Bile acids: a potential role in the pathogenesis of pharyngeal malignancy.

OBJECTIVE: Gastro-oesophageal reflux disease is thought to be a risk factor for head and neck malignancies. Bile acids are one of the principle components of gastric refluxate and have previously been implicated in the development of oesophageal and bowel malignancies. There is clear evidence that bile acids reflux into the laryngopharynx. Despite this, the carcinogenic properties of bile acids in this area are yet to be fully identified. We therefore investigated the potential role of bile acids in pharyngeal malignancy, through the highly conserved process of epithelial-mesenchymal transition (EMT). EMT occurs in invasion and metastasis and is a central process in the development of epithelial carcinoma. DESIGN: Translational research study. METHODS: Human hypopharyngeal squamous carcinoma FaDu cells were challenged with primary (cholic or chenodeoxycholic) and secondary (deoxycholic or lithocholic) bile acids. EMT-relevant proteins TGF-ß1 and MMP-9 were measured in the cell culture supernates at 48 h via ELISA. Cell viability was confirmed >95% via CellTiter-Blue assay. RESULTS: Significantly greater concentrations of TGF-ß1 were measured in the culture supernates of cells treated with cholic acid, deoxycholic acid and chenodeoxycholic acid. MMP-9 levels were increased in deoxycholic acid and lithocolic acid stimulations when compared to control (P < 0.05). CONCLUSION: This is the first demonstration that bile acids induce TGF-ß1 and MMP-9 in pharyngeal cells. TGF-ß1 is considered a master switch for EMT, while MMP-9 is a part of the EMT proteome which degrades basement membranes. This implies a potential role for bile acids in pharyngeal carcinogenesis through the mechanism of EMT and suggests potential novel therapeutic targets.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Anesthetic-like interaction of the sleep-inducing lipid oleamide with voltage-gated sodium channels in mammalian brain.

BACKGROUND: cis-9,10-Octadecenoamide (cOA) accumulates in cerebrospinal fluid during sleep deprivation and induces sleep in animals, but its cellular actions are poorly characterized. In earlier studies, like a variety of anesthetics, cOA modulated gamma-aminobutyric acidA receptors and inhibited transmitter release/burst firing in cultured neurones or synaptoneurosomes. METHODS: Here, radioligand binding ([3H]batrachotoxinin A 20-alpha-benzoate and mouse central nervous system synaptoneurosomes) and voltage clamp (whole cell recording from cultured NIE115 murine neuroblastoma) confirmed an interaction with neuronal voltage-gated sodium channels (VGSC). RESULTS: cOA stereoselectively inhibited specific binding of toxin to VGSC (inhibitor concentration that displaces 50% of specifically bound radioligand, 39.5 microm). cOA increased (4x) the Kd of toxin binding without affecting its binding maximum. Rate of dissociation of radioligand was increased without altering association kinetics, suggesting an allosteric effect (indirect competition at site 2 on VGSC). cOA blocked tetrodotoxin-sensitive sodium currents (maximal effect and affinity were significantly greater at depolarized potentials; P < 0.01). Between 3.2 and 64 microm, the block was concentration-dependent and saturable, but cOA did not alter the V50 for activation curves or the measured reversal potential (P > 0.05). Inactivation curves were significantly shifted in the hyperpolarizing direction by cOA (maximum, -15.4 +/- 0.9 mV at 32 microm). cOA (10 microm) slowed recovery from inactivation, with tau increasing from 3.7 +/- 0.4 ms to 6.4 +/- 0.5 ms (P < 0.001). cOA did not produce frequency-dependent facilitation of block (up to 10 Hz). CONCLUSIONS: These effects (and the capacity of oleamide to modulate gamma-aminobutyric acidA receptors in earlier studies) are strikingly similar to those of a variety of anesthetics. Oleamide may represent an endogenous ligand for depressant drug sites in mammalian brain.

Stereoselective modulatory actions of oleamide on GABA(A) receptors and voltage-gated Na(+) channels in vitro: a putative endogenous ligand for depressant drug sites in CNS.

