The effect of hypo-osmolarity upon transepithelial ion transport in cultured renal epithelial layers (MDCK)

Regulatory volume decrease after exposure to hypo-osmotic media in MDCK epithelial cells results from activation of both K⁺ and Cl^– conductances. Swelling-stimulated ⁸⁶Rb(K) losses were observed only across the basal-lateral membrane and were relatively insensitive to 10 mM Ba²⁺. The effect of hypo-osmotic media upon MDCK epithelia mounted in Ussing chambers has been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (I _sc) followed by inhibition of inward I _sc in both control layers and in layers where inward current (due to transepithelial Cl^– secretion) was first stimulated by 5 M prostaglandin E₁ (PGE₁). The transient stimulation of inward current by hypo-osmotic media was not markedly attenuated by 10 mM Ba²⁺ in PGE₁-stimulated layers. After stimulation of inward (Cl^–-secretory) current to high levels by 10M adrenaline, the predominant effect of basal-lateral exposure to hypo-osmotic media was an inhibition of the inward current. This inhibition was partially reversed by 40M 4,4-diisothiocyanatostilbene-2,2-disulphonate (DIDS). The stimulation, then inhibition, of inward I _sc is likely to be the result of separate swelling-induced K⁺ and Cl^– conductances (respectively) at the basal-lateral membrane. The swelling-stimulated Cl^– conductance is distinct from the apical Cl^– conductance regulated by PGE₁ or adrenaline.     

Activation of the basolateral Cl− conductance by cAMP in rabbit renal proximal tubule S3 segments

The regulatory mechanism of basolateral Cl^– conductance in rabbit renal proximal tubule S3 segments was investigated with conventional and Cl^– sensitive microelectrodes. After the basolateral Cl^–/HCO ₃ ^– exchanger was blocked by 4,4-diisothiocyanatostilbene-2, 2-disulphonic acid (DIDS) we increased the bath K⁺ concentration from 5 mmol/l to 20 mmol/l, which depolarized the cells and thereby increased intracellular Cl^– activity ([Cl^–]_i). This [Cl^–]_i response was enhanced by +63% in the presence of forskolin (20 mol/l), by +40% in the presence of dibutyryl adenosine 3,5-cyclic monophosphate (db-cAMP) (1 mmol/l) and by +44% in the presence of parathyroid hormone (PTH, 10 nmol/l), whereas it was inhibited by a Cl^– channel blocker, indanyl-oxyacetic acid (IAA-94, 0.3 mmol/l). In addition, forskolin, PTH and chlorophenylthio-cAMP enhanced the electrogenic response to removal of bath Cl^– after the blockade of K⁺ conductance, and this activation was also sensitive to IAA-94. On the other hand, 2 mol/l ionomycin and 0.5 mol/l phorbol myristate failed to activate the [Cl^–]_i response to elevation of bath K⁺ concentration and the electrogenic response to Cl^– removal, and ionomycin had no effect even in the absence of DIDS. These results indicate that this basolateral Cl^– conductance can be activated by cAMP, while neither the increase in cytosolic Ca²⁺ nor the activation of protein kinase C has direct effects on this conductance.     

Characterization of the Cl− conductance in the granular duct cells of mouse mandibular glands

We have previously shown that mouse mandibular granular ducts contain a hyperpolarization-activated Cl^– conductance. We now show that the instantaneous current/voltage (I/V) relation of this Cl^– conductance is inwardly rectifying with a slope conductance of 15.4±1.8 nS (n=4) at negative potentials and of 6.7±0.9 nS (n=4) at positive potentials. Thus, the inward rectification seen in the steady-state I/V relation is due, not only to voltage activation of the Cl^– conductance, but also to the intrinsic conductance properties of the channel. We show further that the ductal Cl^– conductance is not activated by including ATP (10 mmol/l) in the pipette solution. Finally, we show that the conductance is not blocked by the addition of any of the following compounds to the extracellular solution: anthracene-9-carboxylate (A9C, 1 mmol/l), diphenylamine-2-carboxylate (DPC, 1 mmol/l), 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 mol/l), 4,4-diisothiocyanato-stilbene-2,2-disulphonate (DIDS, 100 mol/l), indanyloxyacetic acid (IAA-94, 100 mol/l), verapamil (100 mol/l), glibenclamide (100 mol/l) and Ba²⁺ (5 mmol/l). The properties of the ductal Cl^– conductance most nearly resemble those of the ClC-2 channel. Both channel types have instantaneous I/V relations that are slightly inwardly rectifying, are activated by hyperpolarization with a time-course in the order of hundreds of milliseconds, have a selectivity sequence of Br^–>Cl^–>I^–, and are insensitive to DIDS. The only identified difference between the two is that the ClC-2 channel is 50% blocked both by DPC and A9C (1 mmol/l), whereas the ductal Cl^– conductance is insensitive to these compounds.     

pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

Intracellular Cl− concentration in striated intralobular ducts from rabbit mandibular salivary glands

Intralobular striated ducts have been isolated from rabbit mandibular salivary glands and maintained in primary culture for up to 2 days. Such ducts were loaded with the Cl^–-sensitive fluorescent dyeN-(ethoxycarbonylmethyl)-(6-methoxyquinolinium bromide) (MQAE) and intracellular Cl^– concentration ([Cl^–]_i monitored using a fluorescence microscope. Intracellular Cl^– could be rapidly and reversibly emptied from striated duct cells by replacing Cl^– in the superfusing solution with NO ₃ ^– . [Cl^–]_i could be lowered by removal of external Na⁺, exposure to 10 M amiloride or to 10 M 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS). Both amiloride and DIDS were able to inhibit the recovery of [Cl^–]_i after an initial exposure to Na⁺- or Cl^–-free solution. The amiloride derivatives, benzamil (2 M) and N-isobutyl-N-methylamiloride (MIBA), (10 M) also lowered [Cl^–]_i by similar amounts as 10 M amiloride. Varying external K⁺ concentration ([K⁺]_o) also affected [Cl^–]_i. Increasing [K⁺]_o increased [Cl^–]_i, but decreasing [K⁺]_o did not decrease [Cl^–]_i. Instead, [Cl^–]_i was also increased when [K⁺]_o was lowered below the control value. Bumetanide (0.1 mM) lowered [Cl^–]_i by only a small amount, while ouabain (1 mM) had no significant effect on [Cl^–]_i. These data are consistent with current models of electrolyte transport in salivary ducts which include Cl^– channels, Na⁺ channels, and Na⁺/H⁺ exchangers in the apical membrane. The effects of low [K⁺]_o can be interpreted in terms of a K⁺-dependent exit mechanism for Cl^–.     

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 forskolinstimulated 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^– NO ₃ ^– > Br^– > Cl^– ISE^–) differed from the sequence derived from measurements of apical membrane resistance (NO ₃ ^– Br^– Cl^– > SCN^– > I^– 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.     

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 (4.9 mol cm^–2h^–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 mol/l, serosal bath). Maximal rates of serosa-to-mucosa flux of HCO₃ (2.8 mol cm^–2h^–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)-benzole 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 mol/l, serosal bath) and 8-Br-cAMP caused moderate to negligible increases. No significant alterations of apical membrane potential ( –65 mV) and the apparent ratio of membrane resistances (R_a/R_b;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 electrodiffusive pathway for apical anion efflux. In absorption, HCO₃ import from the lumen into the cell is secondary to cAMP-sensitive Na/H exchange.     

Ca2+ influx induced by store release and cytosolic Ca2+ chelation in HT29 colonic carcinoma cells

