Changing expression of chloride channels during preimplantation mouse development.

Plasma membrane chloride channels (ClCs) play important roles in a broad range of cellular processes including cell volume regulation, proliferation, and transepithelial transport, all of which are critical during preimplantation embryonic development. In this study, the molecular and functional expression of voltage-gated ClCs was analyzed throughout preimplantation development of the mouse conceptus. mRNA transcripts for all Clcn genes were detected. Only Clcn1 mRNA showed differential expression in the blastocyst, being detected in the trophectoderm but not in the inner cell mass. CLCN3 protein was detected at low levels in the cytoplasm and plasma membrane in 4-cell embryos and was localized to the apical plasma membrane of the trophoblasts in the blastocyst. Whole-cell patch-clamp recordings demonstrated the presence of a DIDS-sensitive, outwardly rectifying Cl(-) current throughout development, with this conductance being large at the 1-cell, morula and blastocyst stages. A second DIDS-insensitive Cl(-) current, which was inactivated by membrane depolarization, was present in cells differentiating into the trophoblast lineage and during blastocyst expansion. Inhibition of the DIDS-sensitive current and the DIDS-insensitive current, with 9-AC, prevented blastocyst expansion.

Nuclear hormone receptor-dependent regulation of hepatic transporters and their role in the adaptive response in cholestasis.

1. Hepatobiliary transport systems are essential for the uptake and excretion of a variety of organic anions including bile acids and bilirubin. Perturbation of this vital liver function can result in pathological conditions such as cholestasis, where the formation of bile at the canaliculus is impaired resulting in the intrahepatic accumulation of toxic bile constituents. 2. Members of the nuclear hormone receptor superfamily are important mediators of the adaptive response during cholestasis controlling the expression of transporters and other proteins with the aim to limit tissue damage. Bile acids are the endogenous ligands for these nuclear hormone receptors and therefore directly participate in the control of their own transport and metabolism. 3. Adaptive events include repression of bile acid uptake and de novo bile acid synthesis as well as a concomitant induction of alternative efflux routes and bile acid detoxification. Importantly, the adaptation also extends to other organs such as intestine and kidney to facilitate elimination of bile acids from the body. 4. This review provides an overview of the transcriptional regulation of bile acid transporting proteins and metabolizing enzymes mediated by nuclear hormone receptors. Furthermore, the complex networks between nuclear hormone receptors and regulated genes are illustrated and implications of targeting these receptors for the treatment of cholestasis are discussed.

Structure-function relations of biotin derivatives of apamin.

Apamin was biotinylated at various residues so as to produce an apamin derivative that was suitable for labelling apamin sensitive K+ channels. We labelled the sole histidine residue (His-18) of apamin with diazobenzoyl biocytin (DBB) and Lys-4 with NHS-biotin and NHS-XX-biotin. We found that at least two labelled species were produced by DBB. Proton NMR spectroscopy revealed that in addition to labelling the His-18, DBB labelled the Gln-16 and Gln-17 of apamin. Both NHS-biotin and NHS-XX-biotin appeared to specifically label Lys-4. To test the potency of these apamin derivatives, we developed an assay using apamin reversal of the adrenaline induced relaxation of the mouse ascending colon. The biological activity of the His-18 derivative was 46-fold less than that of native apamin. Biotinylation of Lys-4 with NHS-biotin reduced the activity by only 6-fold. The inclusion of a 14-carbon spacer between the Lys-4 and the biotin resulted in a derivative with only a 4-fold reduction in potency.

Proton nuclear magnetic resonance spectroscopy of isolated rabbit fundic glands.

