Second International Ringberg Conference: "Cell Biology and Molecular Basis of Liver Transport".
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Biomedical subjects
F Wehner
Publications and source records attributed to F Wehner.
Osmoregulation in the renal papilla: membranes, messengers and molecules.
This contribution summarizes recent progress in the understanding of the molecular basis of the release of organic osmolytes that occurs when inner medullary cells are confronted with a drop in osmolarity in their environment. For sorbitol release across the basolateral membrane an increase in intracellular calcium seems to be the prominent signal, initiated by G-protein activation, followed by phosphatidylcholine phospholipase activation and generation of arachidonic acid. The increase in betaine permeability is also G-protein dependent but calcium independent, and is restricted to the basal-lateral cell face. Myo-inositol and glycerophosphorylcholine efflux are calcium and G-protein independent and occur both across the apical and basolateral membrane, although to a different extent. Taurine release is also calcium and G-protein independent; a swelling-activated anion channel at the basolateral membrane represents the major efflux pathway.
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Taurine permeation through swelling-activated anion conductance in rat IMCD cells in primary culture.
Whole cell recordings were performed on rat inner medullary collecting duct (IMCD) cells in primary culture. With 140 mmol/l CsCl in bath and pipette we find within 10 min a 60-fold increase in membrane conductance from 0.02 +/- 0.003 to 1.2 +/- 0.1 nS/pF when bath osmolarity is decreased from 600 to 500 mosmol/l. The effect is due to the activation of an outwardly rectifying anion conductance with the anion selectivity SCN- > I- > NO-3 > Br- > Cl- > F- > isethionate > gluconate > or = aspartate > or = glutamate. A relative permeability of the organic osmolyte taurine to Cl- (Ptaurine: PCl-) of 0.15 was detected. With taurine in pipette and bath, the channel exhibits a nearly identical activation and sensitivity profile to a variety of anion channel blockers as under symmetrical Cl- conditions. Furthermore, the 50% inhibitory concentration value for the effect of 5-nitro-2-(3-phenylpropylamino)benzoate on both currents is virtually identical. We conclude that hypotonic stress increases the anion conductance of rat IMCD cells and that this anion conductance mediates taurine efflux.
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Activation of a Cl(-)-conductive pathway in primary cultures of rat inner medullary collecting duct (IMCD) cells under hypotonic stress.
In intracellular recordings with conventional microelectrodes on rat IMCD cells, we find that hypotonic stress depolarizes membrane voltage and decreases cell input resistance. Ion substitution experiments reveal that these effects are largely due to the activation of a prominent Cl- conductance. After block of this conductance with dideoxyforskolin a smaller concomitant increase in K+ conductance becomes detectable.
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Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture.
We studied the ionic mechanisms underlying the regulatory volume increase of rat hepatocytes in primary culture by use of confocal laser scanning microscopy, conventional and ion-sensitive microelectrodes, cable analysis, microfluorometry, and measurements of 86Rb+ uptake. Increasing osmolarity from 300 to 400 mosm/liter by addition of sucrose decreased cell volumes to 88.6% within 1 min; thereafter, cell volumes increased to 94.1% of control within 10 min, equivalent to a regulatory volume increase (RVI) by 44.5%. This RVI was paralleled by a decrease in cell input resistance and in specific cell membrane resistance to 88 and 60%, respectively. Ion substitution experiments (high K+, low Na+, low Cl-) revealed that these membrane effects are due to an increase in hepatocyte Na+ conductance. During RVI, ouabain-sensitive 86Rb+ uptake was augmented to 141% of control, and cell Na+ and cell K+ increased to 148 and 180%, respectively. The RVI, the increases in Na+ conductance and cell Na+, as well as the activation of Na+/K(+)-ATPase were completely blocked by 10(-5) mol/liter amiloride. At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange. When osmolarity was increased from 220 to 300 mosm/liter (by readdition of sucrose after a preperiod of 15 min in which the cells underwent a regulatory volume decrease, RVD) cell volumes initially decreased to 81.5%; thereafter cell volumes increased to 90.8% of control. This post-RVD-RVI of 55.0% is also mediated by an increase in Na+ conductance. We conclude that rat hepatocytes in confluent primary culture are capable of RVI as well as of post-RVD-RVI. In this system, hypertonic stress leads to a considerable increase in cell membrane Na+ conductance. In concert with conductive Na+ influx, cell K+ is then increased via activation of Na+/K(+)-ATPase. An additional role of Na+/H+ exchange in the volume regulation of rat hepatocytes remains to be defined.