Cl^– secretion in HT₂₉ cells is regulated by agonists such as carbachol, neurotensin and adenosine 5-triphosphate (ATP). These agonists induce Ca²⁺ store release as well as Ca²⁺ influx from the extracellular space. The increase in cytosolic Ca²⁺ enhances the Cl^– and K⁺ conductances of these cells. Removal of extracellular Ca²⁺ strongly attenuates the secretory response to the above-mentioned agonists. The present study utilises patch-clamp methods to characterise the Ca²⁺ influx pathway. Inhibitors which have been shown previously to inhibit non-selective cation channels, such as flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=6) inhibited ATP (0.1 mmol·l^–1) induced increases in whole-cell conductance (G _m). When Cl^– and K⁺ currents were inhibited by the presence of Cs₂SO₄ in the patch pipette and gluconate in the bath, ATP (0.1 mmol·l^–1) still induced a significant increase in G _m from 1.2±0.3 nS to 4.7±1 nS (n=24). This suggests that ATP induces a cation influx with a conductance of approximately 3–4 nS. This cation influx was inhibited by flufenamate (0.1 mmol·l^–1, n=6) and Gd³⁺ (10 mol·l^–1, n=9). When Ba²⁺ (5 mmol·l^–1) and 4,4-diisothiocyanatostilbene-2-2-disulphonic acid (DIDS, 0.1 mmol·l^–1) were added to the KCl/K-gluconate pipette solution to inhibit K⁺ and Cl^– currents and the cells were clamped to depolarised voltages, ATP (0.1 mmol·l^–1) reduced the membrane current (I _m) significantly from 86±14 pA to 54±11 pA (n=13), unmasking a cation inward current. In another series, the cation inward current was activated by dialysing the cell with a KCl/K-gluconate solution containing 5–10 mmol·l^–1 1,2-bis-(2-aminoethoxy)ethane-N,N,N,N-tetraacetic acid (EGTA) or 1,2-bis-(2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA). The zero-current membrane voltage (V _m) and I _m (at a clamp voltage of +10 mV) were monitored as a function of time. A new steady-state was reached 30–120 s after membrane rupture. V _m depolarised significantly from –33±2 mV to –12±1 mV, and I _m fell significantly from 17±2 pA to 8.9±1.0 pA (n=71). This negative current, representing a cation inward current, was activated when Ca²⁺ stores were emptied and was reduced significantly (I _m) when Ca²⁺ and/or Na⁺ were removed from the bathing solution: removal of Ca²⁺ in the absence of Na⁺ caused a I _m of 5.0±1.2 pA (n=12); removal of Na⁺ in the absence of Ca²⁺ caused a I _m of 12.8±3.5 pA (n=4). The cation inward current was also reduced significantly by La³⁺, Gd³⁺, and flufenamate. We conclude that store depletion induces a Ca²⁺/Na⁺ influx current in these cells. With 145 mmol·l^–1 Na⁺ and 1 mmol·l^–1 Ca²⁺, both ions contribute to this cation inward current. This current is an important component in the agonist-regulated secretory response.     

The activation of Ca2+-dependent K+ conductance by adrenaline in mouse peritoneal macrophages

Responses to adrenaline in mouse peritoneal macrophages were investigated with perforated and cell-attached patch-clamp recording, and with a combination of the perforated-patch recording and fura-2 fluorescence measurements. Extracellularly applied adrenaline induced a transient outward current (4–10s in duration, 100–500 pA in amplitude) at –40 mV associated with a marked increase in conductance. The adrenaline-induced current [I _o (Adr)] reversed polarity near –80 mV. The reversal potential depended distinctly on the external K⁺ concentration but not on external Cl^– concentration. Removal of external Ca²⁺ did not affect I ^o(Adr) within 2–4 min but subsequent responses to adrenaline were progressively depressed. In contrast, treatment with an intracellular Ca²⁺ chelator, the acetoxymethyl ester of 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid completely abolished I _o(Adr). Furthermore, I _o(Adr) was blocked by bath-applied quinidine and charybdotoxin, but not by tetraethylammonium or apamin. Extracellular application of an ₁-adrenoceptor agonist phenylephrine and of noradrenaline mimicked I _o(Adr). On the other hand, I _o(Adr) was antagonized by a non-selective -adrenoceptor antagonist phentolamine (0.2 M) and an ₁-adrenoceptor antagonist prazosin (0.2 M), but was not affected by an ₂-adrenoceptor antagonist yohimbine (1 M) or a -adrenoceptor antagonist propranolol (1 M). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed the activation of single-channel currents with a conductance of 40 pS during application of adrenaline outside the patch. Parallel measurements of membrane current and fura-2 fluorescence in the same cell demonstrated a correlation between the rise in [Ca²⁺]_i and an increase in K⁺ conductance. Therefore, it is concluded that adrenaline activates a Ca²⁺-dependent K⁺ conductance by release of Ca²⁺ from internal stores through an activation of an ₁-adrenoceptor.     

Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO 3 − /Cl− exchange

We have estimated the changes in cytosolic pH (pH_i) that occur when human platelets are stimulated by thrombin. Changes in pH_i were estimated (i) from the H⁺ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H⁺ efflux that averaged 7.7±1.6 mol/10¹¹ platelets (means±SD) leading to an increase in pH_i, from 7.05±0.04 to 7.45±0.05. Both H⁺ efflux and pH_i changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4-acetamido 4-isothiostilbene-2,2-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO ₃ ^– /Cl^– exchange. Removal of HCO ₃ ^– or Cl^– from the suspending buffer had no effect on the extent of the rise in pH_i. After blockade of Na⁺/H⁺ exchange by 100 M ethylisopropylamiloride (EIPA), thrombin induced a decrease in pH_i the rate of which averaged 0.39 unit/min in HCO ₃ ^– -containing medium, and 0.57 unit/min in HCO ₃ ^– -free medium. The cytosolic buffer capacity for H⁺ was determined by the nigericin/ NH₄Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO ₃ ^– , but only 17.2 mmol/(1xpH) in HCO ₃ ^– -free buffer. The total amount of H⁺ transferred by Na⁺/H⁺ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO ₃ ^– and to 14 mmol/l platelet cytosol in the presence of HCO ₃ ^– , and is in good agreement with the estimated amount of Na⁺ uptake by ADP-stimulated platelets. We conclude that net extrusion of H⁺ from stimulated platelets is predominantly mediated by Na⁺/H⁺ exchange without an apparent contribution of HCO ₃ ^– /Cl^– exchange.     