1H-nuclear magnetic resonance spectroscopy (NMR) was adapted to isolated rabbit fundic glands and identification made of compounds responsible for several observed spectral resonances. A minimum gland concentration of 0.5 mg dry weight or 5 mg wet weight per 0.5 ml was needed for adequate signal-to-noise ratio. At physiological temperature and pH, the glands demonstrated reproducible spectra, stability for accumulation times greater than 30 min and responsiveness to histamine stimulation, as measured by oxygen consumption and aminopyrine uptake. The relatively anaerobic conditions favored use of proton compared to phosphorus NMR, since 1H-NMR allowed significantly shorter spectral accumulation times and therefore did not compromise glandular viability to the same extent as 31P-NMR. The most conspicuous resonance in the gland spectrum was assigned to the -N+(CH3)3 protons of choline and related compounds. In membrane-free lysates, several components of the signal were resolvable and assigned to choline, phosphatidylcholine, phosphocholine and L-alpha-glycerophosphocholine. Thin-layer chromatography verified that phosphatidylcholine and phosphatidylethanolamine were the major phospholipids present in gland lipid. Presumably, they represent the source of the surface-active phospholipids present in gastric juice, which may play a role in gastric cytoprotection.

Insulin secretagogues, but not glucose, stimulate an increase in [Ca2+]i in the fetal rat beta-cell.

Fetal pancreatic islets release insulin poorly in response to glucose; however, the cellular mechanism for this is controversial. By using fura 2 to measure changes in the cytoplasmic free Ca2+ concentration ([Ca2+]i) in beta-cells, we have examined islets from fetal, neonatal, and adult rats to determine the ability of glucose and other secretagogues to cause an increase in [Ca2+]i. The effects of glucose (20 mmol/l), glyceraldehyde (20 mmol/l), leucine (20 mmol/l), arginine (20 mmol/l), and the channel effectors glipizide (50 mumol/l), BAY K8644 (2 mumol/l), diazoxide (300 mumol/l), and verapamil (20 mumol/l) on changes in [Ca2+]i were studied. In both the fetal and the mature islet, glyceraldehyde, leucine, arginine, glipizide, and BAY K8644 caused an increase in [Ca2+]i. In mature islets, glucose also increased [Ca2+]i; however, in the fetal islet, glucose had no effect on [Ca2+]i. The stimulus-induced increases in [Ca2+]i in fetal and adult islets were both significantly inhibited by the addition of either diazoxide or verapamil. Similar results were obtained when insulin secretion was measured. Our data show that various secretagogues are able to stimulate fetal islets and cause an increase in [Ca2+]i. Glucose, however, fails to cause an increase in [Ca2+]i in the fetal islet. Hence, the immature insulin secretory response to glucose by the fetal islet is due to the inability of the fetal beta-cell to translate glucose stimulation into the increase in [Ca2+]i required for exocytosis of the insulin granule.

Diphenylamine-2-carboxylate (DPC) reduces calcium influx in a mouse mandibular cell line (ST885).

Non-selective cation channels are found in many diverse cell types and have been proposed as a potential entry path for Ca2+. ST885 cells contain large numbers of these channels which are active in the resting cell. We have used Fura-2 to monitor changes in intracellular free Ca2+ ([Ca2+]i) in response to step changes in extracellular Ca2+ ([Ca2+]o). We found that DPC, a blocker of the non-selective cation channel in these cells, caused a reduction of approximately 50% in the rate of rise in [Ca2+]i following a step increase in [Ca2+]o. Since our experiments demonstrate that this phenomenon is not due to DPC blockade of Cl- channels, the Na+/Ca2+ exchanger or cyclooxygenase, we conclude that it is attributable to a direct effect of DPC on the non-selective cation channel. It thus appears that the non-selective cation channel is a significant pathway for basal Ca2+ entry in these cells.

Control of intracellular Ca2+ by adrenergic and muscarinic agonists in mouse mandibular ducts and end-pieces.

