ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-C1.16E

The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as shortcircuit current (I_sc). Luminal ATP stimulated a transient increase in the release of mucins and of I _sc corresponding to a secretory Cl^– current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED₅₀ of approximately 40 m. Maximal peak I _sc after stimulation with ATP was approximately 35 A/cm² with an apparent ED₅₀ of about 0.4 mm. ATP-dependent I _sc was at least in part due to Cl^– secretion since removal of Cl^– from the medium reduced the peak I _sc by 40% and the I _sc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP>ADP>AMPadenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P₂-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl^– channels. P₂-receptor-stimulated mucin and I _sc release was strongly inhibited by a 30 min preincubation with the classical K⁺ channel blockers quinine (1 mm), quinidine (1 mm), and Ba²⁺ (3 mm). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K⁺ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P₂ receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.Supported by grants from the National Institutes of Health (DK 39658) to U.H., the Philippe Foundation to D.M., the French Cystic Fibrosis Foundation (AFLM) and L'Association Pour La Recherche Sur Le Cancer to C.L. The authors thank Mr. J. Polack for his efforts and skill with electron microscopy and Dr. George Dubyak for helpful discussions. We also acknowledge the Cystic Fibrosis Center Core grant (DK-27651) for its support of electron and light microscopy.     

ATP-dependent chloride influx into internally dialyzed squid giant axons

Summary Measurements were made of³⁶Cl influx into squid giant axons whose internal solutes were controlled by means of internal dialysis. When the intracellular chloride concentration was 50mm and the internal concentration of adenosine 5-triphosphate (ATP) was 4mm, the average chloride influx was 11.6 pmoles/cm²×sec. When the axons were dialyzed with an ATP-free solution, the average influx fell to 5.1 pmoles/cm²×sec. The effect was fully reversible upon the return of ATP to the dialysis fluid. Chloride-36 influx in the presence and absence of ATP was found to be inversely related to the internal chloride concentration.     

Mode of action of furosemide on the chloride-dependent short-circuit current across the ciliary body epithelium of toad eyes

Summary The effects of furosemide on the chloride-dependent short-circuit current across the toad ciliary epithelium were examined. Under control conditions, the short-circuit current obeyed Michaelis-Menten kinetics against medium chloride concentration, the Michaelis constant (K _m ) for chloride being 90mm and the maximal short-circuit current (V _max) 128 A/cm². Furosemide added to the aqueous side of the epithelium rapidly reduced the short-circuit current; the effect was reversible. The effect of furosemide addition to the stromal side was much smaller and slower than that from the aqueous side. The dose-dependent range of furosemide action was from 0.1 m to 1mm with 50% inhibition occurring at about 3 m. Line-weaver-Burk plot of the short-circuit current against the chloride concentration showed that furosemide decreased the value ofV _max and increased theK _m ; the inhibition being of mixed type. A Hill plot of the dose-response curve yielding a slope of unity suggested one furosemide molecule combines with one chloride transport site. Probenecid, a competitive inhibitor of organic acid transport, reduced the effects of furosemide significantly when added simultaneously. The involvement of organic acid transport system in the mechanism of furosemide action on chloride transport was suggested.Department of Ophthalmology.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

Quantitative analysis of depolarization-induced ATP release from mouse brain synaptosomes: External calcium dependent and independent processes

Summary We and others have shown previously that ATP is secreted from mouse brain synaptosomes following depolarization of the membrane by high [K⁺] ₀ and the time course can be monitored accurately by measuring the light emitted from luciferin-luciferase included in the reaction medium. In the present work we have evaluated the relative importance of [Ca²⁺] ₀ and membrane potential on the ATP secretion process by modelling the time course of ATP release under different conditions. After correction of the records for destruction of released ATP by synaptosomal ecto-ATPase activity, we found that ATP secretion occurs by an apparent first order process. We also established that, in addition to the classical [Ca²⁺] ₀ -dependent mode, ATP secretion also occurred in the absence of extracellular calcium ([Ca²⁺] ₀ < 1 m). Upon lowering the extracellular Ca²⁺ concentration, both the rate and the extent of ATP secretion decreased. To assess the contribution of membrane potential to the release rate we measured ATP secretion at membrane potentials determined by extracellular [K⁺] ₀ (or [Rb⁺] ₀ ) as defined by the distribution of the carbocyanine dye, diSC₃(5). Rate constants computed from measured secretion curves revealed that this parameter was essentially independent of membrane potential in the absence of [Ca²⁺] ₀ . Noise analysis of the light signal showed that the variance increased upon stimulation by high [K⁺] ₀ , suggesting that both modes of secretion are quantal. Thus, we conclude that the rate of ATP secretion from nerve terminals depends upon Ca²⁺ entry but not on membrane potential, per se     