1. cis-9,10-octadecenoamide ('oleamide') accumulates in CSF on sleep deprivation. It induces sleep in animals (the trans form is inactive) but its cellular actions are poorly characterized. We have used electrophysiology in cultures from embryonic rat cortex and biochemical studies in mouse nerve preparations to address these issues. 2. Twenty microM cis-oleamide (but not trans) reversibly enhanced GABA(A) currents and depressed the frequency of spontaneous excitatory and inhibitory synaptic activity in cultured networks. 3. cis-oleamide stereoselectively blocked veratridine-induced (but not K(+)-induced) depolarisation of mouse synaptoneurosomes (IC(50), 13. 9 microM). 4. The cis isomer stereoselectively blocked veratridine-induced (but not K(+)-induced) [(3)H]-GABA release from mouse synaptosomes (IC(50), 4.6 microM). 5. At 20 microM cis-oleamide, but not trans, produced a marked inhibition of Na(+) channel-dependent rises in intrasynaptosomal Ca(2+). 6. The physiological significance of these observations was examined by isolating Na(+) spikes in cultured pyramidal neurones. Sixty-four microM cis-oleamide did not significantly alter the amplitude, rate of rise or duration of unitary action potentials (1 Hz). 7. cis-Oleamide stereoselectively suppressed sustained repetitive firing (SRF) in these cells with an EC(50) of 4.1 microM suggesting a frequency- or state-dependent block of voltage-gated Na(+) channels. 8. Oleamide is a stereoselective modulator of both postsynaptic GABA(A) receptors and presynaptic or somatic voltage-gated Na(+) channels which are crucial for synaptic inhibition and conduction. The modulatory actions are strikingly similar to those displayed by sedative or anticonvulsant barbiturates and a variety of general anaesthetics. 9. Oleamide may represent an endogenous modulator for drug receptors and an important regulator of arousal.

Inhibition of GABA-gated chloride channels by 12,14-dichlorodehydroabietic acid in mammalian brain.

1. 12,14-dichlorodehydroabietic acid (12,14-Cl2DHA) reduced GABA-stimulated uptake of 36Cl- into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABA(A) receptor function. 2. 12,14-Cl2DHA did not affect the binding of [3H]-muscimol to brain membranes but displaced specifically bound [3H]-EBOB. The inhibitory effect on [3H]-EBOB binding was not reversible. 12,14-Cl2DHA reduced the availability of [3H]-EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14-Cl2DHA did not affect the rate of association of [3H]-EBOB with its chloride channel receptor, but increased the initial rate of [3H]-EBOB dissociation. 3. 12,14-Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14-Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells. 4. Ultimately, 12,14-Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABA(A) receptor-chloride channel complex. 5. The depolarizing block seen with 12,14-Cl2DHA in amphotericin-perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials. 6. Our investigation demonstrates that 12,14-Cl2DHA blocks GABA-dependent chloride entry in mammalian brain and operates as a non-competitive insurmountable GABA(A) antagonist. The mechanism likely involves either irreversible binding of 12,14-Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14-Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel.

Volume-sensitive chloride currents in primary cultures of human fetal vas deferens epithelial cells.

Using the patch-clamp technique, we have identified a large, outwardly rectifying, Cl--selective whole-cell current in primary cultures of human vas deferens epithelial cells. Whole-cell currents were time- and voltage-dependent and displayed inactivation following depolarising pulses >/= 60 mV. Currents were equally permeable to bromide (PBr/PCl = 1.05 +/- 0.04), iodide (PI/PCl = 1. 06 +/- 0.07) and Cl-, but significantly less permeable to gluconate (PGluc /PCl = 0.23 +/- 0.03). Currents spontaneously increased with time after establishing a whole-cell recording, but could be inhibited by exposure to a hypertonic bath solution which reduced inward currents by 68 +/- 4%. Subsequent exposure of the cells to a hypotonic bath solution led to a 418 +/- 110% increase in inward current, indicating that these currents are regulated by osmolarity. 4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (100 microM) produced a rapid and reversible voltage-dependent block (60 +/- 5% and 10 +/- 7% inhibition of current, measured at +/- 60 mV, respectively). Dideoxyforskolin (50 microM) also reduced the volume-sensitive Cl- current, but with a much slower time course, by 41 +/- 13% and 32 +/- 16% (measured at +/- 60 mV, respectively). Tamoxifen (10 microM) had no effect on the whole-cell Cl- current. These results suggest that vas deferens epithelial cells possess a volume-sensitive Cl- conductance which has biophysical and pharmacological properties broadly similar to volume-sensitive Cl- currents previously described in a variety of cell types.

