The immunohistochemical localization of superoxide dismutase activity in the avian epithelial growth plate

Summary Superoxide dismutase (SOD) is a scavenger enzyme which catalyses the dismutation (reduction—oxidation) of the superoxide anion (O₂ ^–), a toxic free radical generated during normal cellular respiration. Light microscopy employing immunohistochemistry was utilized for localizing SOD activity in the chick epiphyseal cartilage. Antibodies to mammalian liver CuZn—SOD were prepared and the avidin—biotin—peroxidase technique (ABC complex) was utilized to localize activity for this enzyme in the growth plate cartilage. The localization of enzyme activity varied in accordance with the characteristic zonation pattern of the growth plate (zone of proliferation, zone of maturation, zone of cell hypertrophy and zone of matrix calcification). In the upper regions of the epiphyseal cartilage (the zones of proliferation and maturation), where the vascularity is poor and the oxygen tension low, SOD activity was localized within the chondrocytes. No extracellular activity was observed. However, in the lower regions of the growth plate (the zones of cell hypertrophy and matrix calcification), where both the vascularity and the oxygen tensions are increased, SOD activity was intense in both the chondrocytes and the surrounding extracellular matrix. Thus, the distribution of SOD enzyme activity in this tissue seems to vary in accordance with the level of oxygen present. The significance of the extracellular SOD activity, seen in the lower aspects of the growth plate cartilage, may indicate the sensitivity of matrix components, especially collagen, to toxic free radicals such as the superoxide anion.     

Electron microprobe analysis of the different epithelial cells of toad urinary bladder

Summary The electrolyte composition of toad urinary bladder epithelial cells has been measured using the technique of electron microprobe analysis. Portions of hemi-bladders, which had been mounted in chambers and bathed with a variety of media, were layered with albumin solution on their mucosal surfaces and immediately shock-frozen in liquid propane at –180°C. From the frozen material 1–2m thick cryosections were cut and promptly freeze-dried for 12 hr at –80°C and 10^–6 Torr. Electron microprobe analysis using a scanning electron microscope, an energy dispersive X-ray detector, and a computer programme, to distinguish between characteristic and uncharacteristic radiations, allowed quantification of cellular ionic concentrations per kg tissue wet wt by comparison of the intensities of the emitted radiations from the cells and from the albumin layer. Granular, mitochondrial-rich, and basal cells, and the basal portions of goblet cells, showed a similar composition, being high in K (about 110mm/kg wet wt) and low in Na (about 13mm/kg wet wt). The apical portions of goblet cells were higher in Ca and S and lower in P and K, presumably reflecting the composition of the mucus within them. With Na-Ringer's as the mucosal medium, cells gained Na and lost K, when their serosal surfaces were exposed to ouabain, 10^–2 m. Replacement of mucosal Na by choline virtually prevented these ouabain-induced changes. Cellular ion contents were unchanged when Na in the serosal medium was replaced by choline. No differences in Na and K concentrations were detected between nuclei and cytoplasm. These results provide independent support for the hypothesis that the cellular Na transport pool in toad bladder epithelial cells derives exclusively from the mucosal medium and that no important recycling of Na occurs from the serosal medium to the cells.     

Dopaminergic inhibition of vasopressin-stimulated water flow in the toad bladder: Evidence for local formation of dopamine