Modulation of adrenal cell functions by cadmium salts: 3. Sites affected by CdCl2 during stimulated steroid synthesis

In previous studies cadmium chloride (CdCl₂) nonlethally inhibited Y-1 mouse adrenal tumor cell 20-dihydroxyprogesterone (20DHP) secretion, affecting unstimulated and stimulated steroidogenic pathway sites differently. In addition, dibutyryl cAMP-stimulated 20DHP secretion was unaffected by CdCl₂, while the site of the unstimulated effect was indirectly shown to involve steps between endogenous cholesterol utilization and 20-hydroxycholesterol association with mitochondrial cytochrome P450 side-chain cleavage enzyme. In the present study we determined CdCl₂ effects on plasma membrane sites preceding pre-dbcAMP-stimulation of 20DHP secretion. Y-1 cells were incubated 0.5 h in medium with or without cadmium (using the concentration that inhibited adrenocorticotropin- (ACTH)-stimulated steroid secretion by 50%) together with exogenously added maximally stimulating concentrations of ACTH, cholera toxin, forskolin, or adenosine triphosphate Cholera toxin, forskolin and ATP bypass specific plasma membrane sites involved in the synthesis of intracellular cAMP and activate the steroid hormone biosynthetic pathway. Cadmium effects on ACTH-stimulated endogenous cAMP secretion were also examined. CdCl₂ significantly reduced Y-1 cell 20DHP secretion following exposure to ACTH, cholera toxin, forskolin, and ATP; it also significantly decreased endogenous cAMP secretion into culture medium. These data may be interpreted to suggest that CdCl₂ altered Y-1 cell regulation of adenyl cyclase activity, which reduced cAMP-activated cholesterol uptake by mitochondria as a consequence.Abbreviations ACTH adrenocorticotropin - ATP adenosine triphosphate - ANOVA analysis of variance - CdCl₂ or Cd²⁺ cadmium chloride - cAMP cyclic 3,5-adenosine monophosphate - CTX cholera toxin - dbcAMP dibutyryl cAMP,N,O-dibutyryl-3,5-adenosine monophosphate - EGTA ethylene glycol bis tetraacetic acid - FMEM serum-free Eagle's Minimum Essential Medium with all other supplements - FSK forskolin - Hepes N-2-hydroxyethylpiperazine-N-1,2-ethanesulfonic acid - IC_50' concentration inhibiting stimulated steroid secretion by 50% - IU international unit - MEM Eagle's Minimum Essential Medium - P450scc cytochrome P450 side-chain cleavage enzyme - PREG pregnenolone - PROG progesterone - SEM standard error of the mean - SMEM serum-containing Eagle's Minimum Essential Medium with supplements - 20DHP 20--hydroxy-4-pregnen-3-one     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

Der Einflu\ des Schirmpigmentgehalts auf die Helligkeits- und Kontrastwahrnehmung bei Drosophila-Augenmutanten

