The transepithelial potential difference in the gills of the fiddler crab,Uca tangeri: Influence of some inhibitors

Summary Euryhaline Crustacea living in dilute media, counterbalance the salt loss by active absorption of NaCl across the gill epithelium. To investigate the mechanisms involved in salt absorption, transeptithelial potential difference (PD_te) was measured in isolated, perfused gills of the fiddler crab,Uca tangeri. The influence of some specific inhibitors of epithelial ion transport on the PD_te was tested.With symmetrical conditions on both sides of the epithelium, the posterior gills ofUca tangeri showed a spontaneous PD_te of +5 to +10 mV, that is an active transport potential which was positive on the bath side as referred to the hemolymph side. This potential decreased considerably after application of KCN or 2,4-dinitrophenol (DNP) to the perfusion saline.Omission of K⁺ from the perfusion saline or addition of ouabain led to a reversible drop of the PD_te, suggesting that the absorption of Na⁺ and also of Cl^– is driven by the (Na⁺+K⁺)ATPase located in the basolateral membrane of the epithelial cells.Perfusion of the hemolymph space with saline containing diphenylamine-2-carboxylate (DPC) or the loop diuretic furosemide resulted in a decrease of the PD_te.After application of amiloride to the bath saline the PD_te increased. Half-maximum response to amiloride was reached at a concentration of about 10^–5 mol·l^–1. This suggests that one of the Na⁺ pathways across the apical membrane may consist of Na⁺ channels.Abbreviations PD _te transepithelial potential difference - DPC diphenylamine-2-carboxylate - R _ps resistance of perfusate shunt - R _te transepithelial resistance - R _in input resistance - DNP 2,4-dinitrophenol Parts of this study have been reported at the 1st Congress of Comparative Physiology and Biochemistry, Liège 1984, and at the Vth European Colloquium on Renal Physiology, Frankfurt, 1985     

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crabEriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30‰). We found only negligible CA activity in haemolymph, heart, hypodermis, antennal gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na⁺/K⁺-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosol); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36‰ salinity), we found about 70% of CA associated with the highest level of the Na⁺/K⁺-ATP'ase in the second 100 000 g pellet (membrane fraction), while only 15% were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.     

Das Brutgesch?ft der Zwergrohrdommel(Ixobrychus m. minutus) 1954 im Gebiet der Berliner Havel

Ohne Zusammenfassung     

How to overcome osmotic stress? Marine crabs conquer freshwater. New insights from modern electrophysiology

In the present article we review our findings on split lamella preparations of crab gills mounted in modified Ussing-chambers with respect to mechanistic and ecophysiological aspects. The leaky gill epithelium of shore crabs adapted to brackish water absorbs Na⁺ and Cl^? in a coupled mode, and shows similarities to other salt-absorbing epithelia exposed to moderately diluted media. The results so far obtained for NaCl uptake across the gills of the shore crab are compatible with a transport model where two cell types operate in parallel, one displaying cotransport-like NaCl absorption, similar to that in the thick ascending limb of Henle's loop of the mammalian mephron, and the other one with characteristics of amiloride-sensitive, channel-mediated Na⁺ uptake by frog skin. Although there is no clear evidence for the apical mechanisms in this model, it may serve as a good basis for more detailed studies in the future. The moderately tight gill epithelium of freshwater adapted Chinese crabs absorbs Na⁺ and Cl^? independently from each other, and shows similarities to other salt-absorbing epithelia exposed to freshwater. The characteristics of a positive, Na⁺-dependent short-circuit current with externally Cl^?-free saline indicate that active Na⁺ uptake proceeds in a frog-skin-like mode via apical Na⁺-channels and the basolateral Na⁺/K⁺-pump. The nature of a negative short-circuit current with external Cl^?-saline indicates that active and Na⁺-independent Cl^? uptake is driven by an apical V-type H⁺-pump and proceeds via apical Cl^?/ HCO₃ ^?-exchange and basolateral Cl^?-channels.     

