ANO1 (TMEM16A) in pancreatic ductal adenocarcinoma (PDAC).

Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all cancers. ANO1 (TMEM16A) is a recently identified Ca(2+)-activated Cl(-) channel (CaCC) that is upregulated in several tumors. Although ANO1 was subject to extensive studies in the recent years, its pathophysiological function has only been poorly understood. The aim of the present study is to establish the significance of ANO1 in PDAC behavior and demarcate its roles in PDAC from those of the volume-regulated anion channel (VRAC). We performed qPCR and Western blot measurements on different PDAC cell lines (Panc-1, Mia PaCa 2, Capan-1, AsPC-1, BxPC-3) and compared the results to those obtained in a human pancreatic ductal epithelium (HPDE) cell line. All cancer cell lines showed an upregulation of ANO1 on mRNA and protein levels. Whole-cell patch-clamp recordings identified large Ca(2+) and voltage-dependent Cl(-) currents in PDAC cells. Using siRNA knockdown of ANO1 and three ANO1 inhibitors (T16Ainh-A01, CaCCinh-A01, and NS3728), we found that ANO1 is the main constituent of CaCC current in PDAC cells. We further characterized these three inhibitors and found that they had unspecific effects on the free intracellular calcium concentration. Functional studies on PDAC behavior showed that surprisingly inhibition of ANO1 did not influence cellular proliferation. On the other hand, we found ANO1 channel to be pivotal in PDAC cell migration as assessed in wound healing experiments.

In-vivo study of genotoxic and inflammatory effects of the organo-modified Montmorillonite Cloisite® 30B.

Because of the increasing use of clays and organoclays in industrial applications it is of importance to consider the toxicity of these materials. Recently it was reported that the commercially available Montmorillonite clay, Cloisite(®) 30B, which is surface-modified by organic quaternary ammonium compounds, was genotoxic in vitro. In the present study the in-vivo genotoxic and inflammatory potential of Cloisite(®) 30B was investigated as a follow-up of the in-vitro studies. Wistar rats were exposed to Cloisite(®) 30B twice 24h apart by oral gavage, at doses ranging from 250 to 1000 mg/kg body weight [indicate duration of treatment; Ed.]. There was no induction of DNA strand-breaks in colon, liver and kidney cells and there was no increase in inflammatory cytokine markers in blood-plasma samples. In order to verify the possible absorption of Cloisite(®) 30B from the gastrointestinal tract, inductively coupled plasma mass-spectrometry (ICP-MS) analysis was performed on samples of liver, kidney and faeces, with aluminium as a tracer element characteristic to clay. The results showed that aluminium could be detected in faeces, but not in the liver or kidneys. This indicated that there was no systemic exposure to clay particles from Cloisite(®) 30B. Detection and identification of free quaternary ammonium modifier in the highest dose of Cloisite(®) 30B was carried out by high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS). This analysis revealed a mixture of three quaternary ammonium analogues. The detected concentration of the organomodifier corresponded to an exposure of rats to about 5mg quaternary ammonium analogues/kg body weight.

Anoctamin 6 differs from VRAC and VSOAC but is involved in apoptosis and supports volume regulation in the presence of Ca2+.

Anoctamin 6 (ANO6), also known as TMEM16F, has been shown to be a calcium-activated anion channel with delayed calcium activation. The cellular function of ANO6 is under debate, and different groups have come to different conclusions about ANO6's physiological role. Although it is now quite well established that ANO6 is distinct from the volume-regulated anion channel, it is still unclear whether ANO6 or other anoctamins can be activated by cell swelling. In this study, we suggest that ANO1, ANO6, and ANO10 do not contribute to the volume-activated current in ANO-overexpressing HEK293 cells. Furthermore, knock-down of ANO6 in Ehrlich ascites tumor cells (EATC) and Ehrlich-Lettre ascites (ELA) did not decrease but instead significantly increased swelling-activated membrane currents. Knock-down of ANO6 in EATC did not reduce regulatory volume decrease (RVD) in the absence of extracellular calcium, whereas it significantly reduced RVD in the presence of calcium. Interestingly, we found that knock-down of ANO6 in ELA cells resulted in a decrease in cisplatin-induced caspase-3 activity, confirming earlier findings that ANO6 is involved in apoptosis. Finally, knock-down of ANO1 and ANO6 did not affect the volume-sensitive release of taurine in ELA cells. Thus, our data provide evidence that ANO6 cannot be activated directly by cell swelling unless Ca(2+) is present. We also conclude that ANO6 carries a current during RVD, provided extracellular calcium is present. Thus, swelling activation of ANO6 requires the presence of free calcium.

Is it really organic?--multi-isotopic analysis as a tool to discriminate between organic and conventional plants.

Novel procedures for analytical authentication of organic plant products are urgently needed. Here we present the first study encompassing stable isotopes of hydrogen, carbon, nitrogen, oxygen, magnesium and sulphur as well as compound-specific nitrogen and oxygen isotope analysis of nitrate for discrimination of organically and conventionally grown plants. The study was based on wheat, barley, faba bean and potato produced in rigorously controlled long-term field trials comprising 144 experimental plots. Nitrogen isotope analysis revealed the use of animal manure, but was unable to discriminate between plants that were fertilised with synthetic nitrogen fertilisers or green manures from atmospheric nitrogen fixing legumes. This limitation was bypassed using oxygen isotope analysis of nitrate in potato tubers, while hydrogen isotope analysis allowed complete discrimination of organic and conventional wheat and barley grains. It is concluded, that multi-isotopic analysis has the potential to disclose fraudulent substitutions of organic with conventionally cultivated plants.