The changes in free Ca2+ ([Ca2+]i) in the cells of the secretory end-pieces and intralobular ducts of mouse mandibular glands exposed to adrenergic or cholinergic agonists were measured using fluorescence imaging techniques. [Ca2+]i in both cell types increased in a dose-dependent manner during both adrenergic and cholinergic stimulation. The duct cells responded to noradrenaline and to acetylcholine over the same concentration range (30 nmol/l to 3 mumol/l) although the maximum increase in [Ca2+]i above resting levels evoked by noradrenaline (ca. 137 nmol/l) was about twice that evoked by acetylcholine. The response to acetylcholine was blocked by atropine (0.1 mumol/l) and the response to noradrenaline was blocked by the alpha 1-adrenergic antagonist, prazosin (0.1 mumol/l), but not by the alpha 2-adrenergic antagonist, yohimbine. The alpha-adrenergic agonist, phenylephrine, mimicked the action of noradrenaline but the beta-adrenergic agonist, isoproterenol, had no effect. In contrast to the duct cells, the end-piece cells responded to acetylcholine at much lower concentrations (threshold << 1 nmol/l) than to noradrenaline (threshold ca. 300 nmol/l) and the size of the increase in [Ca2+]i above resting levels evoked by acetylcholine (216 nmol/l) was nearly 5-times greater than for noradrenaline. VIP and substance P failed to evoked a Ca2+ response in either end-piece or duct cells.

An inwardly rectifying K+ channel in human adenomatous parathyroid cells.

The ion channel populations of cells prepared from human adenomatous parathyroid glands have been investigated using standard patch-clamp techniques with pipettes containing isotonic K(+)-rich solutions. The principal channel type observed was an inwardly rectifying K+ channel of 35 pS conductance. In addition, we have frequently observed a lower conductance (12 pS) K+ channel that appeared to conduct current in both directions. In addition to these K+ channels, we have observed non-selective cation channels and possibly CI- channels. Although we have observed occasional current transitions that might have arisen from a large conductance K+ channel similar to that observed in rat and bovine parathyroid cells, we have not been able positively to identify such a channel in human parathyroid cells, either in cell-attached or in excised inside-out patches.

Purinergic responses in HT29 colonic epithelial cells are mediated by G protein alpha -subunits.

Using Fura-2 to measure changes in intracellular calcium ([Ca(2+)](i)), we show that P(2U)receptors in HT29 cells trigger an increase in [Ca(2+)](i)by pertussis toxin-insensitive G proteins. We then use replication-deficient adenoviruses expressing wild-type and dominant negative mutants of G(alpha q)and G(alpha i2), antisense directed against G(alpha q)or the C-terminal fragment of beta-adrenergic receptor kinase (beta ARK-CT) to identify these G proteins. We find the [Ca(2+)](i)response to UTP is not affected by increased expression of the wild-type G(alpha q), wild-type G(alpha i2)or beta ARK-CT, while it is blocked by over-expression of dominant negative G(alpha q). The timecourse of the UTP response is, however, altered by wild-type G(alpha q)and is only weakly inhibited by antisense G(alpha q). This suggests that the P(2U)response is mediated, at least partially, by a G protein distinct from G(alpha q). In contrast, the M(3)muscarinic response is inhibited by over-expression of antisense against G(alpha q), or over-expression of beta ARK-CT, a finding in agreement with our previous observation that the muscarinic response in HT29 cells is mediated by the beta gamma-subunits of G(q). We also find that P(2U)and M(3)receptors do not control identical Ca(2+)stores, suggesting that differential activation of G proteins can lead to Ca(2+)release from distinct stores.

Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells.

Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the alpha-subunit of Gq (Ad-EF1-dnG alpha q) and the other expressing the wild-type alpha-subunit of Gq (Ad-EF1-wtG alpha q). We used an adenovirus expressing green fluorescent protein (Ad-EF1-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca2+]i) or the carbachol responses in these cells. We further show that Ad-EF1-dnG alpha q inhibits the increase in [Ca2+]i produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EF1-wtG alpha q, which indicates that in both HSG and HT29 cells, the increase in [Ca2+]i produced by muscarinic receptor activation is largely mediated by activation of Gq. Neither virus affected the resting level of [Ca2+]i in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.