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Intracellular Ca2+ release and Ca2+ influx during regulatory volume decrease in IMCD cells.
Volume changes and cytosolic Ca2+ concentration ([Ca2+]i) of inner medullary collecting duct (IMCD) cells under hypotonic stress were monitored by means of confocal laser scanning microscopy and fura 2 fluorescence, respectively. Reduction of extracellular osmolality from 600 to 300 mosmol/kgH2O by omission of sucrose led to an increase in cell volume within 1 min to 135 +/- 3% (n = 9), followed by a partial regulatory volume decrease (RVD) to 109 +/- 2% (n = 9) within the ensuring 5 min. In parallel, [Ca2+]i rose from 145 +/- 9 to 433 +/- 16 nmol/l (n = 9) and thereafter reached a lower steady state of 259 +/- 9 nmol/l. Under low-Ca2+ conditions (10 nmol/l) RVD was not impeded and reduction of osmolality evoked only a transient increase of [Ca2+]i by 182 +/- 22 nmol/l (n = 6). Preincubation with 100 mumol/l 8-(N,N-diethylamino)octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8) or 20 mmol/l caffeine, both effective inhibitors of Ca2+ release from intracellular stores, in low Ca2+ as well as in high Ca2+, inhibited the Ca2+ response and abolished RVD. The temporal relationship between Ca2+ release from intracellular stores and Ca2+ entry was analyzed by determining fura 2 quenching, using Mn2+ as a substitute for external Ca2+. Intracellular Ca2+ release preceded Mn2+ influx by 17 +/- 3 s (n = 10). Mn2+ influx persisted during the whole period of exposure to hypotonicity, indicating that there is no time-dependent Ca2+ channel inactivation. Preincubation with TMB-8 or caffeine reduced Mn2+ influx to the control level, indicating that activation of Ca2+ channels in the plasma membrane occurs via intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)
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Electron probe X-ray microanalysis of epithelial cells: aspects of cryofixation.
Content and distribution of diffusible ions in epithelial cells were studied by scanning transmission electron microscopy and energy dispersive electron probe X-ray microanalysis of freeze-dried cryosections from trout kidney, rat liver and Malpighian tubules of Drosophila larvae. Cryofixation of small excised kidney and liver samples by rapid immersion into liquid propane resulted in intracellular K/Na-ratios < 1. In contrast, K/Na-ratios > 7 were obtained after in situ cryofixation by means of a cryopunching device which allows tissue pieces to be frozen during excision from the intact organ. Isolated hepatocytes cryofixed in a small droplet of culture medium had a K/Na-ratio of 3.7. After culturing the hepatocytes, the K/Na-ratio increased to 24. Effects of extracellular media of different composition on the intracellular element content were studied. Malpighian tubules of Drosophila larvae were cryofixed by rapid immersion into liquid propane, and the distribution of K across the cells forming the tubules from the basal to the apical cell membrane was measured. An increasing K gradient was found from the intermediate to the apical cytoplasm. The intracellular K distribution was dependent on ions and transport inhibitors present in the fluid surrounding the Malpighian tubules within the larvae. Content and distribution of ions in epithelial cells sensitively depend on the physiological state immediately before cryofixation. Thus, electron probe X-ray microanalysis of cells and cell functions requires careful selection and control of the cell system to be studied.
