A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Regulation of Cl− secretion by AMPK in vivo

Previous in vitro studies suggested that Cl(-) currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the alpha1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport-metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl(-) secretion by AMPK in vivo. Using AMPKalpha1(-/-) mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl(-) secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKalpha1(-/-) and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl(-) conductance in basolaterally permeabilized colonic epithelium from AMPKalpha1(+/+) but not AMPKalpha1(-/-) mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl(-) secretion in epithelial tissues of AMPKalpha1(-/-) mice, but not in wild-type littermates. There was no effect on Ca(2+)-mediated Cl(-) secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl(-) secretion was enhanced in the colon of AMPKalpha1(-/-) mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl(-) secretion mediated by CFTR is controlled by AMPK in vivo.     

Regulation of the Na+2Cl–K+ cotransporter in isolated rat colon crypts

The Na(+2)Cl(-)K+ cotransporter accepts NH4+ at its K+-binding site. Therefore, the rate of cytosolic acidification after NH4+ addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH4+ led to an initial alkalinization, corresponding to NH3 uptake. This was followed by an acidification, corresponding to NH4+ uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (delta fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na(+)2Cl(-)K+ co-transporter could be amplified if the experiments were carried out in the presence of bath Ba2+ to inhibit NH4+ uptake via K+ channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba2+. In the absence of any secretagogue, preincubation of RCC in a low-Cl- solution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70+/-0.11 to 2.54+/-0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93+/-0.17 to 2.84+/-0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 micromol/l carbachol (CCH) led to a small but significant increase in NH4+ uptake rate from 2.06+/-0.34 to 2.40+/-0.30 U/1000 s (n= 11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 micromol/l and 5 micromol/l, respectively) was from 2.39+/-0.24 to 3.06+/-0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH4+ uptake rate, azosemide (500 micromol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na(+)2Cl(-)K+ cotransporter: (1) lowering of cytosolic Cl- concentration; (2) cell shrinkage; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.     

Nucleotide regulation of paracellular Cl− permeability in natural rabbit airway epithelium

In this study, we demonstrate a novel regulatory mechanism by which mucosal nucleotides via P2Y receptors decrease paracellular Cl⁻ ion permeability in natural rabbit airway epithelium (in addition to a decrease in active Na⁺ absorption). In contrast to primary cultures, the natural airway epithelium is a low-resistance epithelium, and an equivalent circuit model predicts that changes of more than ∼12% in transepithelial conductance (G _t) must include an effect on paracellular conductance (G _s). Mucosal P2Y receptor stimulation with uridine triphosphate (UTP; 200 μM) decreased G _t by up to 50% (average, 24%) and simultaneously decreased the paracellular Cl⁻ permeability (mucosa-to-serosa Cl⁻ flux) by 16%, but had no effect on mannitol permeability. The G _t response to UTP was mimicked and attenuated by ionomycin (1 μM), suggesting a dependence on Ca²⁺ _i. Amiloride (100 μM) and hyperosmolarity (+75 mM mannitol) also decreased G _t, indicating a role of cell shrinkage. Elevation of cAMP with forskolin (8 μM) or isoproterenol (10 μM) increased G _t by 55 and 32%, and forskolin increased paracellular Cl⁻ permeability by 37% without affecting mannitol permeability. The opposite effects of Ca²⁺ _i and cAMP on G _t suggest an autocrine nucleotide signaling sequence where P2Y-dependent decrease in passive, paracellular Cl⁻ transport is succeeded by a reversion of this effect due to P1-receptor-stimulated cAMP formation by adenosine originating from a time-dependent breakdown of mucosal ATP.     

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.     

The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl^– channel (ORDIC) during cAMP-mediated Cl^– secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl^– channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4-dinitro-2,2-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl^– secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC₅₀=0.5±0.3 mol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl^– secretion unless high concentrations were used (IC₅₀=240±10 mol/l). In order to exclude effects of NPPB other than on the apical Cl^– channel, trans-epithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl^– gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl^– current showed a very-low-frequency Lorentzian noise component (f _c=1.4±0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC. As revealed from fura-2 fluorescence measurements, forskolin-stimulated Cl^– secretion occurred in the absence of changes in intracellular Ca²⁺. Thus, we conclude that there is an apical Cl^– channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl^– secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl^– channel was calculated as the conductive site in the apical membrane during forskolin stimulation.     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Adipokine expression and secretion by canine adipocytes: stimulation of inflammatory adipokine production by LPS and TNFα

