The cystic fibrosis mutation (delta F508) does not influence the chloride channel activity of CFTR.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-regulated Cl- channel. In most mammalian cells, the functional consequences of the most common CF mutation, delta F508-CFTR, cannot be assessed as the mutant protein undergoes biosynthetic arrest. However, function can be studied in the baculovirus-insect cell expression system where delta F508-CFTR does not appear to undergo such arrest. Our results show that phosphorylation-regulated Cl- channel activity of delta F508-CFTR is similar to that of wild-type CFTR. This observation was confirmed in comparative studies of purified delta F508-CFTR and CFTR reconstituted in planar lipid bilayers. Therefore, we suggest that this common mutation does not result in a significant alteration in CFTR function.

Lung disease in mice with cystic fibrosis.

The leading cause of mortality and morbidity in humans with cystic fibrosis is lung disease. Advances in our understanding of the pathogenesis of the lung disease of cystic fibrosis, as well as development of innovative therapeutic interventions, have been compromised by the lack of a natural animal model. The utility of the CFTR-knockout mouse in studying the pathogenesis of cystic fibrosis has been limited because of their failure, despite the presence of severe intestinal disease, to develop lung disease. Herein, we describe the phenotype of an inbred congenic strain of CFTR-knockout mouse that develops spontaneous and progressive lung disease of early onset. The major features of the lung disease include failure of effective mucociliary transport, postbronchiolar over inflation of alveoli and parenchymal interstitial thickening, with evidence of fibrosis and inflammatory cell recruitment. We speculate that the basis for development of lung disease in the congenic CFTR-knockout mice is their observed lack of a non-CFTR chloride channel normally found in CFTR-knockout mice of mixed genetic background.

A K(+)-selective channel in the colonic carcinoma cell line: CaCo-2 is activated with membrane stretch.

CaCo-2 is a human colonic carcinoma cell line which becomes differentiated in culture to form a polarized epithelium exhibiting several of the functional characteristics of native colonic tissue. In the present study, CaCo-2 cells have been used for a patch-clamp study of colonic ion conductance pathways. A large, 120 pS K(+)-selective channel was found in cells forming subconfluent monolayers in culture. Unlike Maxi-K+ channels found in other epithelial cells, this channel was not activated with elevations in cytosolic Ca2+. Channel activity was stimulated with membrane depolarization and most markedly with membrane stretch. The application of negative pressure (20 mm-Hg) to both cell-attached and excised, inside-out membrane patches caused a burst of channel activity which disappeared within seconds of suction removal. Single-channel conductance of the pressure-activated channel was decreased when quinine (100 microM) was present in the patch pipette.

Intracellular pH influences the resting membrane potential of isolated rat hepatocytes.

This study in isolated rat hepatocytes sought to determine if the changes in membrane potential associated with intracellular alkalinization or acidification could be attributed to changes in K+ conductance. Intracellular pHi was manipulated using the 'NH4+-pulse' method: inducing intracellular alkalinization with NH4Cl (10 mM), and producing acidification by diluting the NH4+-loaded cells with ammonium ion-free buffer or by adding sodium proprionate. Membrane potential and resistance were measured in freshly isolated rat liver cells using intracellular microelectrodes. The results indicated that intracellular alkalinization was associated with hyperpolarization and decreased membrane resistance, whereas intracellular acidification caused depolarization with increased membrane resistance. As pHi-mediated electrogenic responses have been related to changes in K+ conductance in other epithelial tissues, the influence of K+ transport inhibitors on NH4+-evoked electrical effects was examined. NH4Cl-evoked membrane potential changes were inhibited by the K+ channel blockers, quinine and barium and in potassium depolarized cells (cells bathed in a high K+ medium where [K+]in = [K+]out = 140 mM). Furthermore, Rubidium-86 (86Rb+) efflux from preloaded hepatocytes, a measure of K+ permeability, was enhanced following intracellular alkalinization but inhibited by intracellular acidification. Thus, these results indicate that pHi-evoked electrogenic effects in hepatocytes are mediated through changes in K+ conductance.

Sodium-dependent taurocholate uptake by isolated rat hepatocytes occurs through an electrogenic mechanism.