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells

The present study examines the properties of Cl^–channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl^– channels were larger at positive as compared to negative clamp voltages (V _c): 74±2.6 (V _c > 0) and 47±2.0 pS (V _c < 0) for CF (n= 57) and 69±3.6 (V _c > 0) and 45±2.3 pS (V _c < 0) for N (n=35). The open probability (P _o) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of P _o contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl^–=Br^– =I^– > NO ₃ ^– gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10^–7 mol/l to 10^–5 mol/l. While Cl^– channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl^– channels was 76±3.2 pS for positive clamp voltages (V _c) and 46±3.9 pS for negative V _c (n=8). When the membrane patches were excised from CF cells Cl^– currents appeared spontaneously (n=19). The immediate appearance (within 1 s) of Cl^– channels after excision was observed at positive (n=6) as well as at negative clamp voltage (n=13). Excision activation of CF Cl^– channels was observed at low (< 10^–9 mol/l) or high (10^–3 mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca⁺ activity (< 10^–9–10^–3 mol/l) or pH (6.5–8.5) on the cytosolic side exerted no effects on these Cl^– channels. These results suggest that Cl^– channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic inhibitor leads to an activation of Cl^– channels. The Cl^– channels in excised patches of N and CF cells have identical properties.     

Ca2+- and swelling-induced activation of ion conductances in bronchial epithelial cells

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of –36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl^– conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 mol/l) and ATP (0.1–100 mol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 mol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 mol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ATP > ITP > GTP CTP [,-methylene] ATP 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl^– and K⁺ conductances. The Cl^– conductance was only slightly inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl^– channels, the Ca²⁺-induced Cl^– secretion is due mainly to activation of basolateral K⁺ channels.     

Electrical properties and electrolyte transport in bovine tracheal epithelium: Effects of ion substitutions,transport inhibitors and histamine

The active transport of Na⁺ and Cl^– across the tracheal epithelium of the cow was investigated in vitro, using the short-circuit technique, by means of ion substitutions, transport inhibitors and by measuring²²Na and³⁶Cl fluxes. Under short-circuit conditions, short-circuit current (i _o) was 168±5A cm^–2 (mean±SEM,n=30), i.e. 6.2±0.2 Eq h^–1cm^–2 and resistance (R) was 248±10cm². Net Na⁺ flux toward the submucosa (J _{Na net} ^L-S ) and net Cl^– flux toward the lumen (J _{Cl net} ^S-L ) were of the same magnitude, i.e. 2.7±0.2 and 2.9±0.2 Eq h^–1 cm^–2, respectively. The permeability coefficients were 3.6·10^–6 forP _Na and 7.8·10^–6 cm s^–1 forP _Cl. Under open-circuit conditions, the transepithelial electrical potential difference () was 43±2 mV (lumen negative,n=20).J _{Na net} ^L-S andJ _{Cl net} ^S-L were close to zero.Bilateral substitution of Cl^– with SO ₄ ^2– or isethionate, or administration of furosemide 5·10^–3M or bumetanide 10^–4M in the submucosal bathing medium produced a 40 to 50% decrease ini ₀; furosemide abolishedJ _{Cl net} ^S-L . Bilateral substitution of Na⁺ with choline or Mg²⁺, or addition of ouabain 10^–4M to the submucosal bath abolishedi ₀;J _{Na net} ^L-S andJ _{Cl net} ^S-L were suppressed by ouabain. Amiloride 10^–4M in the luminal bath reducedi ₀ by 23% and diminishedJ _{Na net} ^L-S by 80%. Histamine 10^–4M, added to the submucosal bathing medium, reducedJ _{Na net} ^L-S and increasedJ _{Cl net} ^S-L , under short-circuit conditions. In open-circuit conditions, histamine had little effect on ion fluxes. This substance had no effect on the electrical properties, as shown previously.These results are consistent with the model proposed by Silva et al. [20] for a Cl^–-secreting, Na⁺-reabsorbing epithelium.Supported by the Swiss National Foundation (SNF), grant no. 3.588-0.79     