Adiposity and obesity are increasing in dogs. We have examined here the endocrine function of canine adipose tissue and the regulation of production of inflammation-related adipokines by dog adipocytes. Adiponectin, leptin, IL-6, MCP-1 and TNFα genes were expressed in the main adipose depots of dogs, but there were no major depot differences in mRNA levels. Each adipokine was expressed in canine adipocytes differentiated in culture and secreted into the medium (leptin undetected). IL-6, MCP-1 and TNFα were also expressed and secreted by preadipocytes; adiponectin and leptin were only expressed after adipocyte differentiation. The inflammatory mediators LPS and TNFα had major stimulatory effects on the expression and secretion of IL-6, MCP-1 and TNFα; there was a >5,000-fold increase in IL-6 mRNA level with LPS. IL-6 release into the medium was increased >50-fold over 24 h with LPS and TNFα, while MCP-1 release was increased 23- and 40-fold by TNFα and LPS, respectively. However, there was no effect, or small reductions, in adiponectin and leptin mRNA levels with the inflammatory mediators. Dexamethasone-stimulated leptin gene expression, had no effect on adiponectin expression, but decreased the expression and secretion of IL-6 and MCP-1. The PPARγ agonist rosiglitazone stimulated both adiponectin and leptin expression and inhibited the expression of IL-6, MCP-1 and TNFα; MCP-1 secretion was reduced. These results demonstrate that canine adipocytes express and secrete key adipokines and show that adipocytes of this species are highly responsive to inflammatory mediators with the induction of major increases in the production of inflammation-related adipokines.     

The effect of short-chain fatty acids on Cl− and K+ conductance in rat colonic crypts

The effect of butyrate on membrane potential and membrane currents of colonic enterocytes was studied with the whole-cell patch-clamp method. Superfusion of crypts from the rat distal colon with butyrate-containing solutions induced a membrane depolarization of 16.5±2.3 mV. This response was only observed in the upper third of the crypt. The depolarization was dependent on the presence of Cl^– and was accompanied by an increase in membrane inward current, indicating that it is caused by an increase in Cl^– conductance. Membrane outward current, however, behaved inconsistently. Whereas in most cells an increase was observed, about 25% of the cells responded with a decrease. This unexpected inhibition of the outward current probably represents a decrease of K⁺ conductance caused by the cellular acidification in the presence of butyrate. Ma-noeuvres carried out to acidify the cell interior, like perfusion with acid buffer solutions or inhibition of the Na⁺/H⁺ exchanger by amiloride, mimicked this inhibition of the K⁺ conductance. Orientating cell-attached patch-clamp recordings performed in parallel revealed an activation of previously silent basolateral Cl^– channels by butyrate. They had a linear current/voltage relationship and a single-channel conductance of 20–30 pS. The butyrate-induced depolarization was not dependent on intracellular adenosine 5-triphosphate (ATP) and was also observed when the buffer capacity of the pipette for Ca²⁺ was increased. It was also not inhibited by guanosine-5-O(2-thiodiphosphate) (GDP[S]). In the presence of the lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA), the butyrate response was inhibited and even reversed to a slight hyperpolarization indicating that the butyrate-induced Cl^– channels, but not the K⁺ channels, are stimulated by a leukotriene. The effect of butyrate could be mimicked by administration of leukotriene D₄ (LTD₄₁, 5×10^–7 mol · l^–1). The eicosanoid induced a depolarization, which was completely dependent on the presence of Cl^–. Consequently, LTD₄ is a likely candidate for the lipoxygenase metabolite, which activates basolateral Cl^– channels. This activation seems not to be mediated by a protein phosphorylation or a G-protein.     

The cellular mechanisms of Cl– secretion induced by C-type natriuretic peptide (CNP). Experiments on isolated in vitro perfused rectal gland tubules of Squalus acanthias

