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G Kottra

Publications and source records attributed to G Kottra.

At least 19 recordsLinked to original sources
PubMed1995

Calcium is not involved in the cAMP-mediated stimulation of Cl- conductance in the apical membrane of Necturus gallbladder epithelium.

The permeability properties of the forskolin-stimulated Cl- conductance in the apical membrane of Necturus gallbladder epithelium and the possible participation of intracellular Ca2+ in its stimulation have been investigated. The anion selectivity sequence as derived from biionic potential measurements (SCN- > I- approximately NO3- > Br- > Cl- >> ISE-) differed from the sequence derived from measurements of apical membrane resistance (NO3- approximately Br- approximately Cl- > SCN- > I- approximately ISE-). Accordingly, the conductance was inhibited by SCN- and I- which, from the potential measurements, appeared to be more permeable than Cl-. This finding agrees with observations of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel reported recently. However, none of the commonly used Cl- channel blockers, such as 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and glibenclamide reduced this conductance in Necturus gallbladder. In contrast to the situation in most other epithelia, elevation of intracellular Ca2+ concentration ([Ca2+]i) by ionomycin stimulated only K+ conductance and not that of Cl- in the apical cell membrane. Chelation of intracellular Ca2+ did not prevent the stimulation of Cl- conductance by forskolin. This indicates that [Ca2+]i does not have even a permissive role in the cyclic adenosine monophosphate-(cAMP)-mediated stimulation process, as would have been expected if exocytosis was involved. Further evidence against the involvement of exocytosis in the stimulation process came from the observation that the stimulation was not associated with an increase in apical membrane capacitance and was not suppressed by disruption of the cytoskeleton by preincubation of the tissue with cytochalasin D. The data indicate that Necturus gallbladder epithelium contains homologues of the CFTR Cl- channel which reside permanently in the apical cell membrane and which can be stimulated by a cAMP-dependent phosphorylation process without involvement of cell Ca2+ or exocytosis.

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. I. Microscopic and general electrophysiological observations.

Following the publications by Duffey et al. [Nature 294:451 (1981)] and Palant et al. [Am J Physiol 245: C203 (1983)] it is generally accepted that tight-junction tightness of Necturus gall bladder epithelium is up-regulated by cAMP-mediated and Ca(2+)-mediated stimulation. This conclusion was mainly based on observed increases in transepithelial resistance (Rt). However, since in leaky epithelia Rt cannot be simply equated with the tight junction resistance (Rj), but may include large contributions from the lateral space resistance (Rlis), we asked whether the observed increases in Rt resulted indeed from Rj or whether Rlis also increased. The experiments were performed on Necturus gall bladders using forskolin or the Ca2+ ionophore A23187 as stimulants. Forskolin (2 mumol/l) had a biphasic effect. In the first 5 min Rt decreased from 128 +/- 13 to 119 +/- 14 omega cm2 (P < 0.05, n = 10) which probably reflects stimulation of an apical cell membrane Cl- conductance (see accompanying paper). Subsequently Rt increased in approximately 30 min to 184 +/- 20 omega cm2 and then remained fairly constant. Simultaneously the lateral spaces collapsed. If the spaces were now transiently opened by passing mucosa-positive direct current across the epithelium, Rt fell transiently to 111 +/- 7 omega cm2, but returned gradually to its elevated level when the spaces collapsed again. When the spaces were constantly dilated by a serosa-positive hydrostatic pressure of 1 cm H2O, forskolin neither affected the space width nor increased Rt, and current passage was virtually ineffective, although the cells depolarized in response to forskolin as usual.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Tight-junction tightness of Necturus gall bladder epithelium is not regulated by cAMP or intracellular Ca2+. II. Impedance measurements.