The uptake mechanism for the bile salt, taurocholate, by the liver cell is coupled to sodium but the stoichiometry is controversial. A one-to-one coupling ratio would result in electroneutral transport, whereas cotransport of more than one sodium ion with each taurocholate molecule cause an electrogenic response. To better define the uptake of this bile salt, we measured the effect of taurocholate on the membrane potential and resistance of isolated rat hepatocytes using conventional microelectrode electrophysiology. The addition of 20 microM taurocholate caused transient but significant depolarization accompanied by a significant decrease in membrane resistance. The electrical effect induced by taurocholate mimicked that induced by L-alanine (10 mM), the uptake of which is known to occur through an electrogenic, sodium-coupled mechanism. The sodium dependence of taurocholate-induced depolarization was further confirmed by: (1) replacing Na+ with choline +, and (2) preincubating cells with ouabain (2 mM) or with the Na+-ionophore, gramicidin (25 micrograms/ml); both suppressed the electrogenic response. Further, cholic acid, which inhibits sodium-coupled taurocholate uptake in hepatocytes, inhibited taurocholate evoked depolarization. These results support the hypothesis that sodium-coupled taurocholate uptake by isolated hepatocytes occurs through an electrogenic process which transports more than one Na+ with each taurocholate molecule.

Assessment of the efficacy of in vivo CFTR protein replacement therapy in CF mice.

Cystic Fibrosis (CF) is caused by mutations in the CF gene that lead, for the most part, to mislocalization of the protein product, the cystic fibrosis transmembrane conductance regulatory (CFTR). CFTR is a chloride channel normally situated in the apical membrane of epithelial cells where it contributes to transepithelial ion transport. In this study we demonstrated the feasibility of in vivo transfer of purified CFTR protein via phospholipid liposomes into the apical membrane of nasal epithelia of CFTR knockout mice. Membrane incorporation of immunogold-labeled CFTR could be visualized by electron microscopy and correction of CF-related defects in ion transport measured by nasal potential difference (PD) measurements in about one-third of the animals treated. Although these initial results are promising, effectiveness of this therapeutic approach appears to be limited by the inefficient incorporation of CFTR into the apical epithelial cell membrane.

Phosphorylation-activated chloride channels in human skin fibroblasts.

A chloride-selective channel has been found using patch-clamp electrophysiology in human skin fibroblasts and it exhibits many of the biophysical properties of the Cl- channel found in airway epithelia. As in the case of epithelial Cl- channels, Cl- channels in fibroblasts are activated at depolarized membrane potentials in excised patches, rectifying in an outward direction with a unit conductance of 33 pS at 0 mV. Furthermore, the agonists forskolin and prostaglandin E2 evoke Cl- channel activity in cell-attached patches. The effect of these agonists can be mimicked by direct application of catalytic subunit of protein kinase A with ATP and Mg2+ to the internal membrane surface of excised, inside-out patches. The Cl- channel is also sensitive to inhibition by the stilbene derivative, DIDS. These results indicate that fibroblasts may provide a convenient and available model for the study of epithelial Cl- channel regulation and accelerate efforts to determine the regulatory defect expressed in cystic fibrosis.

Disruption of ClC-2 expression is associated with progressive neurodegeneration in aging mice.

Heterozygous mutations in ClC-2 have been associated in rare cases with increased susceptibility to generalized, idiopathic epilepsy. Initially, it was hypothesized that mutations in ClC-2 may be associated with epilepsy due to a direct role for ClC-2 in the modification of hippocampal neuronal excitability. However, the absence of an overt seizure-susceptibility phenotype in young ClC-2 knockout (KO) mice rendered this hypothesis- implausible. A recent study of older ClC-2 KO mice (>6 months) revealed abnormalities in the myelin of central axons and a subtle defect in the neuronal function in the central auditory pathway. These findings prompted us to re-examine hippocampal neuron morphology and excitability in older ClC-2 KO mice. Interestingly, electrocorticographic recordings obtained in older mice revealed spontaneous interictal spikes which are a marker of perturbed hippocampal neurotransmission with a resultant increase in excitation. This electrophysiological defect was associated with astrocyte activation and evidence of neuronal degeneration in the CA3 region of the hippocampus of these older mice. Together, these findings raise the possibility that ClC-2 expression plays a subtle neuroprotective role in the aging hippocampus.