 We have examined the mechanism whereby C-type natriuretic peptide (CNP), an agonist acting through the second messenger cGMP, enhances NaCl secretion in the rectal gland of Squalus acanthias. Single rectal gland tubules (RGT) were dissected manually, perfused in vitro and equivalent short-circuit current [I _sc=transepithelial voltage/transepithelial resistance (R _te)] as well as basolateral membrane voltage (V _bl) were measured. CNP was added to luminal and basolateral perfusates at concentrations between 1 and 1000 nmol/l and its effects on the above parameters were compared to those of a ”stimulation cocktail” (Stim, containing dibutyryl cAMP, adenosine and forskolin) that maximally enhances cytosolic cAMP, and other agonists and hormones such as guanylin, vasoactive intestinal peptide (VIP), and adenosine. CNP had no effect from the luminal side (n=6). Its effects from the basolateral side consisted of a substantial increase in I _sc (–31.6±7.7 to –316±82.2 μA/cm², n=15). CNP significantly depolarized the luminal membrane from –87.4±1.0 to –82.3±2.6 mV (n=12). V _bl was not changed (n=12) but the fractional conductance for K⁺ was increased (n=3). These effects were qualitatively and even quantitatively comparable to those of other agonists acting via cytosolic cAMP, but were less marked than those caused by Stim (n=64). The effects of VIP and CNP on I _sc were not additive (n=5). The cytosolic Ca²⁺ concentration ([Ca²⁺]_i) was monitored using the fura-2 fluorescence ratio (FFR 340/380 nm) and it was found that CNP, like agonists acting via cAMP, enhances FFR significantly from 1.02±0.05 to 1.32±0.05 (n=8) with a time constant in the 1–2 min in range. Our data suggest that CNP, acting via the second messenger cGMP, induces a marked increase in I _sc in the rectal gland. The concomitant fall in R _te corresponds to increases in the luminal membrane Cl^– conductance and in the basolateral membrane K⁺ conductance. The latter effect is probably due to an increase in [Ca²⁺]_i. Received: 21 December 1998 / Received after revision: 5 February 1999 / Accepted: 9 February 1999     

Increase in intracellular Cl− concentration by cAMP- and Ca2+-dependent stimulation of M1 collecting duct cells

In the lungs of cystic fibrosis (CF) patients, mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) lead to defective Cl^– secretion and hyperabsorption of electrolytes. This may be a an important cause for the defective mucociliary clearance in CF lungs. Previous studies have suggested that inhibition of ENaC during activation of CFTR or by purinergic stimulation could be related to an increase in the intracellular [Cl^–]_i. This was examined in the present study using cultured mouse M1 collecting duct cells transfected with the chloride-sensitive enhanced yellow fluorescent protein YFP_V163S. Calibration experiments showed a linear decrease of YFP fluorescence intensity with increasing [Cl^–]_i (0–100 mM). Activation of CFTR by isobutyl-1-methylxanthine (IBMX, 100 µM) and forskolin (2 µM) increased [Cl^–]_i by 9.6±1.5 mM (n=35). Similarly, ATP (100 µM) increased [Cl^–]_i transiently by 9.5±2.2 mM (n=17). The increase in [Cl^–]_i was reduced by the Na⁺/K⁺/2 Cl^–-cortransporter-1 (NKCC1) blocker azosemide (100 µM), the CFTR blocker SP-303 (50 µM), the blocker of Ca²⁺-activated Cl^– channels DIDS (100 µM) or the ENaC blocker amiloride (10 µM). Changes in YFP_V163S fluorescence were not due to changes in cell volume or intracellular pH. The present data thus demonstrate an increase in [Cl^–]_i following stimulation with secretagogues, which could participate in the inhibition of ENaC.     

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^–.     

Modeling of stimulation–secretion coupling in a chromaffin cell

We constructed a chromaffin cell model for analysis of stimulation–secretion coupling in computer simulation studies. The model includes mechanisms involved in the excitatory synapse, voltage-dependent Na⁺, K⁺ and Ca²⁺ channels, Ca²⁺-activated K⁺ channels (SK type), buffered Ca²⁺ diffusion, Ca²⁺ extrusion, fluorescent Ca²⁺ indicators and Ca²⁺-triggered exocytosis. Calculations of the modeled mechanisms were carried out using the NEURON simulation environment (Hines and Carnevale, Neural Computation 9:1179–1209, 1997). A set of parameter values was determined so as to fit basic experimental results reported in the literature. The model was also applied to simulate our experimental results obtained from chromaffin cells in the perfused rat adrenal medulla. Observed profiles of Ca²⁺responses induced by electrically stimulating the splanchnic nerve with various frequencies (1–50 Hz) were adequately simulated with minor readjustments of parameter values for Ca²⁺influx and extrusion. Secretory responses measured at the same time as the Ca²⁺responses were also simulated with consideration of a time constant to detect catecholamines in the experiment. Similarly, model simulations reproduced both Ca²⁺responses and secretory responses evoked by elevations of the extracellular K⁺ concentration for different periods. The results suggest that the presented model provides a useful tool for analyzing and predicting quantitative relations in various events occurring in stimulation–secretion coupling in chromaffin cells.     

