Kai Graszynski

Freie Universität Berlin, Berlín, Berlin, Germany

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Publications (11)26.44 Total impact

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    ABSTRACT: 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.
    No preview · Article · Feb 1995 · Helgoland Marine Research
  • A. Olsowski · M. Putzenlechner · K. Böttcher · K. Graszynski
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    ABSTRACT: 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.
    No preview · Article · Feb 1995 · Helgoland Marine Research
  • Olaf Strauss · Kai Graszynski
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    ABSTRACT: 1.1. Membrane vesicles have been isolated from the gill epithelium of the crayfish by differential centrifugation followed by Percoll density gradient centrifugation.2.2. In order to characterize the membrane fractions putative “apical” markers like leucine aminopeptidase (EC, nucleoside diphosphatase (EC and alkaline phosphatase were assayed. However, their use may be limited by their high solubility.3.3. After the separation procedure, less than 20% of their initial activities were recovered in the membrane fractions.4.4. Nevertheless, this fractionation procedure led to a splitting of the used marker enzyme activities into two peaks.5.5. Most of the activity of the “apical” marker enzymes was found in the less dense part of the Percoll gradient and the unquestionably basolateral Na+,K+-ATPase in the more dense part.6.6. An amiloride-sensitive and electroneutral Na+/H+-exchanger, as assayed by acridine orange fluorescence-quench, appeared to be localized mainly in the “apical” membrane fraction, according to its distribution within the Percoll gradient which was opposite to the Na+,K+-ATPase but mainly in cofractionation with the “apical” markers.7.7. The activity of the Na+H+-exchanger revealed saturation kinetics with increasing Na+ concentration.8.8. The corresponding Lineweaver-Burk plot showed a Km of 17mmol/l Na+.9.9. With a Hill coefficient of about 1, cooperative effects concerning Na+ seem to be excluded.10.10. Amiloride inhibits this exchanger with a Ki of about 3.5 × 10−5mol/l.
    No preview · Article · Jul 1992 · Comparative Biochemistry and Physiology Part A Physiology
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    Wolfgang Zeiske · Horst Onken · H J Schwarz · Kai Graszynski
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    ABSTRACT: 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.
    Full-text · Article · May 1992 · Biochimica et Biophysica Acta
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    Horst Onken · Kai Graszynski · Wolfgang Zeiske
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    ABSTRACT: 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 (Isc) and conductance (Gtot) of this preparation were measured. Epithelial cells were impaled with microelectrodes through the basolateral membrane and cellular potentials (Vi under open- and Vsc under short-circuit conditions) as well as the voltage divider ratios (Fi, Fo) were determined. With NaCl salines on both sides an outside positive PDte (22±2 mV) and an Isc (-64±13 μA·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 (Vo=-101±5 mV, external bath as reference; Vi=-78±2 mV, internal bath as reference; Vsc=-80±2 mV, extracellular space as reference). The voltage divider ratios of the external (apical membrane plus cuticle) and internal (basolateral membrane) barrier were Fo=0.92±0.01 and Fi=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” Isc under these conditions almost completely depended on external Cl-. Elimination of Cl- in the external bath reversed Isc, and Gtot decreased substantially. Concomitantly, Vsc depolarised and Fo 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, Gtot decreased substantially. Addition of 1 mmol·1-1 acetazolamide to, and omission of NaHCO3 from, the basolateral bath resulted in a decrease of Isc while Gtot remained unchanged. The Cl--channel blocker DPC inhibited Isc almost completely when added to the basolateral saline, whereas Gtot decreased moderately; however, Vsc depolarised without significant change of Fi. Ouabain had no influence on Isc and Gtot. Increasing the basolateral [K+] resulted in a decrease in Isc, while Gtot was not affected. At the same time Vsc largely depolarised and Fi 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 (Isc, Gtot) and cellular (Vsc, Fi) 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.
    Full-text · Article · Jan 1991 · Journal of Comparative Physiology B
  • H.-J Schwarz · K Graszynski