Summary Dopamine administration increases renal excretion of water and Na. It remains uncertain whether these effects of dopamine are the result of a hemodynamic effect or the consequence of a direct cellular action. We investigated the effect of dopamine on water transport by the isolated toad bladderin vitro. Dopamine failed to alter baseline water flow but caused a significant inhibition of arginine vasopressin (AVP) or cyclic adenosine monophosphate (AMP) stimulated water flow. The effect of dopamine on stimulated water flow was not due to activation of adrenergic, adrenergic, or cholinergic receptors. The selective antagonists of dopamine, metoclopramide and apomorphine, prevented the effect of dopamine on AVP-stimulated water flow. These observations suggest the existence of a dopaminergic receptor in the toad bladder.l-Dopa also inhibited AVP-stimulated water flow. The effect ofl-Dopa could be prevented by metoclopramide, thus suggesting thatl-Dopa is converted to dopamine by an aromatic amino acid decarboxylase present in the toad bladder. To investigate this possibility we measured the effect of the decarboxylase inhibitor, carbidopa, on the¹⁴CO₂ production generated by decarboxylation of¹⁴Cl-Dopa in isolated toad bladder epithelial cells. Isolated toad bladder epithelial cells generated significant amounts of¹⁴CO₂ from¹⁴Cl-Dopa. This effect could be blocked by carbidopa, thus suggesting the existence of an aromatic amino acid decarboxylase system in the toad bladder. Carbidopa also prevented the inhibitory effect ofl-Dopa on AVP-stimulated water flow, suggesting thatl-Dopa needs to be converted to dopamine to inhibit water flow. These data suggest the existence of a dopaminergic receptor in the toad bladder. These data also suggest that dopamine can be formed locally in the toad bladder and can thus serve as a local modulator of water transport.     

Intracellular sodium activity and sodium transport inNecturus gallbladder epithelium

Summary Ion-sensitive glass microelectrodes, conventional microelectrodes and isotope flux measurements were employed inNecturus gallbladder epithelium to study intracellular sodium activity, [Na] _i , electrical parameters of epithelial cells, and properties of active sodium transport. Mean control values were: [Na] _i : 9.2 to 12.1mm; transepithelial potential difference, _ms : –1.5 mV (lumen negative); basolateral cell membrane potential, _es : –62 mV (cell interior negative); sodium conductance of the luminal cell membrane,g _Na: 12 mho cm^–2; active transcellular sodium flux, 88 to 101 pmol cm^–2 sec^–1 (estimated as instantaneous short-circuit current). Replacement of luminal Na by K led to a decrease of the intracellular sodium activity at a rate commensurate to the rate of active sodium extrusion across the basolateral cell membrane. Mucosal application of amphotericin B resulted in an increase of the luminal membrane conductance, a rise of intracellular sodium activity, and an increase of short-circuit current and unidirectional mucosa to serosa sodium flux. Conclusions: (i) sodium transport across the basolateral membrane can proceed against a steeper chemical potential difference at a higher rate than encountered under control conditions; (ii) the luminal Na-conductance is too low to accommodate sodium influx at the rate of active basolateral sodium extrusion, suggesting involvement of an electrically silent luminal transport mechanism; (iii) sodium entry across the luminal membrane is the rate-limiting step of transcellular sodium transport and active sodium extrusion across the basolateral cell membrane is not saturated under control conditions.     

Single-file diffusion through the Ca2+-activated K+ channel of human red cells

Summary We have examined the effect of internal and external pH on Na⁺ transport across toad bladder membrane vesicles. Vesicles prepared and assayed with a recently modified procedure (Garty & Asher, 1985) exhibit large, rheogenic, amiloridesensitive fluxes. Of the total²²Na uptake measured 0.5–2.0 min after introducing tracer, 80±4% (mean±se,n=9) is blocked by the diuretic with aK ₁ of 2×10^–8 m. Thus, this amiloridesensitive flux is mediated by the apical sodium-selective channels. Varying the internal (cytosolic) pH over the physiologic range 7.0–8.0 had no effect on sodium transport; this result suggests that variation of intracellular pHin vivo has no direct apical effect on modulating sodium uptake. On the other hand,²²Na was directly and monotonically dependent on external pH. External acidification also reduced the amiloride-sensitive efflux across the walls of the vesicles. This inhibition of²²Na efflux was noted at external Na⁺ concentrations of both 0.2 m and 53mm.These results are different from those reported with whole toad bladder. A number of possible bases for these differences are considered and discussed. We suggest that the natriferic response induced by mucosal acidification of whole toad urinary bladder appears to operate indirectly through one or more factors, presumably cytosolic, present in whole cells and absent from the vesicles.     