In the preceding publication we have reported that, contrary to the prevailing opinion in the literature, the tight-junction tightness of Necturus gall bladder epithelium is not up-regulated by cAMP-mediated or by Ca(2+)-mediated stimulation. This conclusion was based on our observation that the stimulant-induced increase in transepithelial resistance (Rt) occurred only when the lateral intercellular spaces were allowed to collapse, which suggested that the increase reflected primarily or exclusively the increasing resistance of the lateral spaces (Rlis) rather than the postulated increase in tight-junction resistance (Rj). An alternative explanation could have been that the constancy of Rt after space dilatation reflected an increase Rj that was masked by a concomitant fall in apical and basolateral cell membrane resistances Ra and Rbl. To decide between those possibilities we have performed impedance measurements with transepithelial and intracellular microelectrodes on Necturus gall bladder epithelium. Applying previously developed analysis procedures, the measurements readily showed that elevation of intracellular Ca2+ concentration increased Rlis, but left Rj as well as Ra and Rbl quasi constant. Experiments with forskolin, theophylline or isobutylxanthine, on the other hand, were less clear. These stimulants activated an apical Cl- conductance, which drastically reduced Ra and apparently caused low-frequency polarization effects that could not be accounted for by the classical epithelial equivalent circuit. After elimination of the polarization phenomena by uni- or bilateral substitution of Cl- by isethionate or sulphate, however, we were able to demonstrate that Rj remains constant under cAMP-mediated stimulation irrespective of whether the lateral spaces are kept open or are allowed to collapse. We conclude that the tight-junction resistance of Necturus gall bladder epithelium is not controlled by intracellular Ca2+ or by cAMP-mediated stimulation.

Animals↗
PubMed1991

Sodium dependence of the epithelial sodium conductance expressed in Xenopus laevis oocytes.

The epithelial Na+ conductance was expressed in Xenopus laevis oocytes by injection of size-fractionated mRNA of bovine tracheal epithelium. Fractionation was achieved by sucrose density gradient centrifugation. Successful expression was analysed by recording current/voltage (I/V) curves in the presence and absence of amiloride (10 mumol/l). The newly expressed conductance was half-maximally inhibited by 44 nmol/l amiloride and exhibited a selectivity for Na+ over K+ of 140:1. I/V curves obtained at different extracellular Na+ concentrations ([Na+]o) were subjected to a Goldman-fit analysis to obtain the relation between Na+ permeability (PNa) and [Na+]o. The data show that decreasing [Na+]o from 85 mmol/l to 0.85 mmol/l increased PNa by more than threefold, which is thought to reflect Na+ channel inhibition by increasing [Na+]o. This effect clearly exceeded what can be attributed to concentration saturation of single Na+ channel conductance (Palmer and Frindt (1986) Proc Natl Acad Sci USA 83:2767). No correlation of inhibition with intracellular Na+ concentration was observed. Preservation of the [Na+]o-dependent self-inhibition by the newly expressed Na+ conductance suggests that it is an intrinsic property of the Na+ channel protein, probably mediated by an extracellular Na+ binding site.

Amiloride↗
PubMed1990

Barium blocks cell membrane and tight junction conductances in Necturus gallbladder epithelium. Experiments with an extended impedance analysis technique.

The site and concentration dependence of the blocking effect of Ba2+ on Necturus gallbladder epithelium has been investigated. A new approach was used which combines time-dependent electrical cell coupling analysis with intermittently performed measurements of transepithelial and apparent intracellular impedance. From the coupling pulse data the sum of apical and basolateral membrane conductances is obtained, which is then held constant during fitting of the impedance data. This combination technique yields more reliable estimates of apical and basolateral membranes resistances (Ra, Rbl) and of tight junction resistance (Rj) than our previous impedance analysis technique. Using the new approach we have found that luminal Ba2+ concentrations between 0.5 and 1.0 mmol/l increase Ra with saturation-type kinetics without affecting Rbl and Rj, while higher luminal Ba2+ concentrations progressively increase Rj. Corresponding effects were observed under serosal Ba2+. The results validate the new impedance analysis approach and demonstrate that millimolar concentrations of Ba2+ block tight junction conductances. Accordingly, Ba2+ can no longer be considered a tool to exclusively alter cell membrane resistances in epithelia.

Animals↗
PubMed1989

A method to quantify and correct for edge leaks in Ussing chambers.