Drugs transported by P-glycoprotein inhibit a 40 pS outwardly rectifying chloride channel.

P-glycoprotein functions as an ATP-dependent pump for a diverse spectrum of compounds. Recently, it has been shown that P-glycoprotein may be bi-functional and act as a chloride channel as well as a pump. The single channel properties of this conductance are unknown, however, as macroscopic, whole cell currents are inhibited by substrates for P-glycoprotein transport, the single channels underlying this response should also be blocked by these compounds. We found that colchicine, vinblastine, daunomycin and verapamil (50 microM) caused block of a 40 pS outwardly-rectifying chloride channel in cells expressing P-glycoprotein. The inhibitory effect of these compounds appeared specific for the 40 pS chloride channel as a large, 300 pS chloride channel found in the same cells was unaffected by addition of drug. These results suggest that the 40 pS chloride channel may be associated with P-glycoprotein expression.

A nonselective cation channel in rat liver cells is activated by membrane stretch.

A 16-pS channel was studied using patch-clamp electrophysiology in freshly dissociated rat liver cells and rat hepatoma cells. The channel was found to be cation selective and permeable to Na+, K+, and Ca2+. Its gating was unaffected by addition of the calcium ionophore A23187 (5 microM) in the presence of extracellular Ca2+ (2 mM). Ca2+ channel blockers, nifedipine, verapamil, and lanthanum, failed to inhibit the channel. The channel was activated by stretch, applied as suction to the interior of the patch pipette, and by cell swelling, induced by hypotonic shock or organic solute uptake (10 mM L-alanine). Channel activation by cell swelling was transient, lasting approximately 1 min. An elevation in cytosolic Ca2+ was evoked by hypotonic shock, as measured using the fluorescent indicator indo-1/AM. This change in intracellular Ca2+ concentration was dependent on extracellular Ca2+. Inasmuch as the time course for this response corresponded to that of channel activation, it is likely that hypotonic shock stimulated Ca2+ influx through the stretch-activated channel. To determine the role for Ca2+ influx in regulatory volume decrease (RVD), cell volume changes after hypotonic shock were studied using a Coulter counter. RVD was slightly but significantly inhibited by depletion of extracellular Ca2+. On the basis of these results it is proposed that stretch-activated channels in liver cells permit the transient influx of Ca2+, which in turn acts to trigger changes in ion conductance or cytoskeletal components involved in cell volume regulation.

Hepatocellular water and electrolyte secretion.

The study of hepatocellular water and electrolyte secretion has been hampered because of the inaccessibility of the hepatobiliary secretory lumen, the canaliculus. The advent of novel experimental models has allowed the application of electrophysiological techniques to investigate the ionic basis of hepatocellular secretion. The "hepatocyte couplet" isolated from the liver in primary monolayer cultures consists of two hepatocytes which enclose a single canalicular unit. The canaliculus of the couplet appears to behave as it would in vivo, exhibiting both secretory and contractile activity. Intracellular microelectrode recordings from this functional unit have permitted direct electrophysiological assessment of cellular and canalicular potentials and measurement of individual ion conductances across the basolateral membrane surface. Further, the application of patch-clamp electrophysiology to study hepatocellular ion transport pathways has characterized individual channel proteins. Intracellular and (or) patch-clamp electrophysiology has elucidated the ion conductance changes activated by bile salts like taurocholate, neurotransmitters like adrenaline, and hormones such as glucagon. These innovative approaches hold much promise in the future study of the ionic basis of hepatocellular secretion.

L-alanine evokes opening of single Ca2+-activated K+ channels in rat liver cells.

Cellular uptake of neutral amino acids via Na+ cotransporters is known to be associated with an increased membrane K+ conductance mediated by an unknown mechanism that is essential for avoiding excessive cell swelling. We now demonstrate by patch-clamp single-channel current recording that exposure of rat liver cells to L-alanine, but not the poorly transported D-stereoisomer, evokes opening of single K+ channels and that this effect is reversible upon removal of the amino acid. The nature of the conductance pathways opened in the intact cell by L-alanine has been investigated in cell-free excised membrane patches where it can be shown that the K+-selective channels are opened by Ca2+ acting from the inside of the membrane at a concentration as low as 0.1 microM.

cAMP-activated chloride conductance in the colonic cell line, Caco-2.