Ketonkörperstoffwechsel beim Flußkrebs: Stoffwechselwege,Organverteilung und physiologische Bedeutung

Zusammenfassung Beim Flußkrebs wurden sämtliche Enzyme der Acetoacetatsynthese aus Acetoacetyl-CoA nachgewiesen: Thiolase, Hydroxymethylglutaryl-CoA-Synthase und -Lyase. Deacylase-Aktivität war nicht festzustellen.D-(–)-Hydroxybutyrat-Dehydrogenase fehlt in allen untersuchten Organen, ebenso fehlt D-(–)-Hydroxybutyrat im Blut.Unerwarteterweise enthalten alle untersuchten Organe das erste Enzym auf dem Weg der Sterinsynthese aus Hydroxymethylglutaryl-CoA, die Hydroxymethylglutaryl-CoA-Reductase. Ihre spezifische Aktivität beträgt 30–50% der Aktivität in der Mäuseleber. Sie ist ausschließlich in den Microsomen lokalisiert.Besonders hohe spezifische Aktivitäten stellten wir bei dem Acetessigsäureaktivierenden Enzym Succinyl-CoA-Transferase fest.Markiertes Acetoacetat wird in vivo aus ¹⁴C-Acetat, -Leucin, -Glucose und-Palmitat gebildet. Den relativ höchsten Einbau zeigen Acetat und Leucin.Die Enzyme der Ketonkörpersynthese und des -Abbaus kommen in sämtlichen untersuchten Organen gemeinsam vor. Alle untersuchten Organe synthetisieren im Homogenat nach Blockierung des Citratcyclus mit Malonat Acetoacetat. Markiertes Acetoacetat wird von allen untersuchten Organen und Organhomogenaten zu CO₂ abgebaut. Eine Spezialisierung der Organe im Hinblick auf Ketonkörperbildung und -Abbau wie bei den Säugetieren existiert beim Flußkrebs demnach nicht.Augenstielextrakt-Injectionen erhöhen den Glucosegehalt der Hämolymphe, jedoch nicht den Gehalt an Acetoacetat.Während des Hungerns erhöht sich die Acetoacetatkonzentration im Blut erst vom 13. Tag an, zu einem Zeitpunkt, in dem die Kohlenhydrat- und Lipidspeicher bereits erschöpft sind, und die Energie fast ausschließlich aus Protein gewonnen wird. Eine auffällige Pettsäuremobilisation ist bei hungernden Krebsen nicht feststellbar.

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Metabolism of ketone bodies in the crayfish: pathways, tissue distribution, and physiological significance

Summary In the crayfish all enzymes known to act during the synthesis of acetoacetate have been detected: Thiolase, hydroxymethylglutaryl-CoA-synthase and -lyase. Deacylase-activity has not been found.D-hydroxybutyrate-dehydrogenase was absent in all organs tested, and no hydroxybutyrate was found in the hemolymph.Unexpectedly each organ tested contained the first enzyme in the pathway of sterol synthesis, hydroxymethylglutaryl-CoA-reductase. The specific activity amounts to 30–50% of the specific activity in mouse-liver. This enzyme is found only in the microsomes.The acetoacetate activating enzyme, succinyl-CoA-transferase was found to have remarkably high activities in the crayfish.In vivo labelled acetoacetate was synthesized from ¹⁴C-acetate, -leucine,-glucose and -palmitate. The highest rates were observed with acetate and leucine.In the crayfish enzymes for the synthesis and degradation of ketone bodies were detected in these organs: Heart, antennal gland, hepatopancreas, intestine, integument, gills and abdominal muscle. Homogenates of each organ were able to synthesize acetoacetate when malonate was added. Labeled acetoacetate was metabolized to CO₂ by each isolated organ or by homogenates of each organ. Therefore in the crayfish there is no specialized organ for the metabolism of ketone bodies as there is in the mammals.Injections of eyestalk extracts increased the concentration of glucose in the hemolymph but did not influence the level of acetoacetate.In starving crayfish the concentration of acetoacetate in the hemolymph was not elevated until the 13th day. At this time the stores of carbohydrates and lipids were exhausted and energy was obtained from protein. No remarkable mobilization of fatty acids occurred in the starving crayfish.