Ultrastructural study of the Ca2+-dependent ATPase activity in rat adenohypophyseal cells

Summary Ca-dependent ATPase activity in the rat anterior pituitary was demonstrated in 50-m tissue slices of aldehyde-fixed tissue with the medium of Takano et al. (Cell Tissue Res. 243:91. 1986). — The outer surface of the plasma membrane of the parenchymal as well as the folliculo-stellate cells was lined with lead precipitate. The reaction deposit was particularly well localized in intercellular spaces both between two parenchymal cells, and between a parenchymal and a folliculo-stellate cell. A fine reaction deposit was also seen in the endoplasmic reticulum and Golgi apparatus of some parenchymal cells. Elimination of Ca²⁺ from the tissue and the substrate medium drastically reduced the amount of reaction product. If ATP was omitted or replaced by sodium -glycerophosphate, no reaction product was seen. Changing the Ca²⁺ concentration or addition of Mg²⁺ to the standard medium caused a decrease in reaction intensity. Substitution of Mg²⁺ for Ca²⁺ resulted again in well-localized lead deposition which we attribute to the activity of another enzyme. We suggest that the activity we described in the membrane of glandular cells may correspond to the enzyme involved in the long-term regulation of intracellular Ca²⁺ level.     

The nature of the neutral Na+−Cl−-coupled entry at the apical membrane of rabbit gallbladder epithelium: II. Na+−Cl− symport is independent of K+

Summary In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl^– activity is about 25mm, about 4 times higher than intracellular Cl^– activity at the electrochemical equilibrium. It is essentially not affected by 10^–4 m acetazolamide and 10^–4 m 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS) even during prolonged exposures; it falls to the equilibrium value by removal of Na⁺ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl^– and Na⁺ activities are decreased by luminal treatment with 25mm SCN^–; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl^– activity are not significantly different upon Na⁺ or Cl^– entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K⁺ removal or 10^–5 m bumetanide do not affect intracellular Cl^– and Na⁺ activities or Cl^– influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na⁺–Cl^– symport on a single carrier which, at least under the conditions tested, does not cotransport K⁺.     

Concanavalin a receptors in normal and inflamed oesophageal epithelium

Summary We have examined normal and inflamed oesophageal biopsies for the distribution of -d-mannosyl and -d-glucosyl residues using the concanavalin A — horse radish peroxidase — Diamino-benzidine (DAB) technique at the light and electron microscope level. Receptors were found on the epithelial surface and in the neclear membrane and endoplasmic reticulum. A similar distribution was found with the intrusive lymphocytes and polymorphonuclear leucocytes in the inflamed state. Some of the increased intercellular debris from inflamed biopsies contained concanavalin A receptors.     

Ultrastructure of the tubule of the aglomerular teleost Nerophis ophidion

Summary The tubules in the aglomerular kidney of Nerophis ophidion are composed of cells showing different types of specializations of their plasma membrane. All cells possess a luminal brush border composed of microvilli, and show presence of vesicles with 100 Å thick unit membranes — some containing electron dense material —, tubular elements, multivesicular bodies, and plasma membrane invaginations in their apical cytoplasm. These features suggest an absorptive function of the cells. The apical portions of the cells are supplied with typical cilia.Some cells have abundant basilar plasma membrane invaginations usually lacking cytoplasmic organelles. Other cells appear to form interdigitating basilar cytoplasmic processes containing mitochondria; still other cells have smooth basilar cell membranes. These findings are discussed with reference to the known secretory function of the tubules and are compared with tubular fine structure in other species. It was concluded that urine formation by tubular secretion may occur in cells with different types of basilar cell membrane specializations.The occurrence of coated vesicles associated with invaginated basilar plasma membranes indicates transport of proteins (from peritubular blood vessels ?) at these sites.The tubule cells have abundant smooth surfaced endoplasmic reticulum and large and numerous active Golgi zones.Supported by grants from Fonden til Videnskabens Fremme and Therese och Johan Anderssons Minne. Part of this study was done at the Zoologica Stazione, Naples.The assistance of Miss Britt-Marie Petterson and Mr. Magnus Norman is gratefully acknowledged.     