In this study we investigated the properties of adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated Cl- efflux in Caco-2 monolayers by measuring 125I efflux rates from preloaded cells and using patch-clamp electrophysiology. The addition of a cocktail containing 100 microM dibutyryl cAMP (DBcAMP), 10 microM forskolin, and 1 mM 3-isobutyl-1-methylxanthine caused a significant (P less than 0.05) increase in the rate of 125I efflux. Dissipation of cell potential by adding valinomycin (4.5 microM) with 135 mM extracellular KCl reduced the cAMP-evoked 125I efflux. These results suggest that cAMP-stimulated anion efflux occurs through a conductive pore or channel. Whole cell currents evoked with DBcAMP or forskolin were anion selective, PCl greater than PI greater than Pgluconate, and exhibited a linear current-voltage (I-V) relationship. Currents evoked with depolarizing or hyperpolarizing voltage steps showed no evidence of time-dependent activation or inactivation. Single Cl- channels were stimulated in cell-attached patches after treatment with cAMP. Onset of channel activity occurred after 20-30s of cAMP treatment, and the response was long lasting. The I-V relationship for the channel activated in cell-attached patches by cAMP was best fit using two linear regressions. The slope conductance of the channel was 3.2 +/- 0.6 and 7.4 +/- 0.3 pS at hyperpolarizing and depolarizing potentials, respectively. Substitution of 140 mM NaCl with 70 mM NaCl in the patch pipette resulted in a positive shift in reversal potential, indicating that the channel is anion selective.(ABSTRACT TRUNCATED AT 250 WORDS)

Techniques for studying biliary secretion: electrolytes in bile.

A major limitation in understanding bile formation has been technical. The liver and ductular epithelium are relatively inaccessible, necessitating indirect techniques of uncertain validity. This is well seen in attempts to define the role of electrolyte secretion in bile. It is widely agreed that bile salts stimulate a component of canalicular flow and that inorganic electrolyte secretion is stimulated by bile salts. The choleretic efficiency of a bile salt is directly related to the magnitude of the electrolyte effect. But there is no consensus regarding how and where bile salts stimulate electrolyte secretion. Some evidence points to a paracellular route by processes of solvent drag and diffusion. Other studies suggest stimulation of specific transcellular electrolyte pathways. It has been believed that canalicular bile salt-independent bile flow is generated by active blood-to-bile electrolyte transport. Actually, available methods do not permit us to conclude with absolute certainty that there is canalicular bile salt-independent flow, although there is considerable evidence for it. New studies suggest that electrolyte transport in this type of flow is passive and that flow is due to transport of organic anions. Ductular flow does seem to be due to active transport of electrolytes, particularly bicarbonate. Better and more direct techniques are required to settle the controversies in this area.

Calcium-permeable channels in rat hepatoma cells are activated by extracellular nucleotides.

Extracellular ATP is known to cause uptake of Ca2+ by rat liver cells. The specific pathway permitting influx of Ca2+ has not yet been identified. In the present investigations, we studied the properties of ATP-evoked 45Ca2+ uptake in rat hepatoma cell monolayers and then used patch-clamp electrophysiology to identify the channel that may account for this uptake. The results suggest that ATP-stimulated 45Ca2+ uptake occurs as a result of P2-purinergic receptor interaction because uptake was inhibited by Reactive Blue (100 microM), a blocker of this type of receptor. Furthermore, the ability of other adenine nucleotides to stimulate 45Ca2+ uptake was related to the selectivity sequence for binding to the P2-purinergic receptor. ATP-stimulated 45Ca2+ uptake occurs primarily through a conductance pore since it was inhibited by 70% upon dissipation of the membrane potential using the K+ ionophore valinomycin. The calcium channel blockers nifedipine and verapamil failed to inhibit 45Ca2+ uptake, but gadolinium (GdCl3) was an effective blocker. In cell-attached patch-clamp experiments, a single type of channel was activated with ATP (100 microM) addition to the bath in 18 of 32 trials. The current-voltage relationship of the ATP-activated channel is identical to that of the stretch-activated channel previously characterized in this laboratory as a calcium-permeable cation-nonselective channel [Am. J. Physiol. 258 (Cell Physiol. 27): C421-C428, 1990]. There are several lines of evidence which suggest that this cation-nonselective channel may account for ATP-stimulated 45Ca2+ uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium-independent, bicuculline-sensitive [3H]GABA binding to isolated rat hepatocytes.

