁺ based depolarizations. Addition of either Na⁺ or Mg⁺⁺ improves chemorepulsion by providing additional depolarizations. Both the depolarizations and chemorepulsion were blocked by 10 m neomycin, suggesting that the depolarization is necessary for this chemosensory transduction event. At higher concentrations (100 m), lysozyme is a secretagogue. A transient inward current, recorded in Ca⁺⁺ alone solutions with Cs-TEA present, was seen in response to high lysozyme concentrations. The amplitude of this inward current was well correlated with exocytosis. Addition of neomycin (1.0 mm) eliminated both the inward current and exocytosis, suggesting a causal relationship. Neither amiloride or W-7, compounds previously suggested to affect the electrophysiological responses to secretagogues, had any significant effects. The mucopolysaccharide hydrolysis activity of lysozyme was not required for any of these responses. We propose that Paramecium have a high affinity receptor on the body plasma membrane that responds to either lysozyme or a related compound to cause an increase in a novel body Ca⁺⁺ conductance. This receptor-operated Ca⁺⁺ conductance causes membrane depolarization and chemorepulsion at low concentrations and triggers a sufficient Ca⁺⁺ influx at high concentrations to cause exocytosis.We thank Drs. C. Kung and R. Preston for sharing mutants and Drs. H. Machemer, A. Turkewitz and K. Clark for their comments on the first draft of this work. This was supported by NSF grants BNS8916228 and MCB9410756 to TMH and a grant from the American Diabetes Association to BHS.     

Overexpression of a thermostable lipase gene from Pseudomonas fluorescens in Escherichia coli

Summary A thermostable lipase gene from Pseudomonas fluorescens SIK W1 was overexpressed in Escherichia coli BL21 using expression vector pTTY2. The amount of lipase produced by E. coli BL21 with pTTY2 was more than 40% of the total cell proteins when induced with isopropyl--d-thiogalactopyranoside. The lipase was produced as inclusion bodies in the cytoplasm of E. coli. They were solubilized by 8 m urea and refolded into biologically active form. The refolded lipase showed high thermostability; the time required for 90% inactivation of the enzyme (D-value) was 4 h at 95°C and the increment of temperature to reduce heating times by 90% (z _d value) was 76°C.Offprint requests to: J. S. Rhee     

Geitler's “Plattenband” in the diatomSynedra cf.ulna in the light of TEM investigations

The ultrastructure of the diatomSynedra cf.ulna was examined paying special attention to the Plattenband (platelet band). This structure was first described byGeitler in 1948 on the basis of LM observations and denotes a linear array of dictyosomes along the apical axis of the cell. The present investigation confirmsGeitler's observations in all essential details and demonstrates that the dictyosomes are arranged along polarized nuclear extensions running towards the cell poles. Laterally the extensions are accompanied by a number of microtubules. In large cells the total length of the nucleus thus may reach 400 µm and more. Since only the central part of the nucleus is DNA-positive with DAPI and acridine orange, the nuclear nature of the backbone of the Plattenband cannot be recognized by LM techniques. TEM investigation of serial apical and transapical sections, however, prove unambiguously the identity with extended parts of the nucleus.Dedicated to Prof. DrLothar Geitler on the occasion of his 90th birthday.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Characterization of glycoconjugates in an embryonic human epithelial line and changes consequent to adaptation to a hyperosmotic medium

Summary Cell surface and cytoplasmic glycoconjugates were characterized in embryonic human explant cells (a transformed heteroploid line) cultured in iso-osmotic medium (0.137m NaCl) and in hyperosmotic medium (0.274m NaCl) for 10 days in order to study the changes induced in these compounds by hyperosmoticity. Cytochemical and ultracytochemical staining selective for glycoconjugates was carried out. The following results were obtained: (1) glycoproteins, glycosaminoglycans and glycolipids are present on the cell surface and in the cytoplasm of the explant cells; (2) lectin histochemistry combined with glycosidase digestion demonstrated the presence of the disaccharides fucose-N-acetylglucosamine and sialic acid--galactose as terminal sequences; (3) histophotometric evaluation of lectin labelling showed a noticeable decrease in histochemical reactivity of adapted cells; (4) plasma membrane cell coat decreased in adapted cells, which was emphasized by ultracytochemical reactions and a rearrangement of glycolipids in the cytoplasm.     

Cells with microvillous borders in the cerebral ganglion of Leptodora kindtii focke (Crustacea)

Summary Groups of large cells in the cerebral ganglion of Leptodora kindtii join in intricate patterns to enclose lacunar spaces. The cell surfaces bordering on these lacunae are covered by long, densely packed microvilli that all but fill the spaces. Near their brush borders the cells are joined by adhesion plates; for the rest they are separated from each other by glial septa. The possible significance of these structures is discussed.Dedicated to an inspiring teacher and loyal friend, Prof. F. Wassermann on the occasion of his 80th birthday, August 13, 1964.Supported by Grant No. NB-02145 from the United States Public Health Service. The expert assistance of Mrs. Cynthia Jones, Mrs. Sarah Wurzelmann, and Mr. Stanley Brown is gratefully acknowledged.