Chloride channels and cystic fibrosis of the pancreas.

Cystic fibrosis (CF) affects approximately 1 in 2000 people making it one of the commonest fatal, inherited diseases in the Caucasian population. CF is caused by mutations in a cyclic AMP-regulated chloride channel known as CFTR, which is found on the apical plasma membrane of many exocrine epithelial cells. In the CF pancreas, dysfunction of the CFTR reduces the secretory activity of the tubular duct cells, which leads to blockage of the ductal system and eventual fibrosis of the whole gland. One possible approach to treating the disease would be to activate an alternative chloride channel capable of bypassing defective CFTR. A strong candidate for this is a chloride channel regulated by intracellular calcium, which has recently been shown to protect the pancreas in transgenic CF mice. Pharmacological intervention directed at activating this calcium-activated Cl- conductance might provide a possible therapy to treat the problems of pancreatic dysfunction in CF.

Volume-activated chloride currents in pancreatic duct cells.

We have used the patch clamp technique to study volume-activated Cl- currents in the bicarbonate-secreting pancreatic duct cell. These currents could be elicited by a hypertonic pipette solution (osmotic gradient 20 mOsm/l), developed over about 8 min to a peak value of 91 +/- 5.8 pA/pF at 60 mV (n = 123), and were inhibited by a hypertonic bath solution. The proportion of cells which developed currents increased from 15% in freshly isolated ducts to 93% if the ducts were cultured for 2 days. The currents were ATP-dependent, had an outwardly rectifying current/voltage (I-V) plot, and displayed time-dependent inactivation at depolarizing potentials. The anion selectivity sequence was: ClO4 = I = SCN > Br = NO3 > Cl > F > HCO3 > gluconate, and the currents were inhibited to a variable extent by DIDS, NPPB, dideoxyforskolin, tamoxifen, verapamil and quinine. Increasing the intracellular Ca2+ buffering capacity, or lowering the extracellular Ca2+ concentration, reduced the proportion of duct cells which developed currents. However, removal of extracellular Ca2+ once the currents had developed was without effect. Inhibiting protein kinase C (PKC) with either the pseudosubstrate PKC (19-36), calphostin C or staurosporine completely blocked development of the currents. We speculate that cell swelling causes Ca2+ influx which activates PKC which in turn either phosphorylates the Cl- channel or a regulatory protein leading to channel activation.

cAMP-activated chloride channels in a CFTR-transfected pancreatic adenocarcinoma-derived cell line, pANS6.

Pancreatic adenocarcinoma cell lines rarely express the CFTR gene, despite the high levels of CFTR protein that are present in primary pancreatic duct cells. We have attempted to generate a non-CF pancreatic adenocarcinoma cell line that stably produces high levels of CFTR mRNA and protein by transfecting a vector containing the CFTR cDNA, driven by a strong mammalian promoter, into the poorly differentiated pancreatic adenocarcinoma cell line, Panc-1. The pANS6 pancreatic duct cell line expresses substantial levels of CFTR mRNA, but little CFTR protein. Despite this we were able to detect low conductance chloride channels in 40% of patches, stimulated with cAMP, that have similar biophysical properties to CFTR.

Calcium-activated chloride conductance is not increased in pancreatic duct cells of CF mice.