The volume-activated chloride current in endothelial cells from bovine pulmonary artery is not modulated by phosphorylation

We employed the patch-clamp technique to investigate the effects of various phosphorylation pathways on activation and modulation of volume-activated Cl- currents (I _Cl,vol) in cultured endothelial cells from bovine pulmonary arteries (CPAE cells). Half-maximal activation ofI _Cl,vol occurred at a hypotonicity of 27.5 ± 1.2%. Run-down of the current upon repetitive activation was less than 15% within 60 min. Stimulation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) or by (–)-indolactam did not affectI _Cl,vol. Down regulation of PKC activity by a 24-h preincubation of the cells with 0.2 mol/l PMA, or its inhibition by loading the cells with the specific inhibitory 19–31 pseudosubstrate peptide, did not influenceI _Cl,vol. Trifluoperazine and tamoxifen fully blockedI _cCl,vol with concentrations required for half-maximal inhibition of 3.0 and 2.4 mol/1 respectively. This inhibitory effect is probably not mediated by the calmodulin-antagonistic action of these compounds, because it occurs at free intracellular [Ca²⁺] of 50 nmol/l, which are below the threshold for calmodulin activation. The tyrosine kinase inhibitor herbimycin A (1 ol/1) and genistein (100 ol/1) did not affectI _Cl,vol Exposing CPAE cells to lysophosphatidic acid (1mol/1), an activator of p42 MAPkinase and the focal adhesion kinase p125^FAK in endothelial cells, neither evoked a Cl^– current nor affectedI _Cl,vol Neither wortmannin (10 mol/1), an inhibitor of MAP kinases and of PI-3 kinase, nor rapamycin (0.1 mmol/1), which interferes with the p70S6 kinase pathway, affectedI _Cl,vol Exposure of CPAE cells to heat or Na-arsenite, both activators of a recently discovered stress-activated tyrosine phosphorylation pathway, neither activated a current nor affected the hypotonic solution-induced Cl^– current. We conclude that none of the studied phosphorylation pathways is essential for the activation of the Cl^– current induced by hypotonicity.     

Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na⁺ or to Cl^– ions) recorded Na⁺ ( _Na) or Cl^– ( _Cl) activity and transepithelial potential (V _te). Paired control/low-Na⁺ solutions yielded reabsorptive half-times (t _1/2) of 0.68±0.11 min and 7.6±1.8 min respectively; corresponding V _te values were –22.2±4.0 mV and –7.6±1.9 mV. t _1/2 values of control versus low-Cl^– solutions were 0.77±0.32 min and 6.5±1.7 min respectively, whereas respective V _te values were not different from one another: –23.8±4.3 mV versus –18.8±5.5 mV. Nominally K⁺-free solutions or bumetanide, 10 mol/l, did not alter t _1/2 or V _te, with regard to the paired control. Amiloride, 5 mol/l or 2 mmol/l, failed to decrease t _1/2 or to lower V _te; apparently, the role of a Na⁺/H⁺ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t _1/2 by 54% with regard to the control state. Determination of t _1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 mol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na⁺-Cl^– cotransport.     

The “small” conductance chloride channel in the luminal membrane of the rectal gland of the dogfish (Squalus acanthias)

Besides the larger Cl^– channel with a single channel conductance of about 45 pS, a small channel was observed in the luminal membrane of the dogfish rectal gland [9]. In cell excised (inside out) patches with NaCl solution on both sides, the latter channel had a single channel conductance of 11±1 pS (n=21), and its current-voltage relationship was linear in the voltage range+90 to –90 mV. The open state probability increased moderately with negative clamp potentials. Ionic replacement studies revealed a high selectivity of Cl^– over gluconate, sulfate, and iodide, whereas bromide was permeable to some extent. Also the channel is impermeable for Na⁺. The Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate did not affect this small conductance Cl^– channel. It can be concluded that the luminal membrane of stimulated rectal gland cells possesses two types of Cl^– channels, which differ markedly in their characteristics.Supported by Deutsche Forschungsgemeinschaft Gr 480/8 and by NSF and NIH grants to the MDIBL     