Summary The function of the facet-separating pigments in the compound eyes of the fruitfly Drosophila melanogaster with hypernormal (se), normal (+), subnormal (w^a), and missing (w) pigmentation was studied by investigation of: (1) the in-flight optomotor responses to movement of striped patterns with a mean brightness of 300 cd/m², and (2) the retinal action potentials evoked by flashes in a program of .0003 cd/m² average brightness. The pigment deficient mutants (w ^a, w) are less sensitive to the pattern contrast in the bright adapted state, and more sensitive to the flash intensity in the dark adapted state than either the wild-type (+) or the overpigmented mutant(se). These differences are complementary and can be explained by the increased translucency of the pigment cells. Thus the photoreceptors in the equally illuminated eyes of the normal and mutant animals +, se, w ^a, and w are expected to receive light in a ratio of about 11719. However the sensitivity of the receptors as well as the half-peak widths and the density of their visual fields are apparently independent of the eye pigmentation and seem to be equal at common levels of adaptation. The effects of omnidirectional excess light reaching the receptors of the pigment deficient mutants can be simulated in less translucent eyes: when certain amounts of background illumination were combined with the optomotor stimulus in the visual fields of the wild-type receptors it was possible to elicit the predicted mutant behavior.

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Der Kurvenverlauf stimmt qualitativ mit der Lichtempfindlichkeitsverteilung überein, die Washizu, Burkhardt und Streck (1964) aus den Belichtungspotentialen einzelner Sehzellen in den Rasterelementen vergleichbarer Calliphora-Rassen ermittelt haben.Die vorliegende Arbeit ist durch die anregenden Diskussionen mit Prof. W. Reichardt sehr gefördert worden. Dipl.-Physiker H. Wenking verdanken wir die Entwicklung der Meßverstärker, Werkstattleiter H. Braun und seinen Mitarbeitern die sorgfältige Ausführung der feinmechanischen Arbeiten, Herrn E. Freiberg die Anfertigung der Abbildungen und Frl. I. Geiss die Bearbeitung des Textes.     

Effects of catecholamines on electrolyte transport in cortical collecting tubule

Summary We examined the direct effects of isoproterenol (ISO) andl-norepinephrine (NE) on electrolyte transport in isolated rabbit cortical collecting tubules (CCT) perfusedin vitro. The addition of either ISO (10^–6 m) or NE (10^–6 m) to the bath decreased transepithelial potential difference (PD), on average by 51 and 25%, respectively. These effects of ISO and NE were abolished by prior addition of the -adrenergic blocker,l-propranolol. ISO (10^–5 m) had no effect from lumen. Also, osmotic water permeability was not influenced by ISO. Ouabain and ISO had additive effects on PD. Elimination of chloride from both perfusate and bath, or addition of acetazolamide, abolished the effect of ISO on PD. Although isotopic sodium flux from lumen to bath was not influenced by ISO, chemical net chloride absorption increased from 1.1±0.4 to 2.7±0.6 peq·cm^–1·sec^–1 (n=8,p<0.005). In conclusion, both ISO and NE are capable of decreasing PD in rabbit CCT perfusedin vitro. This effect is mediated by -adrenergic receptors and is accompanied by the increase in net chloride absorption. Although the mechanism responsible for this decrease in PD with ISO is unclear, active chloride absorption, active hydrogen secretion, or membrane chloride permeability changes may account for the effects of ISO.     

Adenosine discriminates between the caffeine and adenine nucleotide sites on the sheep cardiac sarcoplasmic reticulum calcium-release channel

Calcium-release channels of sheep cardiac sarcoplasmic reticulum were incorporated into phosphatidylethanolamine bilayers and single channel currents were recorded under voltage-clamp conditions. The effect of adenosine on single channel conductance and gating was investigated, as were the interactions between adenosine and caffeine and adenosine and ,-methylene ATP.Addition of adenosine (0.5–5 mm) to the cytosolic but not the luminal side of the membrane increased the open probability of single calcium-activated calcium-release channels by increasing the frequency and duration of open events, yielding an EC₅₀ of 0.75 mm at 10 m activating Ca²⁺.Addition of 1 mm caffeine potentiated the effects of adenosine at 10 or 100 m-activating cytosolic calcium, but had no effect on the inability of adenosine to activate the channel at 80 pmcalcium, suggesting discrete sites of action on the calcium-release channel for adenosine and caffeine. In contrast, addition of 100 m ,-methylene-ATP decreased single channel open probability in the presence of adenosine, suggesting that these compounds act on the same site on the channel.Activation of single channel opening by adenosine, or by adenosine together with caffeine, had no effect on single channel conductance or the Ca²⁺/Tris⁺ permeability ratio. Channels activated by adenosine were characteristically modified by ryanodine and blocked by m ruthenium red or mm magnesium.These results show that adenosine activates the sheep cardiac sarcoplasmic reticulum Ca²⁺-release channel by increasing the frequency and duration of open events in a Ca²⁺-dependent manner. The receptor site on the channel for adenosine is distinct from that for caffeine but probably the same as that for adenine nucleotides.This work was supported by the British Heart Foundation.     