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Verwendete Abkürzungen HMG-CoA Hydroxymethylglutaryl-CoA - HMG-CoA-Synth Hydroxymethylglutaryl-CoA-Synthase - HMG-CoA-Ly Hydroxy-methylglutaryl-CoA-Lyase - HMG-CoA-Red Hydroxymethylglutaryl-CoA-Eeductase - Succ-T Succinyl-CoA-Transferase - HB-DH Hydroxybutyrat-Dehydrogenase Fräulein C. Kretschmer und Herrn I. Tofaute danke ich sehr für zuverlässige Mitarbeit.     

Intrazelluläre Lokalisation von Enzymen des Flußkrebses

Zusammenfassung Es werden Eigenschaften von Mitochondrien des Flußkrebses untersucht. Als bestes Isolationsmedium erwies sich eine gepufferte 0,1 M KCl-Lösung mit 0,001 M EDTA und 0,001 M ATP. Flußkrebsmitochondrien sind sehr viel empfindlicher gegenüber mechanischer Beanspruchung als Mitochondrien der Mäuseleber.Unterschiede zwischen sehr schonend isolierten und durch inadäquate Isolierungsmethoden geschädigten Mitochondrien werden im elektronenmikroskopischen Bild gezeigt.Die intrazelluläre Verteilung folgender Enzyme wurde mit Hilfe der successiven Enzymextraktion aus unzerstörten Zellen untersucht: Lactatdehydrogenase, Glucose-6-phosphatdehydrogenase, Malatdehydrogenase, Glutamatdehydrogenase, Hydroxyacyl-Coenzym A-dehydrogenase, Ketoacylthiolase, Hydroxymethylglutaryl-Coenzym A-synthase, Succinyl-Coenzym A-transferase und Proteasen der Mitteldarmdrüse.Durch leichte Extrahierbarkeit sind Lactatdehydrogenase und Glucose-6-phosphat-dehydrogenase auch beim Flußkrebs als cytoplasmatische Enzyme charakterisiert.Glutamatdehydrogenase, Hydroxyacyl-Coenzym A-dehydrogenase, Thiolase und Hydroxymethylglutaryl-Coenzym A-synthase werden erst nach agressiver Homogenisation freigesetzt, sind demnach wie bei Säugetieren offenbar partikelgebunden.Succinyl-Coenzym A-transferase und Malatdehydrogenase werden zum Teil in den ersten Extraktionsschritten, zum Teil erst nach der Homogenisation freigesetzt. Die nach der Homogenisation freigesetzte MDH (M-MDH) unterscheidet sich von der in den ersten Extraktionsschritten freigesetzten (C-MDH) durch starke Hemmbarkeit mit überschüssigem Oxalacetat. Bei der Succinyl-Coenzym A-transferase besteht kein Unterschied in der Hemmbarkeit durch Acetoacetyl-CoA oder Succinat zwischen den einzelnen Fraktionen. Die scheinbare K_m für Succinat liegt bei 30 mM.Enzymmessungen an Homogenaten der Mitteldarmdrüse werden gestört durch Proteasen. Diese Proteasen werden zur Hauptsache beim ersten Extraktionsschritt herausgelöst. Es wird angenommen, daß hierbei vor allem Verdauungssaft aus den feinen extrazellulären Tubuli dieses Organs extrahiert wird. Methoden werden geschildert, die zu einer Abschätzung der wahren Aktivitäten anderer Enzyme in Mitteldarmdrüsen-Homogenaten führen.

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Intracellular localization of enzymes in the crayfishEnzymes of fatty acid oxidation, and of ketone body metabolism, dehydrogenases of energy yielding metabolism, and proteases of hepatopancreas