CAMP-dependent activation of ion conductances in bronchial epithelial cells

The cAMP-dependent activation of Cl^– channels was studied in a bronchial epithelial cell line (16HBE14o-) in fast and slow whole-cell, and cell-attached patch-clamp experiments. The cells are known to express high levels of cystic fibrosis transmembrane conductance regulator mRNA and protein. Isoproterenol, forskolin and histamine (all 10 mol/l) reversibly and significantly depolarized the membrane voltage (V _m) and increased the whole-cell Cl^– conductance significantly by 34.0±0.9 (n=3), 18.1±2.7 (n=50), and 25±4.5 (n=37) nS respectively. The effect of histamine was blocked by cimetidine (10 mol, n=5) but not by diphenhydramine (10 mol/l, n=4), which suggests binding of histamine to H₂ receptors. The forskolin-induced current was not inhibited significantly by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/l, n=9) nor glibenclamide (10 mol/l, n=3) and had an anion-permeability sequence of Cl^–= Br^–>I^– (n=9). In cell-attached recordings forskolin (10 mol/l) increased the conductance of the patched membrane from 65.5±13.6 pS to 150.8±33.2 pS (n=30). Although the conductance was increased significantly, clear ion-channel events occurring in parallel with the current activation were not detected in the cell attached membrane. In 4 out of 30 cell-attached recordings single-channel currents were observed. These channels, with a single-channel conductance of about 6 pS, were already active before forskolin was added. No effect of forskolin on the channel amplitude, open probability or kinetics of these channels was observed. From these data we conclude that the cAMP-induced conductance increase in 16HBE14o-cells can be correlated with the activation of very small and not resolvable (probably less than 2 pS) Cl^– channels rather than with the activation of channels with a conductance of 6–10 pS.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

A new double-barrelled,ionophore-based microelectrode for chloride ions

A new Cl^– selective microelectrode based on the ionophore 5,10,15,20-tetraphenyl-21H,23H-porphin manganese(III) chloride is presented which discriminates better against HCO ₃ ^– and several organic anions than electrodes containing the Corning 477913 ion-eschanger. Using a redesigned construction procedure, fine-tip double-barrelled microelectrodes were produced which had slopes of –52.4±0.6 mV (SE,n=24), resistances of about 7·10¹¹ and a selectivity coefficient logK _{Cl, HCO3} ^pot of –1.40±0.03. Some electrodes showed a small unexplained sensitivity to pH>7.6. When used to puncture cells of isolated S3 segments of rabbit renal proximal tubule during perfusion with HCO ₃ ^– Ringer solution, the electrodes gave a membrane potential of –69.8±1.5 mV and an intracellular Cl^– activity, [Cl^–]_i, of 35.3±2.6 mmol/l. Upon switching bath and lumen perfusions to Cl^–-free solutions the residual [Cl^–]_i dropped to 1.20±0.03 mmol/l, while in similar measurements with ion-exchanger electrodes the residual [Cl^–]_i dropped only to 10.9±0.5 mmol/l. These observations demonstrate the superiority of the new electrode and prove that previously determined high [Cl^–]_i values in Cl^–-free ambient solutions reflect interference problems rather than non-exchangeable intracellular chloride.     

β-Adrenoceptor-mediated depolarization of the resting membrane in guinea-pig papillary muscles: changes in intracellular Na+, K+ and Cl− activities

Effects of -adrenergc stimulation on the membrane potential and intracellular Na⁺, K⁺ and Cl^– activities were examined in isolated guinea-pig ventricular muscles using conventional and ion-selective elctrodes. Isoproterenol in concentrations of 30 nM–1 M produced a transient depolarization followed by a slight hyperpolarization in electrically stimulated or quiescent papillary muscles. The negative logarithm of the concentration producing 50% maximum effect (pD₂) for the membrane-depolarizing effect of isoproterenol was smaller than that for the positive inotropic effect, suggesting that a higher level of cAMP accumulation is required to produce the transient depolarization. Whereas the isoproterenol(1 M)-induced depolarization was not blocked by tetrodotoxin (10 M), nifedipine (10 M), Cs⁺ (5 mM), Ba²⁺ (0.3 mM), amiloride (1 mM) or ouabain (10 M), it was significantly attenuated by anthracene-9-carboxylic acid (1 mM), a Cl^–-channel blocker. Intracellular K⁺ activity increased, whereas intracellular Na⁺ activity slightly decreased during the transient depolarization. Intracellular Cl^– activity significantly decreased during the isoproterenol-induced depolarization of the resting membrane. These results suggest that an inward current resulting from outward Cl^– movement, rather than inward Na⁺ movement, may be involved in the -adrenoceptor-mediated membrane depolarization.