Crypt base cells show forskolin-induced Cl− secretion but no cation inward conductance

Whole-cell patch-clamp studies in base cells of isolated colonic crypts of rats pretreated with dexamethasone were performed to examine the effects of stimulation by forskolin (10 mol/1). The experiments were designed in order to distinguish between two postulated effector mechanisms: the activation of a non-selective cation channel and the activation of Cl^– channels. As shown in an accompanying report, forskolin depolarizes the membrane voltage (V _m) by some 40–50 mV and enhances the whole-cell membrane conductance (G _m) substantially in these cells. In this report all experiments were performed in the presence of forskolin. A reduction of the bath Na⁺ concentration from 145 to 2 mmol/1 led to a hyperpolarization ofV _m by some 20–30 mV This hyperpolarization occurred very slowly suggesting that the hyperpolarization produced by the low-Na⁺ solution was caused indirectly and not by a change in the equilibrium potential for Na⁺,E _Na ⁺. A complete kinetic analysis of the effect on voltage of bath Na⁺ revealed a saturation-type relation with a high apparent affinity for Na⁺ of around 5–10 mmol/1. A reduction in bath Cl^– concentration from 145 to 32 mmol/1 caused a depolarization ofV _m from –34 ± 3 to –20 ± 4 mV (n = 13) in the presence of a high bath Na⁺ concentration, but had the opposite effect at low (5 mmol/1) Na⁺ concentrations:V _m was hyperpolarized from –46 ± 4 to –62 ± 6 mV (n = 13). If the effect of Na⁺ onV _m was caused by a non-selective cation channel the opposite would have been expected. To test directly whether the Na⁺2Cl^–K⁺ cotransporter was responsible for the effects of changes in bath Na⁺ onV _m, the effects of increasing concentrations of several loop diuretics were examined. Furosemide, piretanide, torasemide and burnetanide (up to 0.1–0.5 mmol/1) all hyperpolarizedV _m, albeit only by less than 10 mV. Another subclass of loop diuretics containing a tetrazolate in position 1 [e.g. azosemide, no. 19A and no. 20A from Schlatter E, Greger R, Weidtke C (1983) Pflüger Arch 396: 210–217] were much more effective. Azosemide hyperpolarizedV _m from –46 ± 3 to –74 ± 2 mV (n = 18) and reducedG _m from 11 ± 1 to 4 ± 1 nS (n = 14). These data indicate that forskolin stimulates Cl^– secretion in these cells by a mechanism fully compatible with the current scheme for exocrine secretion involving the Na⁺2Cl^–K⁺ cotransporter.     

Acetazolamide inhibition of basolateral Cl−/HCO 3 − exchange in rabbit renal proximal tubule S3 segment

Cell pH (pH_i) and cell membrane potential (V _b) were measured in isolated S3 segments of rabbit renal proximal tubule with double-barrelled microelectrodes to search for a possible effect of the carbonic anhydrase inhibitor, acetazolamide (ACZ), on Cl^–/HCO ₃ ^– exchange in the basolateral cell membrane. ACZ was found to retard and reduce the pH_i response to bath Cl^– removal reversibly with half-maximal inhibition at 0.42 mmol/l and a rather flat concentration dependence (Hill coefficient 0.36). To determine whether the retardation resulted from inhibition of cytoplasmic carbonic anhydrase, which might have delayed the attainment of HCO ₃ ^– /CO₂ equilibrium, we have measured the response of pH_i to step changes in PCO₂ in the presence and absence of ACZ. ACZ greatly retarded the pH_i response to CO₂ steps; however, the concentration dependence differed (half-maximal inhibition at 18 mol/l) and even at maximal ACZ concentrations the response to CO₂ steps was more than twice as fast as the response to Cl^– replacement. Since, in addition, the ACZ inhibition of Cl^–/HCO ₃ ^– exchange could not be overcome by increasing PCO₂ we conclude that the ACZ effect on Cl^–/HCO ₃ ^– exchange in rabbit proximal tubule S3 segments does not result from inhibition of cytosolic or membrane-bound carbonic anhydrase, but from a direct interaction with the exchanger molecule.     

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.     