Summary Properties of the mitochondria from the crayfish have been investigated. Buffered 0,1 M KCl with 0,001 M ATP proved to be the best medium for the isolation. Mitochondria of the crayfish are much more sensitive to mechanical stress than mitochondria of mouse-liver.Differences between carefully isolated mitochondria of the crayfish and those damaged by an inadequate method are demonstrated electron microscopically.The intracellular localization of the following enzymes has been investigated by successively extracting them from non-disrupted cells: Lactate-dehydrogenase, glucose-6-phosphate-dehydrogenase, glutamate-dehydrogenase, hydroxyacyl-coenzyme-A-dehydrogenase, ketoacyl-coenzyme A-thiolase, hydroxymethylglutarylcoenzyme A-synthase, succinyl-coenzyme A-transferase and proteases of the hepatopancreas.Lactate-dehydrogenase and glucose-6-phosphate-dehydrogenase are characterized as cytoplasmatic enzymes because of their easy extraction.Glutamate-dehydrogenase, hydroxyacyl-coenzyme A-dehydrogenase, thiolase and hydroxymethylgmtaryl-coenzyme A-synthase are soluble only after agressive homogenization and therefore are believed to be particle-bound.Succinyl-coenzyme A-transferase and malate-dehydrogenase are partly extracted in the first extraction step and partly after homogenization. The malate-hydrogenase delivered after homogenization (M-MDH) is different from the enzyme extracted in the first steps (C-MDH) in its inhibition by excess oxaloacetate. There is no difference between the fractions of succinyl-coenzyme A-transferase in the inhibition by excess acetoacetyl-coenzyme A or succinate. The apparent K _mfor succinate is 30 mM.Measurements of enzymes in homogenates of the hepatopancreas are disturbed by proteases. These proteases are mainly removed in the first extraction-step. It is believed that in this step for the most part extracellular gastric juice of the fine extracellular tubuli of hepatopancreas is extracted. Methods for estimating the real activities of enzymes in homogenates of the hepatopancreas have been described.

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Herrn Prof. Dr. G. Kümmel und Fräulein H. Claassen danke ich sehr für die elektronenmikroskopische Präparation und die freundliche Überlassung der elektronenmikroskopischen Aufnahmen. Fräulein C. Kretschmer sei gedankt für ihre zuverlässige Mitarbeit beim biochemischen Teil dieser Arbeit.     

Enzyme des Fettsäureabbaus in den Organen des Flußkrebses Orconectes limosus Rafinesque

Zusammenfassung In den Organen des Flußkrebses wurden drei Enzyme der Fettsäureoxydation festgestellt: Crotonase (E.C.: 4.2.1.17), Hydroxyacyl-CoA-dehydrogenase (HAD. E.C.: 1.1.1.35) und Ketoacyl-thiolase (KAT, E.C.: 2.1.3.9).Eigenschaften der HAD und KAT bei unseren Testbedingungen werden geschildert. Im großen und ganzen ähneln die Enzyme in ihren Eigenschaften den Wirbeltierenzymen. Im Gegensatz zu den Wirbeltierenzymen zeigt die HAD des Flußkrebses auch mit NADPH₂ Aktivität (50–75% der Aktivität mit NADH₂). Mit 10^–3 und 2·10^–3 M o-Phenanthrolin wird nur die Aktivität gegen NADPH₂, nicht die gegen NADH₂, gehemmt.Bei unseren Präparationsbedingungen befinden sich 80–90% der Gesamtaktivität der HAD und KAT im 20000 g-Überstand.Hohe spezifische Aktivitäten der HAD und KAT findet man in Herz, Antennendrüse und Darm, niedrige vor allem im Schwanzmuskel.HAD und KAT sind auch in den Organen des Flußkrebses proportionskonstante Enzyme. Durch Quotienten aus Aktivitäten von Schlüsselenzymen des Fettsäureabbaus, der Glykolyse und des Citratcyclus wurden die Organe hinsichtlich ihres energieliefernden Stoffwechsels charakterisiert.

