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doi: 10.1007/s002329900493pmid: 10051685
Fast inactivation of the Ca2+ release-activated Ca2+ current (I CRAC) was studied using whole cell patch-clamp recordings in rat basophilic leukemia (RBL-1) cells. Application of hyperpolarizing voltage steps from the holding potential of 0 mV revealed that I CRAC declined in amplitude over tens of milliseconds during steps more negative than −40 mV. This fast inactivation was predominantly Ca2+-dependent because first, it could be more effectively suppressed when BAPTA was included in the recording pipette instead of EGTA and second, replacing external Ca2+ with Sr2+ resulted in less inactivation. Recovery from inactivation was faster in the presence of BAPTA than EGTA. The extent of fast inactivation was independent of the whole cell I CRAC amplitude, compatible with the notion that the inactivation arose from a local feedback inhibition by permeating Ca2+ ions only on the channel it permeated. Ca2+ release from stores did not affect fast inactivation, nor did FCɛRI receptor stimulation. Current clamp recordings showed that the majority of RBL cells had a membrane potential close to −90 mV following stimulation of FCɛRI receptors. Hence fast inactivation is likely to impact on the extent of Ca2+ influx through CRAC channels under physiological conditions and appears to be an important negative feedback process that limits Ca2+ increases.
doi: 10.1007/s002329900494pmid: 10051686
Membrane phospholipids represent a potential influence on the enzymatic properties of the Na,K-ATPase. Little is known concerning the effects of the fatty acid environment surrounding the enzyme on the kinetic properties of the Na,K-ATPase. We used the most obvious difference among the α isoforms of rat, their affinities for digitalis glycosides, to examine the relationship between the lipid environment and the Na,K-ATPase. Specific membrane environments that differ in their fatty acid composition were produced by drug-induced diabetes, as well as variations in diet. The α1 isoforms in various tissues were then characterized by their resistance to ouabain in Na,K-ATPase-enriched membrane microsomal fractions. The Na,K-ATPase activity in nerves and hearts were altered by diabetes and partially restored in nerves after a fish oil diet. Evaluation of enzyme kinetics (dose-response curves for ouabain) in membrane preparations allowed us to correlate the ouabain affinity of α1 isoform with fatty acid composition. The affinity of the α1 isoform for ouabain was significantly increased with accretions in the total amount of fatty acids of the n-6 series (P < 0.0001). Our observations provide a partial explanation for the observed difference in isoform properties among tissues. Moreover, these results underline the interaction between membrane fatty acids and the glycoside binding site of the Na,K-ATPase α1 subunit.
Urbach, V. ; Leguen, I. ; O'Kelly, I. ; Harvey, B.J.
doi: 10.1007/s002329900495pmid: 10051687
Using spectrofluorescence imaging of fura-2 loaded renal A6 cells, we have investigated the generation of the cytosolic Ca2+ signal in response to osmotic shock and localized membrane stretch. Upon hypotonic exposure, the cells began to swell prior to a transient increase in [Ca2+] i and the cells remained swollen after [Ca2+] i had returned towards basal levels. Exposure to 2/3rd strength Ringer produced a cell volume increase within 3 min, followed by a slow regulatory volume decrease (RVD). The hypotonic challenge also produced a transient increase in [Ca2+] after a delay of 22 sec. Both the RVD and [Ca2+] i response to hypotonicity were inhibited in a Ca2+-free bathing solution and by gadolinium (10 μm), an inhibitor of stretch-activated channels. Stretching the membrane by application of subatmospheric pressure (-2 kPa) inside a cell-attached patch-pipette induced a similar global increase in [Ca2+] i as occurred after hypotonic shock. A stretch-sensitive [Ca2+] i increase was also observed in a Ca2+-free bathing solution, provided the patch-pipette contained Ca2+. The mechanosensitive [Ca2+] i response was by gadolinium (10 μm) or Ca2+-free pipette solutions, even when Ca2+ (2 mm) was present in the bath. Long-term (>10 min) pretreatment of the cells with thapsigargin inhibited the [Ca2+] i response to hypotonicity. These results provide evidence that cell swelling or mechanical stimulation can activate a powerful amplification system linked to intracellular Ca2+ release mechanisms.
Fischer, H. ; Seelig, A. ; Beier, N. ; Raddatz, P. ; Seelig, J.
doi: 10.1007/s002329900496pmid: 10051688
The NHE-1 isoform of the Na+/H+ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na+/H+ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na+/H+ antiporter. The Na+/H+ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K M = 30 ± 4 mm for Na+. Addition of either one of the four inhibitors decreased the acidification rate. The IC50 values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC50 values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na+/H+ antiporter and allows a quantitative kinetic analysis of the proton excretion rate.