Na(+)-H+ exchange in sheep parotid endpieces. Apparent insensitivity to amiloride.

We have used microspectrofluorimetry with the pH-sensitive dye, BCECF, to examine the control of intracellular pH in the secretory endpieces of the sheep parotid gland. Unstimulated endpieces in HCO3(-)-free media have a cytosolic pH of 7.5 +/- 0.03 (n = 69) which is maintained by a Na(+)-dependent proton extrusion process that can be partially supported by Li+ but not by Cs+, and is not affected by changes in extracellular Cl-, HCO3- or K+. It is not blocked by SITS or DIDS, which inhibit Na(+)-(n)HCO3- co-transport and CL(-)-HCO3- exchange, nor is it sensitive to the amiloride analogs, MIA and EIPA, which inhibit Na(+)-H+ exchangers, although very high concentrations of amiloride itself (1 mmol/l) have a (probably non-specific) inhibitory effect. It seems likely that sheep parotid secretory endpieces do contain a Na(+)-H+ exchanger that drives secretion of a HCO3(-)-rich juice, and that its insensitivity to amiloride and its analogs explains why these drugs do not block fluid secretion by the intact sheep parotid gland.

Intracellular Ca2+ release by flufenamic acid and other blockers of the non-selective cation channel.

We report in this paper using measurement of intracellular free Ca2+ with fura-2, that flufenamic acid and several related blockers of the 25 pS Ca(2+)-activated non-selective cation channel cause release of Ca2+ from an intracellular store other than the endoplasmic reticulum, possibly from mitochondria. A new compound, 4'-methyl-DPC, is found to be as effective in blocking non-selective cation channels as other flufenamate analogs but, like the parent compound, the non-selective cation channel blocker DPC, it does not cause release of Ca2+ from intracellular stores. DPC and 4'-methyl-DPC are thus the most suitable of the available blockers of non-selective cation channels for use in studies on the role of these channels in normal cell function.

Feedback inhibition of epithelial Na(+) channels in Xenopus oocytes does not require G(0) or G(i2) proteins.

Regulation of amiloride-sensitive epithelial Na(+) channels (ENaC) is a prerequisite for coordination of electrolyte transport in epithelia. Downregulation of Na(+) conductance occurs when the intracellular Na(+) concentration is increased during reabsorption of electrolytes, known as feedback inhibition. Recent studies have demonstrated the involvement of alphaG(0) and alphaG(i2) proteins in the feedback control of ENaC in mouse salivary duct cells. In this report, we demonstrate that Na(+) feedback inhibition is also present in Xenopus oocytes after expression of rat alpha,beta, gamma-ENaC. Interfering with intracellular alphaG(0) or alphaG(i2) signaling by coexpression of either constitutively active alphaG(0)/alphaG(i2) or dominant negative alphaG(0)/alphaG(i2) and by coinjecting sense or antisense oligonucleotides for alphaG(0) had no impact on Na(+) feedback. Moreover, no evidence for involvement of the intracellular G protein cascade was found in experiments in which a regulator of G protein signaling (RGS3) or beta-adrenergic receptor kinase (betaARK) was coexpressed together with alpha,beta, gamma-ENaC. Although some experiments suggest the presence of an intracellular Na(+) receptor, we may conclude that Na(+) feedback in Xenopus oocytes is different from that described for salivary duct cells in that it does not require G protein signaling.

P2X(1) receptor membrane redistribution and down-regulation visualized by using receptor-coupled green fluorescent protein chimeras.