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Taurocholate depolarizes rat hepatocytes in primary culture by increasing cell membrane Na+ conductance.
Rat hepatocytes in primary culture were impaled with conventional microelectrodes. Addition of 5-100 mumol/l taurocholate led to a slowly developing depolarization that was maximal at 50 mumol/l (10.5 +/- 1.5 mV, n = 15) and not reversible. The effect was Na+ dependent and decreased in cells preincubated with 1 mumol/l taurocholate. Increasing external K+ tenfold depolarized the cells by 12.3 +/- 2.3 mV under control conditions and by 6.3 +/- 1.2 mV with 50 mumol/l taurocholate present (n = 7). Depolarization by 1 mmol/l Ba2+ was 7.6 +/- 0.8 mV and 6.0 +/- 0.7 mV (n = 9) before and after addition of taurocholate, respectively. Cable analysis and Na+ substitution experiments reveal that this apparent decrease in K+ conductance reflects an actual increase in Na+ conductance: in the presence of taurocholate, specific cell membrane resistance decreased from 2.8 to 2.3 k omega x cm2 x Na+ substitution by 95% depolarized cell membranes by 8.9 +/- 2.9 mV (n = 9), probably due to indirect effects on K+ conductance via changes in cell pH. With taurocholate present, the same manoeuvre changed membrane voltages by -0.8 +/- 2.6 mV. When Na+ concentration was restored to 100% from solutions containing 5% Na+, cells hyperpolarized by 3.5 +/- 3.6 mV (n = 7) under control conditions and depolarized by 4.4 +/- 2.9 mV in the presence of taurocholate, respectively. In Cl- substitution experiments, there was no evidence for changes in Cl- conductance by taurocholate. These results show that taurocholate-induced membrane depolarization is due to an increase in Na+ conductance probably via uptake of the bile acid.
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The anion transport inhibitor DIDS increases rat hepatocyte K+ conductance via uptake through the bilirubin pathway.
1. In confluent primary cultures of rat hepatocytes, membrane effects of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) were recorded with conventional microelectrodes. In addition, cell pH and cell Ca2+ were monitored by use of the fluorescent dyes BCECF and fluo-3, respectively. Uptake of DIDS was determined by measuring intracellular DIDS fluorescence between 470 and 520 nm (excitation wavelength 390 nm). 2. In the presence of 0.2 mM DIDS, membrane voltages hyperpolarized from -44.0 +/- 1.8 to -73.1 +/- 1.9 mV (n = 16). This change was monophasic and occurred with a time constant of 170 +/- 25 s. The effect was only partly reversible. 3. Cable analysis revealed a concomitant decrease in the specific cell membrane resistance from 3.2 to 1.5 k omega cm2. 4. In ion substitution experiments, a 10-fold elevation of external K+ (from 2.5 to 25 mM) depolarized cell membranes by 6.2 +/- 1.5 mV (n = 5). In the presence of 0.2 mM DIDS, this membrane response was increased 5-fold to 32.2 +/- 4.1 mV. 5. Replacement of Cl- by 99% with gluconate depolarized the cells by 9.3 +/- 1.9 mV. In contrast, with 0.2 mM DIDS present, Cl- removal led to a membrane hyperpolarization of 5.9 +/- 0.9 mV (n = 4). 6. DIDS had no effect on cytosolic pH or Ca2+. 7. To determine the sidedness of the DIDS effect, i.e. to analyse if the increase in K+ conductance is mediated by uptake of the compound, DIDS was added in the presence of different substrates of hepatocellular anion transport. Taurocholate (50 microM) and frusemide (0.5 mM), which are both taken up via the sinusoidal multi-specific bile acid transporter, did not change DIDS-induced membrane hyperpolarization. 8. In contrast, 0.5 mM bromosulphthalein (BSP), a substrate of the bilirubin transporter, competitively inhibited the membrane hyperpolarization elicited by various concentrations of DIDS (0.1-1.0 mM). 9. Hepatocellular uptake of BSP is known to be, in part, Cl- dependent and to be competitively inhibited by Indocyanine Green. When 0.2 mM DIDS was added to a superfusate, in which 99% of Cl- had been exchanged by gluconate, the velocity of membrane hyperpolarization was decreased by 45%. In the presence of Indocyanine Green (0.1 mM) DIDS-induced membrane hyperpolarization was reduced to approximately 20%. 10. Addition of 0.2 mM DIDS to hepatocyte monolayers led to a time-dependent increase in cell fluorescence which was absent at 4 degrees C and which was completely blocked by 0.5 mM BSP.(ABSTRACT TRUNCATED AT 400 WORDS)
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Artifactual expression of maxi-K+ channels in basolateral membrane of gallbladder epithelial cells.