To determine whether hepatocytes possess specific receptor sites for gamma-aminobutyric acid (GABA), a potent amino acid neurotransmitter, [3H]GABA, was added to sodium-free suspensions of Percoll-purified hepatocytes derived from collagenase-perfused rat livers under various experimental conditions and in the presence or absence of specific GABA receptor agonists (muscimol) and antagonists (bicuculline). The effects of GABA, muscimol, and bicuculline on hepatocyte resting membrane potentials were also determined. Specific binding of [3H]GABA to hepatocytes was a consistent finding. GABA-hepatocyte interactions were reversible and temperature dependent. Muscimol and bicuculline inhibited binding in a dose-dependent manner (IC50, 30 nM and 50 microM, respectively), whereas strychnine (1.0-100 microM), a nonspecific central nervous system stimulant, had no appreciable effect. Both GABA and muscimol (100 microM) caused significant hyperpolarization of hepatocyte resting membrane potential (delta PD 5.4 +/- 3.1 and 22.2 +/- 16.2 mV, respectively, means +/- SD, P less than 0.0005). Bicuculline (100 microM) inhibited the effect of muscimol (P less than 0.05). The results of this study suggest that specific GABA receptor sites exist on the surface of isolated rat hepatocytes. The presence of such sites raises the possibility that, in addition to adrenergic and cholinergic innervation, hepatic function may be influenced by GABA-ergic neurotransmitter mechanisms.

Effect of sodium taurocholate on amiloride-inhibitable sodium uptake and on intracellular pH of isolated rat hepatocytes.

Evidence is accumulating that bile salts affect cellular mechanisms for the transport of inorganic electrolytes. We investigated the effect of amiloride on 22Na+ uptake rates in the presence and absence of sodium taurocholate (NaTC) and the effect of NaTC on intracellular pH (pHi) in isolated rat hepatocytes. Initial 22Na+ uptake rates (nanomoles per milligram of protein per minute) were significantly suppressed by amiloride (0.6 mmol/L), although the effect was small. NaTC significantly increased 22Na+ uptake rates. The amiloride-sensitive portion of 22Na+ uptake was significantly increased in the presence of NaTC (0.36 +/- 0.07 SEM nmol/mg protein/min vs. 0.20 +/- 0.07 nmol/mg protein/min, P less than 0.02). The pHi was estimated by using the pH probes carbon 14-labeled 5,5'-dimethyloxazolidinedione and acridine orange. NaTC caused intracellular alkalinization. Amiloride caused intracellular acidification, and the reduction of pHi by amiloride was enhanced in the presence of NaTC, although this enhancement is difficult to interpret because of the large effects of amiloride on pHi relative to those of NaTC. Our results indicate that NaTC affects sodium uptake by isolated hepatocytes probably by stimulating the Na+-H+ antiport.

Bile salt dependent bile flow in the rabbit: evidence for the importance of an amiloride inhibitable pathway.

Bile salt dependent flow and electrolyte secretion in response to two bile salts were studied in awake rabbits. It was found that sodium glycodeoxycholate had a much greater choleretic and cholioneretic efficiency than sodium taurocholate. The effect of the bile salts on flow and electrolyte secretion was not linear across the range of bile salt secretion rates studied. When amiloride was administered significant decreases in choleretic and cholioneretic efficiencies occurred, but furosemide had no effect. It is concluded that bile salts stimulate electrolyte transport via amiloride inhibitable cellular processes, and that this electrolyte transport is in part responsible for bile salt dependent bile flow.