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    ABSTRACT: 1.1. A half platelet preparation from Chinese crab (Eriocheir sinensis) gill is described which allows electrophysiological investigations of ion transport by gill epithelial monolayer when mounted in a modified Ussing chamber.2.2. The resistance of these preparations equals half that of complete gill platelets (containing the gill epithelium and cuticle twice) indicating that cell damage during preparation of half platelets is negligible.3.3. The transepithelial resistance (resistance of cuticle subtracted previously) was determined to be about 140 Ω cm2 when both sides are bathed with identical salines.4.4. Similarities to the results obtained with perfused complete gills demonstrates the reliability of this preparation.5.5. When identical salines are applied on both sides of the epithelium an outside positive transepithelial potential difference (PDte) up to 40 mV was measured.6.6. The occurrence of such a high PDte under symmetric conditions and its sensitivity to CN− suggests the PDte to be generated by active transport processes.
    No preview · Article · Jan 1989 · Comparative Biochemistry and Physiology Part A Physiology
  • Peter Krippeit-Drews · Gisela Drews · Kai Graszynski
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    ABSTRACT: 1. Transepithelial potential differences (PDte) and acidification rates of the bath chamber were measured on isolated perfused posterior gills of the fiddler crabUca tangeri adapted to dilute seawater. 2. The PDte decreased to almost zero when Na+ was substituted by choline or when ouabain was added to the perfusion saline in high concentrations (10 mmoll–1). Thus, the rheogenic NaCl-transport across the gill epithelium seems to be totally Na+-dependent. 3. When Cl– was replaced by gluconate, a bath positive PDte occurred which was insensitive to ouabain. This PDte could also be observed when, in addition to Cl– removal, Na+ was replaced by TMA+. 4. Bath acidification under normal conditions could be abolished by ouabain, indicating that there is H+ excretion via electrically silent Na+/H+ exchange. In contrast, bath acidification under Cl–-free conditions is only partially blocked by ouabain. 5. It is concluded that under Cl–-free conditions a rheogenic H+-pump in the apical membrane is responsible for the ouabain-insensitive bath acidification as well as for the PDte.
    No preview · Article · Dec 1988 · Journal of Comparative Physiology B
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    Horst Onken · Kai Graszynski
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    ABSTRACT: 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 (PDte). Substitution of Na+ by choline on both sides of the epithelium resulted in a substantial hyperpolarization of the PDte, while substitution of Cl– by gluconate reverses PDte 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 PDte 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 PDte, while the Cl–-channel blocker, diphenylamine-2-carboxylic acid (DPC), showed no effect. The PDte 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 PDte with an IC50 of 3.710–5 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 (PDte decrease). Ouabain did not reduce the PDte 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.
    Full-text · Article · Dec 1988 · Journal of Comparative Physiology B
  • Barbara Kosiol · Thomas Bigalke · Kai Graszynski
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    ABSTRACT: 1.1. A simple and fast method with highly reproducible results is described for the purification of gill epithelium plasma membranes of Orconectes limosus.2.2. The isolation of a plasma membrane fraction was indicated by 18-fold enrichment of the specific (Na+, K+)-ATPase activity and only slight contamination by mitochondria.3.3. With sufficient excess of free Mg2+ the apparent Km for ATP and MgATP was 0.44 mmol/l ATP.4.4. Maximal (Na+, K+)-ATPase activity was achieved at 100–200 mmol/l Na+ with an apparent Ka of 15.6 mmol/l and at 20 mmol/l K+ with an apparent Ka of 2.8 mmol/l.5.5. Ninety-four per cent of (Na+, K+)-ATPase activity was inhibited by 5 mmol/l ouabain with Ki 7.0 × 10−5 mol/l.
    No preview · Article · Dec 1988 · Comparative biochemistry and physiology. B, Comparative biochemistry

    No preview · Article · Dec 1988 · Comparative Biochemistry and Physiology Part A Physiology
  • Gisela Drews · Kai Graszynski
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    ABSTRACT: 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 (PDte) was measured in isolated, perfused gills of the fiddler crab,Uca tangeri. The influence of some specific inhibitors of epithelial ion transport on the PDte was tested. With symmetrical conditions on both sides of the epithelium, the posterior gills ofUca tangeri showed a spontaneous PDte 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 PDte, 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 PDte. After application of amiloride to the bath saline the PDte 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.
    No preview · Article · Apr 1987 · Journal of Comparative Physiology B