Electrophysiology of cultured human lens epithelial cells

Summary The lens epithelial K⁺ conductance plays a key role in maintaining the lens ionic steady state. The specific channels responsible for this conductance are unknown. We used cultured lens epithelia and patch-clamp technology to address this problem. Human lens epithelial explants were cultured and after 1–4 passages were dissociated and used in this study. The cells from which we measured had a mean diameter of 31±1 m (sem,n=26). The resting voltage was –19±4 mV (sem,n=10) and the input resistance was 2.5±0.5 G (sem,n=17) at –60 mV. Two currents were prominent in whole-cell recordings. An outwardly rectifying current was seen in nearly every cell. The magnitude of this current was a function of K⁺ concentration and was blocked by 3mm tetraethylammonium. The instantaneous current-voltage relationship was linear in symmetric K⁺, implying that the outward rectificiation was due to gating. The current showed complex activation and inactivation kinetics. The second current seen was a transient inward current. This current had kinetics very similar to the traditional Na⁺ current of excitable cells and was blocked by 0.1 m tetrodotoxin. In single-channel recordings, a 150-pS K⁺ channel and a 35-pS nonselective cation channel were seen but neither account for the macroscopic currents measured.     

Characterization and delignification activity of a thermostable α-l-arabinofuranosidase from Bacillus stearothermophilus

Bacillus stearothermophilus L1 was isolated by enrichment culture using an alkaline extract of pulp as the carbon source at 65°C and pH 9.0. The bacterium produced extracellular xylanase and -l-arabinofuranosidase (EC 3.2.1.55). The xylanase activity was high when the cells were grown in the presence of d-xylose, whereas the arabinofuranosidase activity was high when grown in media containing l-arabinose. The arabinofuranosidase was purified 59-fold with an 80% yield by DEAE Sephacel and Sephadex G-100 chromatography. The purified enzyme had an apparent molecular mass of 110 000 kDa and consisted of two subunits of 52 500 kDa and 57 500 kDa. Using p-nitrophenyl--l-arabinofuranosidase as the substrate, the enzyme had a Michaelis constant (K _m) of 2.2 × 10^–4 m, maximum reaction velocity (V_max) of 11o mol min^–1 mg^–1, temperature optimum of 70°C and pH optimum of 7.0 (50% activity at pH 8.0). The enzyme was specific for the furanoside configuration. The purified enzyme partially delignified softwood Kraft pulp. Treatment of the pulp with 38 units ml^–1 of -l-arabinofuranosidase at 65°C for 2 h at pH 8.0 and 9.0 led to lignin releases of 2.3% and 2.1%, respectively. The enzyme acted synergistically with a thermophilic xylanase in the delignification process, yielding a 19.2% release of lignin. Correspondence to: Eugene Rosenberg     

Effect of hypertonicity and colchicine on intramembranous particle aggregation in toad urinary bladder

Summary Colchicine, an agent which disrupts microtubules, inhibits the vasopressin (VP)-induced increase in water permeability as well as intramembranous particle (IMP) aggregation in the luminal plasma membrane of granular cells of toad urinary bladder. However, the hydroosmotic response induced by serosal hypertonicity is not affected by colchicine. The present investigation was initiated to establish whether serosal hypertonicity is associated with IMP aggregation and whether the aggregation, if present, is altered by colchicine. The experimental half of paired hemibladders from the toad, Bufo marinus, treated with 0.1 mM colchicine for 4 h prior to exposure to serosal mannitol (240 mM) demonstrated no significant difference in osmotic water How (Jv) (1.03 × 0.18 vs. 1.13 ± 0.22l · min^–1 · cm^–2; p>0.20) when compared with control hemibladders. Similarly, comparison of control and colchicine-treated bladders revealed no difference in the number of IMP aggregation sites per area of membrane (17.8 ± 2.0 vs. 24.7 ± 3.5/100^m; p>0.10), the relative area of membrane occupied by these sites (0.30 ± 0.06 vs. 0.39 ± 0.07%; p>0.10) or the mean size of the aggregates (17.0 ± 1.4 vs. 15.8 ± 1.0 × 10³ m²; p > 0.20). These results indicate that in toad bladder the increase in J_v induced by serosal hypertonicity is associated with IMP aggregation. Secondly, an intact microtubule system is not required to induce the hydroosmotic or the aggregation responses. If, as has been proposed, the cellular actions of VP and serosal hypertonicity share a common pathway to bring about an increase in osmotic water permeability and cause IMP aggregation in the luminal membrane of the granular cell, the present results suggest that the pathway begins at a step subsequent not only to the generation of cAMP, but also beyond the involvement of the microtubule system.This work was supported in part by U.S. Public Health Service Grant AM 13845. Dr. Dratwa was supported through a U.S. Public Health Service International Research Fellowship F05TW2447. The authors gratefully acknowledge the technical assistance of Mrs. Helen Parks, Mr. Isaiah Taylor, Mrs. Betty Waller, and Mrs. Jessie Calder     