A technique is presented which allows edge leaks in Ussing chambers to be detected and quantified. It is based on the fact that in leaky chambers direct current or low frequency alternating current passes preferentially around the edge of the tissue, while high frequency alternating current shunts the cell membranes and distributes homogeneously through the entire chamber. By measuring the current density in the center of the chamber with low and high frequency alternating current and using correction factors which account for the shape of the electrical field as a function of chamber geometry and of estimated tissue resistance, edge leaks can be quantified. This technique allows correct transepithelial resistance values to be obtained from leaky measurements, and enables the question of whether a given discrepancy between cellular and transepithelial resistances reflects leaky tight junctions or a leaky chamber or both to be answered.

Animals↗
PubMed1988

Renal nerves in exaggerated water and sodium excretion by hypertrophied kidney of anesthetized rats.

The effect of acute renal denervation (RD) on water (V), sodium (UNaV), and potassium excretion (UKV) from the hypertrophied and control kidney was studied in 5-sec-butyl-5-ethyl-2-thiobarbituric acid (Inactin)-anesthetized male rats 7 days after unilateral nephrectomy (Nx) or sham operation (SNx). V, UNaV, and UKV from the hypertrophied kidney were similar before and after RD or sham RD. In contrast, in SNx rats, left RD resulted in an ipsilateral increase in V (from 2.76 +/- 0.39 to 5.31 +/- 0.99 microliters.min-1.g-1), UNaV (from 109 +/- 36 to 857 +/- 331 nmol.min-1.g-1), and UKV (from 144 +/- 44 to 807 +/- 130 nmol.min-1.g-1; P less than 0.05 in all cases). Moreover, renal parameters from the hypertrophied kidney, subjected to either RD or sham RD, were not different from values after RD in SNx rats (V: Nx, sham RD = 5.72 +/- 1.10; Nx, RD = 5.23 +/- 0.66; SNx, RD = 5.31 +/- 0.99 microliters.min-1.g-1; UNaV: Nx, sham RD = 896 +/- 319; Nx, RD = 821 +/- 262; SNx, RD = 857 +/- 331 nmol.min-1.g-1; UKV: Nx, sham RD = 782 +/- 127; Nx, RD = 860 +/- 82; SNx, RD = 807 +/- 130 nmol.min-1.g-1). In additional experiments, integrated renal nerve activity (RNA) to the kidney in Nx and SNx rats was 4.0 +/- 0.3 and 10.7 +/- 0.9 microV (P less than 0.05), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. I. Experimental procedures.

A method has been developed to determine rapidly and simultaneously the resistance of the tight junctions, the resistance of the lateral intercellular space, and the resistances and capacitances of the apical and basolateral cell membrane in leaky epithelia by alternating current spectroscopy. The present paper describes the experimental procedures. Multi-frequency sine-wave currents are simultaneously applied across the tissue and the transepithelial and intracellular voltage responses are recorded with shielded microelectrodes, digitized, and stored in a computer. Using Fast Fourier Transform techniques the frequency-dependent transepithelial impedance and an apparent basal cell membrane impedance are then calculated from the voltage and current waveform. By fitting appropriate model circuits to the data the above listed individual resistances can be deduced. The model calculations and the fit results are described in the subsequent paper [14]. In the present design the analysis covers the frequency range between 2.5 Hz and 12.5 kHz. The minimal measuring time is in the order of 1-2s.

Animals↗
PubMed1984

Rapid determination of intraepithelial resistance barriers by alternating current spectroscopy. II. Test of model circuits and quantification of results.