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Enzymes of fatty acid oxidation in the organs of the crayfish orconectes limosus Rafinesque

Summary Three enzymes of fatty acid oxidation have been detected in the organs of the crayfish: Crotonase (E.C.: 4.2.1.17),Hydroxyacyl-CoA-dehydrogenase (HAD, E.C.: 1.1.1.35) and Ketoacyl-thiolase (KAT, E.C.: 2. 1. 3. 9.).The properties of HAD and KAT have been investigated at the conditions of our tests. In general the enzymes of the crayfish resemble to those of vertebrates. In contrary to the HAD of vertebrates the HAD of the crayfish shows activity not only with NADH₂ but also with NADPH₂ (50–75% of the activity with NADH₂). The activity with NADPH₂ is inhibited by 10^–3 and 2·10^–3 M o-phenanthroline, the activity with NADH₂ is not.In our preparations 80–90% of the total activity was localized in the 20 000 g — supernatant.High specific activities of HAD and KAT are found in the heart, antennal gland and the intestine, low predominantly in the abdominal muscle.As in vertebrates and insects HAD and KAT in the different organs have constant proportions in their activities. The organs of the crayfish are characterized with regard to their energy supply by help of quotients of the activities of keyenzymes of fatty acid oxidation, glykolysis and citrate cycle.

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Fräulein C. Kretschmer, Herrn H. Förstel und meiner Frau danke ich für zuverlässige Mitarbeit.     

Invertebrate epithelial Na+ channels: amiloride-induced current-noise in crab gill.

Epithelial sheets (including cuticle) from posterior gills of the freshwater-adapted euryhaline crab Eriocheir sinensis were obtained according to the method of Schwarz and Graszynski ((1989) Comp. Biochem. Physiol. 92A, 601-604; (1989) Verh. Dtsch. Zool. Ges. 82, 211 and (1989) Arch. Int. Physiol. Biochim. 97, C45). With external NaCl-saline, the outward-directed short-circuit current (Isc) could hardly be influenced by external amiloride up to 100 mumol/l but was, on the contrary, strictly dependent on apical Cl- (Onken, Graszynski and Zeiske (1991) J. Comp. Physiol. B 161, 293-301). In absence of external chloride an inward-directed, amiloride-inhibitable Isc was observed which depended on external Na+ (thus, Isc approximately INa) in a two-step, saturating mode. The Isc-block by amiloride obeyed saturation kinetics (half-maximal at less than or equal to 1 mumol/l, suggesting apical Na(+)-channels). Only for Na+ concentrations below 100 mmol/l we found an indication for a competitive interaction between Na+ and amiloride at the channel. Current fluctuation analysis revealed the presence of an amiloride-induced relaxation (Lorentzian) component in the Isc-noise (so-called 'blocker-noise'). The Lorentzian parameter-shifts with increasing amiloride concentration indicate first-order kinetics of the blocker with its apical receptor. Using a 'two-state' blocking model we calculated, for amiloride concentrations between 2 and 5 mumol/l, a mean single-channel current of 0.46 pA and a mean channel density of 250.10(6) cm-2.     

Na+-independent,electrogenic Cl- uptake across the posterior gills of the Chinese crab (Eriocheir sinensis): Voltage-clamp and microelectrode studies