Membrane structural and functional responses to vasopressin in toad bladder

Summary Freeze-fracture electronmicroscopy demonstrates that vasopressin stimulation of isolated toad bladder results in a striking morphologic alteration of epithelial membrane structure. This alteration is characterized by the aggregation of intramembranous particles in orderly linear arrays at multiple sites in the luminal membranes of granular cells specifically. The size of these aggregates varies considerably, in terms of area, over a range from 0.5 to 70×10^–3 m². The median aggregate size is about 10.5×10^–3 m². Since the extent of vasopressin-associated particle aggregation, in terms of frequency of sites per area of membrane or cumulative area of membrane occupied by them, closely correlates with induced changes in transport function, as measured by osmotic water flow, the aggregates themselves appear to be of physiologic significance in the mechanism of action of vasopressin. This hypothesis is supported by the observations that sites of aggregation occur (a) in response to serosal exposure to hormone specifically, (b) independently of an osmotic gradient, and (c) following stimulation with cyclic adenosine monophosphate.     

Binding of3H-phenamil,an irreversible amiloride analog,to toad urinary bladder: effects of aldosterone and vasopressin

Summary Phenamil, an analog of amiloride, has previously been shown to bind specifically to sodium channels in toad bladder (J.L. Garvin et al.,J. Membrane Biol. 87:45–54, 1985). In this paper,³H-phenamil was used to measure sodium channel density in both isolated epithelial cells and intact bladders. From the specific binding to intact bladders, a channel density of 455±102 channels/m² was calculated. No correlation between specific binding and the magnitude of irreversible inhibition of shortcircuit current was found. Pretreatment of intact bladders with 1 mg/ml trypsin reduced specific binding to isolated cells by 82±5%. In isolated cells, neither aldosterone nor vasopressin had any significant effect on specific phenamil binding. It is inferred that phenamil binds to both open and closed channels which may be either in the mucosal membrane or in the submembrane space. Finally, and rather surprisingly, we found that³H-phenamil binds irreversibly to the basolateral membrane at concentrations as low as 4×10^–7 m. Therefore, care must be used in interpreting binding studies with amiloride or its analog at such concentrations.     

The subcellular distribution of transferrin in rat choroid plexus studied with immunogold labelling of ultracryosections

Summary Choroid plexus epithelium from third ventricle choroid plexus of 2–3-week-old rats was examined for transferrin-like immunoreactivity. In 5 µm paraffin sections most epithelial cells exhibited a pronounced immunoperoxidase staining for transferrin. The ultrastructure of the epithelium in question was examined by conventional electron microscopy. Immunolabelling of ultracryosections with IgG-gold, protein-A gold or protein-A gold—antiprotein-A protein-A gold showed an intense labelling of the basal extracellular space. The lateral intercellular space and the luminal surface showed a more variable labelling; no labelling of the tight junction zone was seen. Intracellularly a distinct labelling of the uptake and disposal pathway (the endosomal—lysosomal system) was observed, but also the synthetic machinery (rough endoplasmic reticulum, stacked Golgi membranes) showed a characteristic labelling. Thus it seems likely that both uptake and synthesis of transferrin occur in choroid plexus epithelial cells.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nm, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 m) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (K _d = 7.2 m; V = 1005.5 U mg^–1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (K _d = 2.2 m; V = 1036.7 U mg^–1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.     