The P2X(1) purinergic receptor subtype occurs on smooth muscle cells of the vas deferens and urinary bladder where it is localized in two different size receptor clusters, with the larger beneath autonomic nerve terminal varicosities. We have sought to determine whether these synaptic-size clusters only form in the presence of varicosities and whether they are labile when exposed to agonists. P2X(1) and a chimera of P2X(1) and green fluorescent protein (GFP) were delivered into cells using microinjection, transient transfection or infection with a replication-deficient adenovirus. The P2X(1)-GFP chimera was used to study the time course of P2X(1) receptor clustering in plasma membranes and the internalization of the receptor following prolonged exposure to ATP. Both P2X(1) and P2X(1)-GFP clustered in the plasma membranes of Xenopus oocytes, forming patches 4-6 microm in diameter. Human embryonic kidney 293 (HEK293) cells, infected with the adenovirus, possessed P2X(1) antibody-labeled regions in the membrane colocalized with GFP fluorescence. The ED(50) for the binding of alpha,beta-methylene adenosine triphosphate (alpha,beta-meATP) to the P2X(1)-GFP chimera was similar to native P2X(1) receptors. ATP-generated whole-cell currents in oocytes or HEK293 cells expressing either P2X(1) or P2X(1)-GFP were similar. Exposure of HEK293 cells to alpha, beta-meATP for 10-20 min in the presence of 5 microM monensin led to the disappearance of P2X(1)-GFP fluorescence from the surface of the cells. These observations using the P2X(1)-GFP chimera demonstrate that P2X(1) receptors spontaneously form synaptic-size clusters in the plasma membrane that are internalized on exposure to agonists.

A nucleotide receptor that mobilizes Ca2+ in the mouse submandibular salivary cell line ST885.

1. We have identified a Ca(2+)-mobilizing receptor that responds to extracellular ATP (0.1-1000 microM) in a salivary epithelial cell line (ST885). The activated receptor also stimulates Ca2+ entry from the extracellular fluid. 2. The receptor shows an agonist selectivity profile consistent with the behaviour of the nucleotide receptor class. In particular, the pyrimidine nucleotide, UTP, was equipotent with ATP. 3. Analysis of concentration-response data for the ATP and UTP-activated receptor using the Hill equation yielded EC50 values (concentrations of agonist inducing a half-maximal response) for ATP of 4.2 microM and for UTP of 4.6 microM. 4. ATP and UTP induced cross-desensitization and were not additive when administered simultaneously at maximal concentration. These findings are consistent with the hypothesis that both nucleotides act upon a common receptor. 5. The ATP analogue, 2-methylthio ATP, mobilized Ca2+ ions with higher potency (EC50 = 0.23 microM) but lower efficacy than either ATP or UTP. However, 2-methylthio ATP was not a partial agonist of the ATP/UTP receptor. At maximal concentration (30 microM), it failed to antagonize the effects of near maximal concentrations of ATP or UTP. We conclude that 2-methylthio ATP acts upon a distinct receptor.

Pharmacological characterization of the nucleotide receptors that mobilize Ca2+ ions in human parathyroid cells.

We have used the fluorescent probe fura-2 to perform agonist studies of the receptor(s) that mobilizes Ca2+ ions in response to extracellular ATP in human parathyroid cells. Extracellular ATP induced Ca2+ responses in both normal and adenomatous parathyroid cells. Activation resulted in an initial small transient response during which Ca2+ ions were released from intracellular stores, followed by a prominent plateau response during which Ca2+ ions entered the cells from the extracellular fluid. The responses exhibited moderate desensitization upon repeated stimulation with ATP, and the ratio of the plateau to the peak response remained constant for any given group of activated cells. The baseline intracellular calcium concentration was 100 +/- 4.3 nM (mean +/- S.E.M., n = 3). Following maximal activation by extracellular ATP it rose to a peak of 684 +/- 45.7 nM (n = 3) and a plateau level of 415 +/- 9.9 nM (n = 3). We examined the effects of a variety of nucleotide species. The order of potency was: adenosine, AMP < alpha, beta-methylene ATP < ADP < ATP approximately UTP. In the concentration range 1-1000 microM, UTP (the concentration of agonist inducing a half-maximal response, EC50 = 2.4 microM) was slightly more potent than ATP (EC50 = 3.6 microM), and the two nucleotides evoked similar maximal responses. In the concentration range 0.01-1.0 microM, however, there was a clear difference in the behaviour of the two nucleotides. In particular, ATP, but not UTP, evoked responses that suggested the presence of a second receptor of higher potency but markedly lower efficacy.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ion transport inhibition on rat mandibular gland secretion.