Summary Single gill lamellae from posterior gills of Chinese crabs (Eriocheir sinensis) were isolated, separated into halves and mounted in a modified Ussing chamber. Area-related short-circuit current (I_sc) and conductance (G_tot) of this preparation were measured. Epithelial cells were impaled with microelectrodes through the basolateral membrane and cellular potentials (V_i under open- and V_sc under short-circuit conditions) as well as the voltage divider ratios (F_i, F_o) were determined.With NaCl salines on both sides an outside positive PD_te (22±2 mV) and an I_sc (-64±13 A·cm^-2) with a polarity corresponding to an uptake of negative charges (inward negative) were obtained. Trough-like potential profiles were recorded across the preparation under open- as well as short-circuit conditions (V_o=-101±5 mV, external bath as reference; V_i=-78±2 mV, internal bath as reference; V_sc=-80±2 mV, extracellular space as reference). The voltage divider ratios of the external (apical membrane plus cuticle) and internal (basolateral membrane) barrier were F_o=0.92±0.01 and F_i=0.08±0.01, respectively. To investigate a Cl^--related contribution to the above parameters, Na⁺-free solutions in the external bath (basolateral NaCl-saline) were used. Inward negative I_sc under these conditions almost completely depended on external Cl^-. Elimination of Cl^- in the external bath reversed I_sc, and G_tot decreased substantially. Concomitantly, V_sc depolarised and F_o increased. Cl^--dependent current and conductance showed saturation kinetics with increasing external [Cl^-]. Addition of 20 mmol·1^-1 thiocyanate to the external bath had similar, although less pronounced, effects as Cl^- substitution. Equally, external SITS (1 mmol·1^-1) inhibited the current and, concomitantly, G_tot decreased substantially. Addition of 1 mmol·1^-1 acetazolamide to, and omission of NaHCO₃ from, the basolateral bath resulted in a decrease of I_sc while G_tot remained unchanged. The Cl^--channel blocker DPC inhibited I_sc almost completely when added to the basolateral saline, whereas G_tot decreased moderately; however, V_sc depolarised without significant change of F_i. Ouabain had no influence on I_sc and G_tot. Increasing the basolateral [K⁺] resulted in a decrease in I_sc, while G_tot was not affected. At the same time V_sc largely depolarised and F_i decreased. Addition of the K⁺-channel blocker Ba⁺⁺ (5 mmol·1^-1) to the basolateral solution resulted in a two-step alteration of the transepithelial (I_sc, Gtot) and cellular (V_sc, F_i) parameters. The results are discussed with regard to (i) the mechanisms responsible for active transbranchial Cl^- uptake, and (ii) the technical improvement of being able to perform transport studies with crab gill preparations in an Ussing chamber.Abbreviations DMSO dimethylsulfoxide - DPC diphenylamine-2-carboxylate - F _{o, i} voltage divider ratio for external (o) and internal (i) barrier, respectively - G _Cl conductance related to the external [Cl^-] - G _tot total tissue conductance - I _Cl short-circuit current related to the external [Cl^-] - I _sc short-circuit current - PD _te transepithelial potential difference - R _ME resistance of the microelectrode - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - V _{o, i} open-circuit voltage across the external (o) and internal (i) barrier, respectively - V _sc intracellular potential under short-circuit conditions     

Active Cl− absorption by the Chinese crab (Eriocheir sinensis) gill epithelium measured by transepithelial potential difference

Summary Isolated posterior gills from Chinese crabs (Eriocheir sinensis) acclimated to tap-water were perfused and bathed with full, physiological saline (containing Na⁺ and Cl^–). Under these conditions they developed an outside positive transepithelial potential difference (PD_te). Substitution of Na⁺ by choline on both sides of the epithelium resulted in a substantial hyperpolarization of the PD_te, while substitution of Cl^– by gluconate reverses PD_te to outside negative values.The magnitudes of the potential differences were strongly related to the adaptation media (artificial seawater or tap-water).The KCN-sensitive, outside positive PD_te was shown to be strongly dependent on Cl^–. Application of thiocyanate and 4-acetamido-4-isothiocyanato-stilbene-2,2 disulfonic acid (SITS) to the bath solution resulted in a reduction of the PD_te, while the Cl^–-channel blocker, diphenylamine-2-carboxylic acid (DPC), showed no effect. The PD_te was markedly reduced by acetazolamide, an inhibitor of carbonic anhydrase (CA), and these results are discussed with reference to the presence of a Cl^–/HCO ₃ ^– -exchanger in the apical membrane.Chloride was shown to pass the basolateral membrane via Cl^–-channels: Diphenylamine-2-carboxylic acid (DPC) reduced the PD_te with an IC₅₀ of 3.7×10^–5 mol/l when added to the perfusion saline. A basolateral K⁺-channel and its linkage to Cl^– uptake could be demonstrated by using the K⁺-channel blocker, Ba²⁺, or increased K⁺ concentrations in the perfusion saline (PD_te decrease). Ouabain did not reduce the PD_te under nominally Na⁺-free conditions, indicating that the Cl^– transport is independent of the Na⁺/K⁺-ATPase. In this paper we shall discuss the possible energy sources and linkages between pH regulation and active Cl^– absorption under these experimental conditions.Abbreviations A9C anthracene-9-carboxylic acid - CA carbonic anhydrase - DMSO dimethylsulfoxide - DPC diphenylamino-2-carboxylic acid - PD _te transepithelial potential difference - SITS 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid - TEA tetraethyl-ammoniumchloride