Calcium-activated anion secretion is elevated in the pancreatic ductal epithelium of transgenic cf/cf mice which lack the cystic fibrosis transmembrane conductance regulator (CFTR). To elucidate whether this effect is due to increased activity of calcium-activated chloride channels, we have studied the relationship between CFTR and calcium-activated chloride currents in pancreatic duct cells isolated from Cambridge cf/cf mice. CFTR chloride currents activated by cAMP were detected in 59% (29/49) of wild-type cells and in 50% (20/40) of heterozygous cells. However, we could not detect any CFTR currents in the homozygous cf/cf cells (0/25). The maximum CFTR current density measured at a membrane potential of 60 mV was 23.5 +/- 2.8 pA/pF (n = 29) in wild-type cells, and about half that value, i.e. 12.4 +/- 1.6 pA/pF (n = 20) in heterozygotes (P = 0.004). Calcium-activated chloride currents were detected in 73% (24/33) of wild-type, 75% (21/28) of heterozygous and in 58% (7/12) of homozygous cf/cf cells. There was no significant difference between the steady-state calcium-activated current densities in the three genotypic groups; the current measured at 60 mV being 527 +/- 162 pA/pF (n = 24) from wild-type, 316 +/- 35 pA/pF (n = 21) from heterozygote and 419 +/- 83 pA/pF (n = 7) from homozygous cells. Our data suggest that lack of CFTR does not enhance the calcium-activated chloride conductance in murine pancreatic duct cells.

An enzyme-linked immunosorbent assay for lactose synthase (galactosyltransferase) in serum and its application as a tumor marker in ovarian carcinoma.

This assay for lactose synthase (galactosyltransferase, EC 2.4.1.22) in serum involves two sequential incubations: serially diluted standard or sample antigen is reacted with a fixed amount of antibody; unbound antibody is then adsorbed to wells of antigen-coated microtiter plates and determined by a second antibody directed against the first antibody and coupled to phosphatase. The standard curve is linear for galactosyltransferase concentrations of 10 to 600 micrograms/L. The within-assay CV of a serum sample was 9.3% (SD 4.1%), the between-assay was 3.8% (SD 2.4%). Serum galactosyltransferase concentrations computed from three different dilutions yielded CVs of 6.5% (SD 5.7%, n = 14). We evaluated the method's accuracy by recovery analysis and by comparing enzyme activity in serum with that of purified galactosyltransferase from human milk. The normal reference interval, as estimated from data on 27 healthy blood donors, was 60-436 micrograms/L (mean 224, SD 101 micrograms/L). We applied the assay to samples of serum from ovarian carcinoma patients grouped according to tumor burden. We also determined galactosyltransferase in ascites fluid and found these values useful for diagnosis, whereas determinations in serum may serve mainly for patient monitoring.

Electrophoretic comparison of cellular and soluble galactosyltransferase (lactose synthetase A protein) using specific antibodies.

Rabbit antisera against soluble human milk galactosyltransferase (GT) having anti-GT activity, as demonstrated by inhibition of enzyme activity were used for a comparative study of the molecular sizes of galactosyltransferase. For this purpose, affinity-purified antibodies were used for the identification of milk, serum and effusion galactosyltransferase from native or partially purified preparations resolved by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) by the immune replica technique. Milk galactosyltransferase migrated as a 55-kilodalton (kD) protein, serum and effusion GT slightly faster. Cross-reactive enzyme forms of 110 kD and 20 kD were detected in milk only. In order to establish a relationship between intracellular and soluble galactosyltransferase, HeLa cells were metabolically labeled by [35S]-methionine, cells lysed, subjected to immunoprecipitation and the precipitate analyzed by SDS-PAGE/fluorography: a single band corresponding to the intracellular form of GT have similar mobility as the milk enzyme was detected. These results indicate a close structural similarity between soluble and cellular galactosyltransferase as judged by immunological cross-reactivity and electrophoretic mobility.

A solid-phase ELISA for human galactosyltransferase.

Monospecific rabbit anti-human milk galactosyltransferase antibodies were used to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) for the human milk enzyme. The assay was based on competition with galactosyltransferase-phosphatase conjugate. The detection limit of the standard curve was 30 ng/ml. Galactosyltransferase was quantified in dialyzed milk samples by ELISA with variation coefficients of less than 7.7% and between 6.8 and 18.8% within and between assays, respectively. The average content was 61 +/- 19 microgram/ml. The assay proved suitable for quantitation of partially purified enzyme preparations in body fluids.