The effects of substituting gluconate for extracellular Cl, and of treatment with various ion transport blockers, on cytosol pH (pHi) and secretion by the acetylcholine stimulated rat mandibular gland were studied in vitro. Gluconate replacement increased pHi from 7.12 +/- 0.02 to 7.27 +/- 0.04, caused secretory rate to fall by 75%, and increased salivary HCO3 from 14 +/- 0.9 mmol/L to 67 +/- 1.5 mmol/L. Furosemide (1 mmol/L), which blocks Na-K-2Cl symports and Cl-HCO3 antiports, had effects similar to those of gluconate replacement, except that secretion was reduced only by 59%. Bumetanide (1 mmol/L), which blocks only Na-K-2Cl symports, caused a 67% reduction in secretion rate, but it had little effect on pHi and caused only a small rise in salivary HCO3 concentration. SITS (1 mmol/L), which blocks Cl-HCO3 antiports, increased pHi to 7.26 +/- 0.03 and induced a small rise in the secretory rate. Methazolamide and acetazolamide (1 mmol/L), both of which inhibit carbonic anhydrase and may also block anion channels, increased pHi to 7.43 +/- 0.02 and 7.20 +/- 0.03, respectively, but had no effect on secretory rate, and reduced salivary HCO3 slightly. Ba (3 mmol/L), tetraethylammonium (10 mmol/L), and decamethonium (5 mmol/L) all caused marked but reversible reductions in secretory rate, consistent with the known actions of these agents on K channels. Ba, however, also appeared to act as a Ca antagonist, an action that it seemed to share with Mn ions (5 mmol/L).(ABSTRACT TRUNCATED AT 250 WORDS)

An outwardly rectifying chloride channel in a human submandibular gland duct cell line (HSG).

We have used the single channel patch-clamp technique to study the ion channels in the plasma membrane of the HSG human submandibular gland duct cell line. When the pipette contained an isotonic KCl-rich solution and the bath an isotonic NaCl-rich solution, the predominant channel type seen in excised inside-out patches was a Cl- channel with an outwardly rectifying I-V relation that had a conductance of 12 pS at positive pipette potentials and 43 pS at negative pipette potentials. The channel was only seen in excised patches and its open probability was not significantly increased by membrane depolarization. The channel selectivity sequence (relative to Cl-) was estimated from reversal potential measurements to be SCN- > NO3- > I- approximately Cl- approximately Br- > acetate. In inside-out patches the channel was blocked by addition of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) (100 mumol/l) to the bath but not by 9-anthracene carboxylic acid (9-AC) (100 mumol/l). The channel was not activated by increases in the free Ca2+ concentration on the cytosolic surface.

Control of Na+ transport in salivary duct epithelial cells by cytosolic Cl- and Na+.

The duct cells of the mandibular glands of mice (and many other mammalian salivary glands) absorb NaCl from an isotonic, Na+-rich primary saliva, formed by the gland's secretory endpieces, utilising an amiloride-sensitive Na+ channel in the apical (luminal) domain of the plasma membranes. The present study focuses on the mechanisms whereby the apical membrane Na+ conductance is controlled so that the rate of Na+ influx from lumen to cytosol via the Na+ channels is matched to the rate of Na+ extrusion from cytosol to interstitium via the basolateral Na+-K+-ATPase (so called homocellular regulation or epithelial cross-talk). Our results show that the apical membrane Na+ conductance is not controlled by a sensor of extracellular (luminal) Na+, as has been previously believed, but by sensors of cytosolic Na+ and Cl- which down-regulate the Na+ channels when the cytosolic concentration of either ion increases. These effects of cytosolic Na+ and Cl- are mediated, respectively, by G proteins of the Gi and Go subclasses.