doi: 10.1007/s002329900497pmid: 10051689
The most frequently observed K+ channel in the tonoplast of Characean giant internodal cells with a large conductance (ca. 170 pS; Lühring, 1986; Laver & Walker, 1987) behaves, although inwardly rectifying, like animal maxi-K channels. This channel is accessible for patch–clamp techniques by preparation of cytoplasmic droplets, where the tonoplast forms the membrane delineating the droplet. Lowering the pH of the bathing solution, that virtually mimicks the vacuolar environment, from an almost neutral level to values below pH 7, induced a significant but reversible decrease in channel activity, whereas channel conductance remained largely unaffected. Acidification (pH 5) on both sides of the membrane decreased open probability from a maximum of 80% to less than 20%. Decreasing pH at the cytosolic side inhibited channel activity cooperatively with a slope of 2.05 and a pK a 6.56. In addition, low pH at the vacuolar face shifted the activating voltage into a positive direction by almost 100 mV. This is the first report about an effect of extraplasmatic pH on gating of a maxi-K channel. It is suggested that the Chara maxi-K channel possesses an S4-like voltage sensor and negatively charged residues in neighboring transmembrane domains whose S4-stabilizing function may be altered by protonation. It was previously shown that gating kinetics of this channel respond to cytosolic Ca2+ (Laver & Walker, 1991). With regard to natural conditions, pH effects are discussed as contributing mainly to channel regulation at the vacuolar membrane face, whereas at the cytosolic side Ca2+ affects the channel. An attempt was made to ascribe structural mechanisms to different states of a presumptive gating reaction scheme.
doi: 10.1007/s002329900498pmid: 10051690
The depolarization-activated, high-conductance ``maxi'' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K+, Na+, Ca2+ and Ba2+ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model), which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary conductance vs. activity relationships for K+, Na+ and Ca2+, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative cation permeabilities with solution composition, and the complex effects of Ba2+ and Ca2+ on K+ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input to electrocoupling models allowing the relationships between membrane voltage, Ca2+ influx and Ca2+ signaling to be studied theoretically.
Kaysen, J.H. ; Campbell, W.C. ; Majewski, R.R. ; Goda, F.O. ; Navar, G.L. ; Lewis, F.C. ; Goodwin, T.J. ; Hammond, T.G.
doi: 10.1007/s002329900499pmid: 10051691
The rotating wall vessel has gained popularity as a clinical cell culture tool to produce hormonal implants. It is desirable to understand the mechanisms by which the rotating wall vessel induces genetic changes, if we are to prolong the useful life of implants. During rotating wall vessel culture gravity is balanced by equal and opposite hydrodynamic forces including shear stress. The current study provides the first evidence that shear stress response elements, which modulate gene expression in endothelial cells, are also active in epithelial cells. Rotating wall culture of renal cells changes expression of select gene products including the giant glycoprotein scavenger receptors cubulin and megalin, the structural microvillar protein villin, and classic shear stress response genes ICAM, VCAM and MnSOD. Using a putative endothelial cell shear stress response element binding site as a decoy, we demonstrate the role of this sequence in the regulation of selected genes in epithelial cells. However, many of the changes observed in the rotating wall vessel are independent of this response element. It remains to define other genetic response elements modulated during rotating wall vessel culture, including the role of hemodynamics characterized by 3-dimensionality, low shear and turbulence, and cospatial relation of dissimilar cell types.
Marunaka, Y. ; Niisato, N. ; O'Brodovich, H. ; Post, M. ; Tanswell, A.K.
doi: 10.1007/s002329900500pmid: 10051692
The aim of the present study was to investigate the roles of Ca2+ and protein tyrosine kinase (PTK) in the insulin action on cell volume in fetal rat (20-day gestational age) type II pneumocytes. Insulin (100 nm) increased cell volume in the presence of extracellular Ca2+ (1 mm), while cell shrinkage was induced by insulin in the absence of extracellular Ca2+ (<1 nm). This insulin action in a Ca2+-containing solution was completely blocked by co-application of bumetanide (50 μm, an inhibitor of Na+/K+/2Cl− cotransporter) and amiloride (10 μm, an inhibitor of epithelial Na+ channel), but not by the individual application of either bumetanide or amiloride. On the other hand, the insulin action on cell volume in a Ca2+-free solution was completely blocked by quinine (1 mm, a blocker of Ca2+-activated K+ channel), but not by bumetanide and/or amiloride. These observations suggest that insulin activates an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl− cotransporter in the presence of 1 mm extracellular Ca2+, that the stimulatory action of insulin on an amiloride-sensitive Na+ channel and a bumetanide-sensitive Na+/K+/2Cl− cotransporter requires Ca2+, and that in a Ca2+-free solution insulin activates a quinine-sensitive K+ channel but not in the presence of 1 mm Ca2+. The insulin action on cell volume in a Ca2+-free solution was almost completely blocked by treatment with BAPTA (10 μm) or thapsigargin (1 μM, an inhibitor of Ca2+-ATPase which depletes the intracellular Ca2+ pool). Further, lavendustin A (10 μm, an inhibitor of receptor type PTK) blocked the insulin action in a Ca2+-free solution. These observations suggest that the stimulatory action of insulin on a quinine-sensitive K+ channel is mediated through PTK activity in a cytosolic Ca2+-dependent manner. Lavendustin A, further, completely blocked the activity of the Na+/K+/2Cl− cotransporter in a Ca2+-free solution, but only partially blocked the activity of the Na+/K+/2Cl− cotransporter in the presence of 1 mm Ca2+. This observation suggests that the activity of the Na+/K+/2Cl− cotransporter is maintained through two different pathways; one is a PTK-dependent, Ca2+-independent pathway and the other is a PTK-independent, Ca2+-dependent pathway. Further, we observed that removal of extracellular Ca2+ caused cell shrinkage by diminishing the activity of the amiloride-sensitive Na+ channel and the bumetanide-sensitive Na+/K+/2Cl− cotransporter, and that removal of extracellular Ca2+ abolished the activity of the quinine-sensitive K+ channel. We conclude that the cell shrinkage induced by removal of extracellular Ca2+ results from diverse effects on the cotransporter and Na+ and K+ channels.
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