Requirement of HCO 3 − for Cl−-absorption in seawater-adapted eel intestine

The role of HCO ₃ ^– /CO₂ buffer in Cl^– absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl^– absorption, expressed as short/circuit current (I _sc), was measured in either 20 mM HCO ₃ ^– /1% CO₂ Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO ₃ ^– /CO₂ with HEPES/O₂ was without effect on I _sc and transepithelial voltage (V _t), whereas bilateral removal of HCO ₃ ^– /CO₂ reduced I _sc and V _t by 50%, indicating that the presence of HCO ₃ ^– /CO₂ buffer at one side of the epithelium is sufficient to keep Cl^– absorption at the maximum rate. We examined in further detail the individual components of the HCO ₃ ^– /CO₂ system that stimulates Cl^– absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO₂ to the luminal or serosal solution (final pH=7.6 in the chamber) had no effect on I _sc and V _t, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO ₃ ^– /1% CO₂ Ringer or 20 mM HCO ₃ ^– alone. Stimulation of I _sc induced by unilateral (luminal or serosal) HCO ₃ ^– /CO₂ was inhibited by luminal or serosal 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid (SITS) (0,25 mM) or by serosal Na⁺ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of I _sc induced by luminal addition of HCO ₃ ^– /CO₂, whereas it was without effect when HCO ₃ ^– /CO₂ was added serosally or bilaterally. We reached the following conclusions, (a) Cl^– absorption is stimulated by HCO ₃ ^– /CO₂ buffer via an increase in intracellular HCO ₃ ^– concentration and/or pH_i changes consequent to the HCO ₃ ^– uptake mediated by HCO ₃ ^– transport systems operating on both cell membranes, (b) A Na⁺-dependent SITS-inhibitable HCO ₃ ^– transport mechanism operates at the basolateral membrane, (c) The transfer of HCO ₃ ^– through the luminal membrane is mediated by the carbonic anhydrase enzyme located on the brush-border membranes of the enterocyte: the movement of HCO ₃ ^– , via a SITS-sensitive transport system, occurs most likely in form of OH^–, which originates from the dehydration reaction of HCO ₃ ^– catalysed by the carbonic anhydrase. (d) There is no apparent amiloride-sensitive Na⁺/H⁺ antiporter on either cell membrane.This work was supported by a research grant from Ministero dell'Università e della Ricerca Scientifica e Tecnologica — Progetto di interesse nazionale e di rilevante interesse per lo sviluppo della Scienza     

Blockers of volume-activated Cl− currents inhibit endothelial cell proliferation

Volume-activated Cl^– currents (I_Cl,vol) and cell growth have been measured in cultured endothelial cells from bovine pulmonary artery (CPAE) in the absence and presence of compounds which block these currents. The anti-oestrogen drug tamoxifen, which efficiently arrests the growth of breast cancer cells (l), inhibits both I_Cl,vol and cell proliferation with IC₅₀ of 3.8 and 4.8 mol/l respectively. NPPB and quinine, which also block I_Cl,vol, inhibit the growth of CPAE cells as well. Current and cell growth were closely correlated under all these conditions. We conclude that I_Cl,vol might be involved in the control of endothelial cell growth and thus might be important for the modulation of vascularisation and vascular remodelling.     

Distribution of cGMP-dependent and cGMP-independent Ca2+-activated Cl− conductances in smooth muscle cells from different vascular beds and colon

In the present patch-clamp study we have, for the first time, shown the tissue distribution of a recently characterized cGMP-dependent Ca²⁺-activated Cl^– conductance [18] in smooth muscle cells freshly isolated from different regions: aorta, pulmonary artery, tail artery, femoral artery, femoral vein, middle cerebral artery, renal artery, portal vein, superior mesenteric artery, mesenteric small artery and colon. The cGMP-dependent Cl^– conductance has properties distinct from those of the classical Ca²⁺-activated Cl^– conductances; their different sensitivities to niflumic acid and zinc were here utilized to distinguish them. They were found to be co-expressed in different patterns in smooth muscle cells of different origins. The cGMP-dependent conductance was greater in myocytes from cerebral artery and femoral vein and was greater in the renal artery, aorta, mesenteric small artery, femoral artery and the superior mesenteric artery. The presence of the cGMP-dependent Ca²⁺-activated Cl^– current in smooth muscle cells isolated from the colon demonstrates that this conductance is not limited to the vasculature. The classical Ca²⁺-activated Cl^– conductance was strongly expressed in smooth muscle cells from the portal vein and the tail artery, and noticeably higher in the pulmonary artery.