To patch clamp the basolateral cell membrane, sheets of Necturus gallbladder epithelium were stripped of the subepithelial tissue layers and affixed apical side down on cover slips coated with Cell-Tak [F. Wehner, L. Garretson, K. Dawson, Y. Segal, and L. Reuss. Am. J. Physiol. 258 (Cell Physiol. 27): C1159-C1164, 1990]. In 90% of the patches we observed K+ channels identical to the maxi-K+ channels previously demonstrated in the apical membrane (Y. Segal and L. Reuss. J. Gen. Physiol. 95: 791-818, 1990). To ascertain whether these channels were present in the native tissue, we carried out intracellular-microelectrode studies. We tested for activation of basolateral membrane K+ conductance by depolarization or by elevation of intracellular Ca2+ and for tetraethylammonium sensitivity of the basolateral membrane voltage and fractional resistance. The results were negative, indicating that maxi-K+ channels are not expressed in the basolateral membrane of the "intact" epithelium. Using the same intracellular-microelectrode protocol on the apical membrane, we demonstrated the presence of an apical K+ conductance attributable to maxi-K+ channels. Additional experiments revealed a Ba(2+)-sensitive basolateral K+ conductance in the native epithelium. We conclude that in the stripped preparation there is artifactual expression of maxi-K+ channels. In addition, the native basolateral membrane K+ channels either are not expressed in this preparation or have a low conductance and cannot be discerned from the background noise.
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Osmolarity reduction transiently increases K+ conductance of confluent rat hepatocytes in primary culture.
Rat hepatocytes in confluent primary cultures were impaled with conventional microelectrodes. Reducing extracellular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of cell membranes (maximum after 5 min) from -40 +/- 4 to -51 +/- 2 mV (n = 7). This hyperpolarization is blocked by 1 mmol/l Ba2+ and 0.5 mmol/l quinine. In ion substitution experiments, increasing K+ 10-fold (from 2.7 to 27 mmol/l) depolarizes membrane voltage by 9 +/- 2 mV in normosmotic solutions. In hyposmotic solutions this depolarization is increased to 20 +/- 1 mV at the time of maximum hyperpolarization and decreases thereafter to 8 +/- 2 mV (n = 4). Cable analysis reveals a transient decrease of specific membrane resistance that exactly parallels the increase in membrane voltage response to high K+. In addition, electrical coupling between cells continuously decreases under hyposmotic conditions, indicating that intercellular communication is affected. Reducing Cl- 100-fold (from 116.5 to 1.2 mmol/l; HCO(3-)-free solutions) depolarizes hepatocytes by 24 +/- 3 mV under normosmotic conditions. In hyposmotic solutions, this effect is increased to 39 +/- 4 mV at maximum hyperpolarization and decreases again to 26 +/- 3 mV (n = 8). This transient increase in the voltage response to Cl- removal is abolished by 0.5 mmol/l quinine (n = 5) and 1 mmol/l Ba2+ (n = 5), suggesting that it is indirect via changes in K+ conductance. This concept is corroborated by ion substitution experiments (HCO(3-)-free conditions), which show that under hyposmotic conditions voltage response to high K+ is considerably decreased in low Cl- solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
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Ba2+ release from soda glass modifies single maxi K+ channel activity in patch clamp experiments.