The impedance of Necturus gallbladder epithelium was measured with transepithelial and intracellular microelectrodes in different transport states. The data are analysed with five electrical equivalent circuits, which differ with respect to the configuration of the paracellular shunt path (lumped vs distributed model of the lateral space), and of the apical cell membrane (non-ideal capacitance or surface amplification by micro-tubular infoldings). Least square fits indicate: 1. that the lumped model cannot represent the epithelium properly, even under control conditions; 2. that the distributed model, which considers the lateral intracellular space separately as a cable-like structure, describes the data well, both under control conditions and during collapse of the lateral spaces; and 3. that the above indicated variations of the apical membrane configuration improve the fits, but have little effect on the magnitude of the calculated circuit parameters. Quantitatively the analysis of 214 measurements on 25 gallbladders under control conditions yields the following results: The resistances of the tight junctions, of the lateral intercellular space, and of the apical and basal cell membrane are Rj = 123, Rlis = 35.5, Ra approximately 3,500, and Rb = 225 (all in omega cm2), and the capacitances of the cell membranes are Ca = 4.95 and Cbl = 26.5 (mu F/cm2). In oxygen deficiency and after cessation of chamber perfusion transport decreased, the spaces collapsed, and Rlis increased to approximately 130 omega cm2. Although the accuracy of the estimated Ra values is still limited, the analysis shows that it is possible to determine Rj and Rlis separately and to follow their changes in response to experimental maneuvers.

Animals↗
PubMed1983

Intratubular and peritubular capillary hydrostatic and oncotic pressures after chronic renal sympathectomy in the anaesthetized rat.

The possible role of peritubular capillary physical forces in the diuretic-natriuretic effects of chronic renal denervation was investigated in Inactin-anaesthetized nondiuretic control (C) and unilaterally denervated (D) rats. Micropuncture techniques were combined with measurement of intratubular and peritubular capillary hydrostatic pressures and afferent and efferent arteriolar plasma oncotic pressures were determined, as well. Compared to data of C rats and of innervated kidneys, marked denervation diuresis and natriuresis were seen without changes in GFR. Both late proximal and early distal (F/P)In values were significantly lower in D kidneys with similar SNGFR. Afferent (pia) and efferent (pi e) arteriolar oncotic pressures were unchanged by denervation (C-pi a = 23.3 +/- 0.79, pi e = 29.9 +/- 0.87 mm Hg; D-pi a = 23.2 +/- 0.94, pi e = 29.8 +/- 1.04 mm Hg). Proximal intratubular hydrostatic pressure was moderately but significantly higher in D kidneys (C = 11.9 +/- 0.5, D = 13.7 +/- 0.3 mm Hg, P less than 0.01), while peritubular capillary pressures were: efferent arteriole (C = 13.9 +/- 0.5, D = 13.4 +/- 0.6 mm Hg, NS). It is concluded that the tubular effects of chronic renal sympathectomy are not dependent on changes in Starling forces of the peritubular environment.

Animals↗
PubMed1983

Functional properties of the paracellular pathway in some leaky epithelia.

We here review the functional properties of the paracellular pathway of leaky epithelia such as gallbladder and renal proximal tubule. These epithelia are characterized by leaky terminal bars between adjacent cells which allow small ions, non-electrolytes and water to leak from lumen to interstitial fluid or back. In the past 10 years a great deal of information has been obtained about the properties of the misnamed 'tight' junctions in the terminal bars, by assuming that the overall permeation pattern reflected predominantly the junctional permeation properties. Although recent trans- and intraepithelial impedance analyses indicate that this assumption is not always justified (the contribution of the lateral intercellular space to the paracellular shunt resistance is not negligible, when the spaces are collapsed) it seems that the major conclusions are correct. The properties of the terminal junctions may thus be summarized as follows. (1) Large molecules such as horseradish peroxidase are not able to pass. (2) Passage of lipophilic substances is insignificant, as these substances permeate by the cellular route. (3) Depending on the tissue, ion permeation is either governed by channels with negative fixed charges, or positive fixed charges, or both. As inferred from ion selectivity patterns the channels of different epithelia are either wide and highly hydrated or narrow and poorly hydrated, thus allowing more or less water molecules to pass besides the ions. In narrow channels single-file diffusion may occur. (4) Besides the selective channels a free solution shunt seems to be present in some epithelia. (5) When applied in millimolar concentrations 2,4,6-triaminopyrimidinium and amiloride block negatively charged junctional channels. However these substances do not simply turn leaky epithelia into tight epithelia, because they have additional effects on the cell membranes. (6) As observed in cell cultures, formation of tight junctions requires connecting particles to be present on the cell surface--which seems to be controlled by the cytoskeleton-- and requires the presence of calcium ions as ligands. (7) Cellular control over paracellular permeability may be exerted through changes of intracellular calcium concentration.