Isolation of an abnormally phosphorylated erythrocyte membrane band 3 glycoprotein from patients with myotonic muscular dystrophy

Summary A fraction of erythrocyte Band 3 (M _r , 93,000) glycoprotein that demonstrates decreased autophosphorylation in membranes from myotonic muscular dystrophy patients is demonstrated. Sequential affinity chromatography of Triton X-100 solubilized erythrocyte membrane proteins separated three specifically retained glycoprotein fractions on a Ricin Communis I-Sepharose 4B column. One fraction contains a portion of the major sialoglycoprotein (apparentM _r , 78,000) and is specifically eluted from the column by 10mm NaCl and 100mm d-galactose (10/100). The two other glycoprotein fractions are eluted by 100mm NaCl, 10mm d-galactose (100/10) and 100mm NaCl, 100mm d-galactose (100/100). The composition of both fractions contains greater than 95% Band 3 (apparentM _r , 93,000) glycoprotein.The quantities of glycoprotein in each fraction obtained from erythrocytes of myotonic dystrophy patients did not differ from the quantities obtained from control erythrocytes. Following endogenous protein kinase incubations of ghosts with [-³²P]ATP, the specific [³²P] phosphorylation of the 10/100 and 100/10 fractions are identical. The 100/100 fraction, which makes up approximately 3% of the total erythrocyte membrane protein, demonstrates a different pattern for myotonic dystrophy patients; specific phosphorylation was reduced by 50% relative to activity in control experiments. These findings are consistent with previous experiments that demonstrated decreased autophosphorylation of the glycoprotein portion of Band 3 (Roses & Appel, 1975,J. Membrane Biol. 20: 51) and are consistent with the autosomal dominant mode of inheritance in this disease.     

Free Ia Eα chain expression in the E α + :E β − : recombinant strain A.TFR5

Molecular and biochemical techniques have been used to explore the reasons behind low E chain expression in the E ⁺ E ^– I-region recombinant strain, A.TFR5. A.TFR5 (A ^f E ^k, ap5), a recombinant between A.CA (A ^f E ^f) and A.TL (A ^k E ^k), carries the E ^k subregion. Previous results have shown that it expresses the E chain, but at reduced levels relative to E ⁺ E ⁺ strains. No E chains were detected, which is consistent with the A.TFR5E gene being derived from the A.CA parent, which carries the null E ^f allele. In this paper, the defect in E-chain expression is explored. Restriction fragment length polymorphism analysis has localized the recombination event in A.TFR5 approximately 30 kb upstream of E, in the region of the large intervening sequence of E. Northern blot analysis of total RNA from A.TFR5 shows normal amounts of the E message, but no E message. Two-dimensional gel analysis of 15 min pulse-labeled A.TFR5, A.CA, and A.TL E immunoprecipitates shows decreased levels of the intracellular E chain in A.TFR5 relative to A.TL. However, analysis of total cell extracts shows normal levels of this protein. A glycoprotein fraction isolated from total cell extracts of 5 h labeled cells contains normal amounts of intracellular E, but decreased amounts of the mature cell-surface protein. These data suggest that in the absence of E, the E chain (1) takes on an altered conformation that is not as efficiently recognized by alloantibodies, and (2) is found in normal levels as the partially glycosylated intracellular precursor, but is not processed and/or transported efficiently to the cell surface.     

Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium,nucleotides and kinases

Agonists that elevate calcium in T₈₄ cells stimulate chloride secretion by activating K_BIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (I _SC) techniques. Open probability (P ₀) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 m–5 mm), ATP (0.1 mm) + protein kinase A (PKA; 180 nm), or isobutylmethylxanthine (IBMX; 1 mm). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nm). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using -toxin. Finally, protein kinase C (PKC) inhibited single K_BIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.This work was supported by the Canadian Cystic Fibrosis Foundation and the Medical Research Council of Canada. J.W.H. is a Chercheur-Boursier of the Fonds de la recherche en santé du Québec.     

Ionic dependence of bumetanide binding to the rabbit parotid Na/K/Cl cotransporter

Summary The Na/K/Cl-dependent component of the binding of the loop diuretic bumetanide to basolateral membrane vesicles from the rabbit parotid is studied. A Scatchard analysis indicates that this binding is due to a single high-affinity site withK _D =3.2±0.3 m (n=9) at 100mm sodium, 100mm potassium and 5mm chloride. When KCl-dependent²²Na transport and tracer [³H]-bumetanide binding are monitored simultaneously as a function of (unlabeled) bumetanide concentration it is found that theK _0.5 for bumetanide inhibition of both processes are identical indicating that the high-affinity bumetanide binding site studied here is identical with a bumetanide-inhibitory site on the Na/K/Cl cotransport system previously identified in this preparation (R.J. Turner, J.N. George and B.J. Baum,J. Membrane Biol. 94:143–152, 1986). High-affinity bumetanide binding exhibits a hyperbolic dependence on both [Na] and [K] consistent with Na/bumetanide and K/bumetanide binding stoichiometries of 11 andK _0.5 values of approximately 33mm for sodium and 23mm for potassium. In contrast, the dependence on [Cl] is biphasic, with bumetanide binding increasing from 0 to 5mm chloride and decreasing toward baseline levels thereafter. Scatchard analysis of this latter inhibitory effect of chloride indicates a competitive interaction with bumetanide in agreement with earlier indications that bumetanide inhibits Na/K/Cl cotransport at a chloride site. However, studies of the effects of various anions on bumetanide binding and²²Na transport show a poor correlation between the specificities of these two processes, suggesting that the inhibitory chloride site is not a chloride transport site.     

Transepithelial transport in cell culture:d-Glucose transport by a pig kidney cell line (LLC-PK1)

Summary The pig kidney cell line LLC-PK₁ cultured on a collagen coated membrane filter formed a continuous sheet of oriented asymmetrical epithelial cells joined by occluding junctions. A transepithelial electrical potential (PD) and short-circuit current (SCC) were dependent on the presence of Na and sugar in the apical bathing solution. In the presence of 5.5mm d-glucose, a PD of 2.8 mV, apical surface negative, a SCC of 13 A cm^–2 and transepithelial resistance of 211 ohm·cm² were recorded. The SCC was promptly reduced by the addition of phlorizin to the apical bath but unaffected when placed in the basolateral bath. The effect on SCC of various sugars was compared by the concentrations required for half-maximal SCC: 0.13mm -methyl-d-glucoside, 0.28mm d-glucose, 0.65mm -methyl-d-glucoside, 0.77mm 6-deoxy-d-glucose, 4.8mm d-galactose, and 29mm 3-O-methyl-glucose. When [Na] was reduced, the concentration ofd-glucose required for half-maximal SCC increased. Isotopically labeled³H and¹⁴Cd-glucose were used to simultaneously determine bidirectional fluxes; a resultant net apical-to-basolateral transport was present and abolished by phlorizin. The transported isotope cochromatographed with labeledd-glucose, indicating negligible metabolism of transported glucose. The pig kidney cell line, LLC-PK₁, provides a cell culture model for the investigation of mechanisms of transepithelial glucose transport.     