Glasses used to fabricate patch pipettes may release components which affect ion channels (Cota, G., and C.M. Armstrong. 1988. Biophys. J. 53:107-109; Furman, R.E., and J.C. Tanaka. 1988. Biophys. J. 53:287-292; Rojas, L., and C. Zuazaga. 1988. Neurosci. Lett. 88:39-44). The gating properties of maxi K+ channels from Necturus gallbladder epithelium depend on whether borosilicate glass (BG) or blue tip hematocrit glass (SG) is used to construct the patch pipettes. The data are consistent with solubilization from SG of a component which exerts voltage-dependent, cytosolic-side specific block, closely resembling "slow block" by Ba2+ ions. Ringer's solution preincubated with SG, but not with BG, blocked inside-out maxi K+ channels when used as bathing solution. Mass spectrometry revealed that Ba2+ is released by the glass from fast and slow-release compartments (SG contains 3% wt/wt BaO), and is the only ion found in the solution at concentrations consistent with the observed channel block. Additionally, SG released O2-, Na+, Ca2+, and Mg2+, all to micromolar concentrations. These elements do not interfere with maxi K+ channels but they could in principle alter the properties of other ion channels. Thus, screening for channel-modifying substances released by the glass may be necessary for the adequate interpretation of patch-clamp results.
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Electric properties of rat liver cell cultures on gas-permeable membranes.
In rat hepatocytes grown on gas-permeable membranes (Petzinger et al. In Vitro Cell. Dev. Biol. 24: 491-499, 1988), cellular and canalicular potentials as well as input resistances were measured using two-channel microelectrodes. In HCO3(-)-containing solutions, we found -30.9 +/- 0.4 (SE) (n = 141) and -13.9 +/- 1.4 mV (n = 22) for cell and canalicular membrane potentials, respectively. There was no dependence of these parameters on the age of the primary culture. Canalicular input resistance, however, increased from 13.3 +/- 2.0 M omega (n = 4) at day 1 after seeding to 36.1 +/- 5.0 M omega (n = 9) at day 2 and stabilized thereafter, while cell input resistance continuously decreased from 37.0 +/- 3.3 M omega at 1 h (n = 6) to 5.2 +/- 2.1 M omega (n = 27) at 3 days after preparation. In ion substitution experiments there were no changes in the transference numbers for K+, Na+, or Cl- that could account for this effect. Cable analysis, however, revealed that the decrease in input resistance reflects a time-dependent increase in electrical coupling between cells. We conclude that rat liver cells on gas-permeable membranes are highly suited for the quantitative analysis of cell-to-cell interaction. In addition, cells and canaliculi are readily accessible with two-channel microelectrodes, making this preparation a promising tool for electrophysiological analysis of hepatocellular transport mechanisms.
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Naloxone-insensitive transport effects of loperamide in guinea-pig gallbladder epithelium.
The effects of the antidiarrheal drug, loperamide, on HCO3 and Na transport across guinea-pig gallbladder epithelium were investigated using Ussing-chamber methods. Under basal conditions, mucosal loperamide (10(-4) mol/l) moderately lowered both the absorptive (JHCO3ms) and the secretory HCO3 flux (JHCO3sm) (pH-stat method), most likely by changing paracellular HCO3 flow. Exposure to serosal prostaglandin E1 (10(-6) mol/l) abolished Na absorption and turned HCO3 secretion electrogenic. The associated short-circuit current (Isc) was inhibited by loperamide in a concentration-dependent manner; mucosal addition (threshold at 3 x 10(-6) mol/l) of the drug was more effective. Inhibition of Isc was related to a decrease in JHCO3sm, but exceeded the drop in JHCO3net. The effects on JHCO3sm and Isc were mimicked by [Met5]enkephalin. Naloxone (10(-6) mol/l) was unable to influence the effects of loperamide and [Met5]enkephalin on Isc. There were no pro-absorptive effects of loperamide on unidirectional Na fluxes. We conclude that antisecretory properties of loperamide are solely due to inhibition of electrogenic HCO3 secretion, an effect unrelated to opiate receptor binding.