Animals↗
PubMed1982

A simple method for constructing shielded, low-capacitance glass microelectrodes.

A new simple method is presented to produce shielded low-capacitance microelectrodes. A metal-shield is vapour-deposited on the inner surface of a glass pipette which is slid over the microelectrode proper and insulated at the tip by dipping in polystyrene. The unshielded protruding tip can be as small as 10 micrometers. A special advantage is the low capacitance between electrode and shield of approximately 0.16 microF/cm shield length.

Glass↗
PubMed1982

Square wave pulse analysis of cellular and paracellular conductance pathways in Necturus gallbladder epithelium.

In search for a rapid and reliable method to identify and quantitatively determine cell membrane resistances and paracellular shunt resistances in epithelia we have developed appropriate techniques to measure transepithelial and intracellular potential transients in response to transepithelially applied square wave constant current pulses. Model considerations indicate that in a unilayered, homogeneous epithelium with open lateral spaces the transient potential response across each cell membrane should obey a single exponential function in case the tight junction resistance is high, as in a tight epithelium, whereas in a leaky epithelium it should consist of a superposition of two exponentials with equal sign at the membrane with the higher intrinsic time constant and of two exponentials of different sign (overshoot with recline) at the membrane with the lower intrinsic time constant. The latter predictions were experimentally verified in a study on Necturus gallbladder epithelium and equivalent circuit parameters for the cell membrane resistances and capacitances as well as for the resistance of the shunt path were calculated from the data by curve fitting procedures. The resistances of the apical and basal cell membrane and of the shunt path averaged 1220, 201 and 91 omega cm2 respectively while the apical and basal cell membrane capacitances were 8.0 and 26.3 micro F/cm2 respectively. The fact that the resistance values are 4-15 times lower than estimates derived previously from 2D-cable analysis relates to a better preservation of the transport function under the present incubation conditions as verified by a new series of cable analysis data. The capacitances agree well with estimates of the surface amplification of the cell membranes from electronmicrographs, thus confirming the validity of the interpretation of the observed voltage transients.

Animals↗
PubMed1981

Renorenal vasomotor reflex.

The acetylcholine induced contralateral renorenal vasomotor reflex has been re-examined. In dogs under pentobarbital anaesthesia blood flow of the contralateral kidney as measured by electromagnetic flow probe did not change after an infusion or bolus injection of Ach. Basal sympathetic nervous activity of the contralateral kidney increased only slightly and these changes correlated with the decrease of arterial blood pressure. In conclusion, the renorenal vasomotor reflex could not be evoked under the present experimental conditions.

Acetylcholine↗
PubMed1981

Effect of renal denervation on free flow proximal tubular potential difference in the rat.

Proximal transtubular potential difference (PD) was measured using the semi-microelectrode technique in control (C, n = 10) and unilaterally denervated (D, n = 10) rats. Acute renal sympathectomy resulted in a twofold and fourfold increase in diuresis and sodium excretion, respectively, with no change in GFR. PD (mean +/- S.E.M.) in the earliest accessible proximal convolutions (EPT) of group C was +/- 0.27 +/- 0.08 mV (n = 16), while in group D it was -0.16 +/- 0.07 mV (n = 18) (p less than 0.01). PD in mid (MPT) and late (LPT) proximal segments was unchanged by denervation (MPT: C = 0.94 +/- 0.05, n = 21; D = 0.98 +/- 0.04, n = ns. LPT: C = 1.04 +/- 0.11, n = 17; D = 0.95 +/- 0.06, n = 18 NS.). The shift to the negative of PD in EPT caused by denervation lends support to our earlier suggestion that active transport of solutes and organic substances is depressed by sympathectomy in the proximal tubule. The unaltered PD in MPT and LPT seems to indicate that passive forces promoting reabsorption in this part of the nephron are probably preserved.

Animals↗