Purification and properties of glutamine synthetase from Nocardia asteroides

Glutamine synthetase (GS, EC 6.3.1.2) from Nocardia asteroides was purified to homogeneity by ammonium sulfate precipitation, Sephadex G-150, and DEAE-Sepharose chromatography. The native molecular weight of the purified enzyme was determined to be 720 kDa. SDS-PAGE analysis of the purified preparation revealed a single band corresponding to 59 kDa, indicating the possible presence of 12 identical subunits. The divalent cations Mn^2- and Mg²⁺ were found to be essential for optimal transferase and biosynthetic activity, respectively. The optimal pH and temperature for both activities of the enzyme were found to be 7.2 and 50°C. Amino acids such as l-alanine, glycine, and aspartate inhibited the GS activity. The K _m values for the substrates of the biosynthetic reaction ATP, glutamate, and ammonium chloride were found to be 400 m, 7.7mm, and 200 m, respectively. Addition of ammonium chloride to the nitrogen-limited culture resulted in a decrease of GS transferase and biosynthetic activities. Phosphodiesterase treatment of the extract from ammonia-shocked cultures showed an increase in GS transferase activity. The results indicate the possible regulation of GS by covalent modification.     

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport

Summary The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with aK _D of 7.5 m and those slowly accessible, but rapidly accessed in the presence of valinomycin with aK _D of 2.5 m. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number ofK _D of those sites. The time course of this increase correlates with H⁺ uptake and Rb⁺ extrusion by those vesicles and H⁺ carriers such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC¹⁴N and valinomycin there is transient uptake of SCN^–. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H⁺ gradient.     

Concentration Dependence of Sodium Permeation and Sodium Ion Interactions in the Cyclic AMP-gated Channels of Mammalian Olfactory Receptor Neurons

The dependence of currents through the cyclic nucleotide-gated (CNG) channels of mammalian olfactory receptor neurons (ORNs) on the concentration of NaCl was studied in excised inside-out patches from their dendritic knobs using the patch-clamp technique. With a saturating concentration (100 μm) of adenosine 3′, 5′-cyclic monophosphate (cAMP), the changes in the reversal potential of macroscopic currents were studied at NaCl concentrations from 25 to 300 mm. In symmetrical NaCl solutions without the addition of divalent cations, the current-voltage relations were almost linear, reversing close to 0 mV. When the external NaCl concentration was maintained at 150 mm and the internal concentrations were varied, the reversal potentials of the cAMP-activated currents closely followed the Na⁺ equilibrium potential indicating that P _Cl/P _Na≈ 0. However, at low external NaCl concentrations (≤100 mm) there was some significant chloride permeability. Our results further indicated that Na⁺ currents through these channels: (i) did not obey the independence principle; (ii) showed saturation kinetics with K _ms in the range of 100–150 mm and (iii) displayed a lack of voltage dependence of conductance in asymmetric solutions that suggested that ion-binding sites were situated midway along the channel. Together, these characteristics indicate that the permeation properties of the olfactory CNG channels are significantly different from those of photoreceptor CNG channels. Received: 7 November 1996/Revised: 24 March 1997     

Noradrenaline induced secretion of nonelectrolytes through frog skin

Summary Addition of noradrenaline (4×10^–5 m) to the inner bathing fluid in the skin of the frogRana esculenta results in increased unidirectional fluxes of urea, thiourea, N-methyl-thiourea, N-N-dimethylthiourea and mannitol. Fluxes towards the external medium ( ₀) undergo a much greater increase than those moving in the opposite direction ( _i ). The effect of noradrenaline on ( ₀) is higher for urea and thiourea than mannitol, while its effect on ( ₀) thiourea derivatives is related to lipid solubility. This phenomenon does not occur for ( _i ) of the same molecules.FCCP (10^–6 m) pretreatment strongly inhibits the noradrenaline effect on ( ₀). In skin pretreated whith colchicine (2×10^–5 m) both urea fluxes are increased to the same extent by noradrenaline. Noradrenaline is concluded to exert two separate effects: (1) a change in permeability in both directions; (2) a secretion of nonelectrolytes towards the external fluid. Such secretion is most probably associated with the hormone-induced secretion of fluid and electrolytes, perhaps mediated by an exocytotic mechanism.