Alprostadil↗
Transcellular bicarbonate transport in rabbit gallbladder epithelium: mechanisms and effects of cyclic AMP.
HCO3 permeation through rabbit gallbladder epithelium has been investigated in vitro using voltage-clamp, pH-stat and microelectrode techniques. Mucosa-to-serosa flux of HCO3 (approximately 4.9 mumol cm-2 h-1) was dependent on luminal Na and inhibited by amiloride (1 mmol/l, luminal bath), methazolamide (0.1 mmol/l, both sides), and ouabain (30 mumol/l, serosal bath). Maximal rates of serosa-to-mucosa flux of HCO3 (approximately 2.8 mumol cm-2 h-1) required serosal Na and mucosal Cl. This flux was inhibited by ouabain, 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (1 mmol/l, serosal bath), and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (0.1 mmol/l, luminal bath). Ineffective were methazolamide (0.1 mmol/l, both sides) and amiloride (1 mmol/l, serosal bath). 8-Br-cAMP (1 mmol/l, serosal bath) largely inhibited the absorptive and moderately stimulated the secretory flux. In tissue conductance, short-circuit current, and transmural voltage prostaglandin E1 (1 mumol/l, serosal bath) and 8-Br-cAMP caused moderate to negligible increases. No significant alterations of apical membrane potential (approximately -65 mV) and the apparent ratio of membrane resistances (Ra/Rb; approximately 1.9) were found. Cell membranes responded to luminal Cl removal mostly with a slow hyperpolarization that was mitigated by 8-Br-cAMP or, in some cases, converted into a small, transient depolarization. Our results are best explained by transcellular HCO3 transport in both directions. In secretion, basolateral HCO3 entry occurs by some form of co-transport with Na, and apical exit by Cl/HCO3 exchange. cAMP opens no major electro-diffusive pathway for apical anion efflux. In absorption, HCO3 import from the lumen into the cell is secondary to cAMP-sensitive Na/H exchange.
8-Bromo Cyclic Adenosine Monophosphate↗
Electrophysiological methods for studying ion and water transport in Necturus gall bladder epithelium.
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Animals↗
A nonenzymatic preparation of epithelial basolateral membrane for patch clamp.
A preparation has been developed that permits patch clamping of the basolateral membrane of Necturus gallbladder epithelial cells with a high success rate. The epithelium is separated from the underlying tissues mechanically, without enzymatic treatment. Its apical surface is attached to a plastic cover slip, and the basolateral surface, facing up, is cleaned with a suction pipette under microscopic observation. With this cleaning procedure, the success rate in obtaining gigaohm seals increases from less than 1% to approximately 10% of the attempts. The cells appear to retain their structural and functional integrity, as evidenced by electron-microscopic appearance and magnitude of cell membrane voltages. Major advantages of the preparation are that the basolateral membrane domain is preserved and that enzymatic treatment, which could potentially alter membrane proteins, is not necessary.
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[Importance and function of the melanocyte-stimulating hormone in malignant melanoma. Importance of MSH].
According to the present state of findings there are melanocyte-stimulating hormones (MSH) alpha, beta, gamma and delta with hormone alpha-MSH special physiological importance for man. Our study of special literature shows that the secretion of MSH is affected by exogene factors, different biorhythms and some diseases. Numerous investigation with melanocytes and melanoma cell cultures clearly show the impact of MSH on the function, regulation and the proliferation of pigment cells. The results presented contribute to the discussion of possibilities of improving diagnostics or therapy of malignant melanoma.
Biomarkers, Tumor↗