Mechanism of action ofVibrio cholerae enterotoxin

Summary The characteristics of the cholera toxin-stimulated adenylate cyclase of toad (Bufus marinus) and rat erythrocyte plasma membranes have been examined, with special emphasis on the response to purine nucleotides, fluoride, magnesium and catecholamine hormones. Toad erythrocytes briefly exposed to low concentrations of cholera toxin (40,000 to 60,000 molecules per cell) and incubated 2 to 4 hr at 30°C exhibit dramatic alterations in the kinetic and regulatory properties of adenylate cyclase. The approximateK _m for ATP, Mg⁺⁺ increases from about 1.8 to 3.4mm in the toxinstimulated enzyme. The stimulation by cholera toxin increases with increasing ATP, Mg⁺⁺ concentrations, from 20% at low levels (0.2mm) to 500% at high concentrations (greater than 3mm). Addition of GTP, Mg⁺⁺ (0.2mm) restores normal kinetic properties to the toxin-modified enzyme, such that stimulation is most simply explained by an elevation ofV _max. GTP enhances the toxin-treated enzyme activity two-to fourfold at low ATP concentrations, but this effect disappears at high levels of the substrate. At 0.6mm ATP and 5mm MgCl₂ the apparentK _a for GTP, Mg⁺⁺ is 5 to 10m. The control (unstimulated) enzyme demonstrates a very small response to the guanyl nucleotide. 5-ITP also stimulates the toxin-treated enzyme but cGMP, guanine, and the pyrimidine nucleotides have no effect. Cholera toxin also alters the activation of adenylate cyclase by free Mg⁺⁺, decreasing the apparentK _a from about 25 to 5mm. (–)-Epinephrine sensitizes the toad erythrocyte adenylate cyclase to GTP and also decreases the apparentK _a for free metal. Sodium fluoride, which cause a 70- to 100-fold activation of enzyme activity, has little effect on sensitivity to GTP, and does not change the apparentK _a for Mg⁺⁺; moreover, it prevents modulation of these parameters by cholera toxin. Conversely, cholera toxin severely inhibits NaF activation, and in the presence of fluoride ion the usual three- to fivefold stimulation by toxin becomes a 30 to 60% inhibition of activity. The toxin-stimulated enzyme can be further activated by catecholamines; in the presence of GTP the (–)-epinephrine stimulation is enhanced by two- to threefold. The increased catecholamine stimulation of toad erythrocyte adenylate cyclase induced by cholera toxin is explained primarily by an increase in the maximal extent of activation by the hormones. Rat erythrocyte adenylate cyclase is also modified by cholera toxin. In the mammalian system the apparent affinity for the hormone appears to be increased. Cholera toxin thus induces profound and nearly permanent changes in adenylate cyclase by a unique process which mimics the stimulation by hormones in important ways, and which also accentuates the normal hormonal response. The relevance of these findings to the mechanism of action of cholera toxin is considered.Part of this work was reported at the 1974 meeting of the Federation of American Societies for Experimental Biology (Bennett & Cuatrecasas, 1974).     

Intracellular K+ activities and cell membrane potentials in a K+-transporting epithelium,the midgut of tobacco hornoworm (Manduca sexta)

Summary Transbasal electrical potential (V _b) and intraepithelial potassium chemical activity ((K⁺) _i ) were measured in isolated midgut epithelium of tobacco hornworm (Manduca sexta) using double-barrelled glass microelectrodes. Values ofV _b ranging from +8 to –48 mV (relative to blood side) were recorded. For all sites, (K⁺) _i is within a few millivolts of electrochemical equilibrium with the blood side bathing solution. Sites more negative than –20 mV show relatively high sensitivity ofV _b to changes in blood side K⁺ concentration: 43% of these sites can be marked successfully with iontophoresed Lucifer yellow CH dye and shown to represent epithelial cells of all three types present in the midgut. In about half of successful marks, dye-coupling of several adjacent cells is seen. Low potential sites — those withV _b less negative than –20 mV —typically do not show high sensitivity ofVb to changes of external K⁺, but rather (K⁺) _i rapidly approaches the K⁺ activity of blood side bathing solution. These sites can seldom be marked with Lucifer yellow (4% success). The mean (K⁺) _i of the high potential sites is 95±29 (sd)mm under standard conditions, a value which is in accord with published values for the whole tissue.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.