Validation of methods for the detection and quantification of engineered nanoparticles in food.

The potential impact of nanomaterials on the environment and on human health has already triggered legislation requiring labelling of products containing nanoparticles. However, so far, no validated analytical methods for the implementation of this legislation exist. This paper outlines a generic approach for the validation of methods for detection and quantification of nanoparticles in food samples. It proposes validation of identity, selectivity, precision, working range, limit of detection and robustness, bearing in mind that each "result" must include information about the chemical identity, particle size and mass or particle number concentration. This has an impact on testing for selectivity and trueness, which also must take these aspects into consideration. Selectivity must not only be tested against matrix constituents and other nanoparticles, but it shall also be tested whether the methods apply equally well to particles of different suppliers. In trueness testing, information whether the particle size distribution has changed during analysis is required. Results are largely expected to follow normal distributions due to the expected high number of particles. An approach of estimating measurement uncertainties from the validation data is given.

Survival of invasive bladder cancer patients, 1998-2009; a central and northern Denmark population-based cohort study.

OBJECTIVE: Invasive bladder cancer (IBC) is a common urological malignancy accounting for 4%-5% of all cancers in Denmark. Our aim was to examine possible changes in short- and long-term survival of patients with IBC during 1998-2009. STUDY DESIGN AND SETTING: Data on all patients (N = 4032) with an incident diagnosis of IBC within a population of 1.8 million were retrieved from the Danish National Registry of Patients from 1998 to 2009. We computed survival after 1, 3, and 5 years, stratified by age and gender, and estimated mortality rate ratios (MRR) using Cox proportional hazard regression to compare mortality over time, controlling for age and gender. Data on tumor stage or histology were not included. RESULTS: During the study period, the annual numbers of incident IBC patients remained stable. The median age was 74 years in each of the four 3-year periods in the study. The survival was relatively stable during the first three periods, whilst data from the last period showed modest improvement. The overall 1-year survival increased from 68% in 1998-2000 to 70% in 2007-2009, corresponding to an age and gender adjusted MRR of 0.89 (95% confidence interval [CI] 0.76-1.03). The 3- and 5-year survival was predicted to increase from 44% to 49% and from 35% to 40% respectively. This corresponded to a 3-year age and gender adjusted MRR of 0.87 (95% CI 0.77-0.98) and a 5-year MRR of 0.88 (95% CI 0.79-0.99). The 1-, 3-, and 5-year survival increased for men in all age groups (<70 years, 70-79 years, ≥80 years) and in women only in the 70-79-year age group. CONCLUSION: The survival of IBC patients increased slightly in northern and central Denmark in the 1998-2009 period.

Migration of nanosized layered double hydroxide platelets from polylactide nanocomposite films.

Melt-extruded L-polylactide (PLA) nanocomposite films were prepared from commercially available PLA and laurate-modified Mg-Al layered double hydroxide (LDH-C12). Three films were tested for total migration as well as specific migration of LDH, tin, laurate and low molecular weight PLA oligomers (OLLA). This is the first reported investigation on the migration properties of PLA-LDH nanocomposite films. The tests were carried out as part of an overall assessment of the suitability of such films for use as food contact materials (FCM). Total migration was determined according to a European standard method. All three films showed migration of nanosized LDH, which was quantified using acid digestion followed by inductively coupled plasma mass spectrometric (ICP-MS) detection of (26)Mg. Migration of LDH from the films was also confirmed by examining migrates using transmission electron microscopy (TEM) and was attributed indirectly to the significant PLA molecular weight reduction observed in extruded PLA-LDH-C12 films. Migration of tin was detected in two of the film samples prepared by dispersion of LDH-C12 using a masterbatch technique and migration of the laurate organomodifier took place from all three film types. The results indicate that the material properties are in compliance with the migration limits for total migration and specific lauric acid migration as set down by the EU legislation for FCM, at least if a reduction factor for fresh meat is taken into consideration. The tin detected arises from the use of organotin catalysts in the manufacture of PLA.

Combining asymmetrical flow field-flow fractionation with light-scattering and inductively coupled plasma mass spectrometric detection for characterization of nanoclay used in biopolymer nanocomposites.

It is expected that biopolymers obtained from renewable resources will in due course become fully competitive with fossil fuel-derived plastics as food-packaging materials. In this context, biopolymer nanocomposites are a field of emerging interest since such materials can exhibit improved mechanical and barrier properties and be more suitable for a wider range of food-packaging applications. Natural or synthetic clay nanofillers are being investigated for this purpose in a project called NanoPack funded by the Danish Strategic Research Council. In order to detect and characterize the size of clay nanoparticulates, an analytical system combining asymmetrical flow field-flow fractionation (AF(4)) with multi-angle light-scattering detection (MALS) and inductively coupled plasma mass spectrometry (ICP-MS) is presented. In a migration study, we tested a biopolymer nanocomposite consisting of polylactide (PLA) with 5% Cloisite30B (a derivatized montmorillonite clay) as a filler. Based on AF(4)-MALS analyses, we found that particles ranging from 50 to 800 nm in radius indeed migrated into the 95% ethanol used as a food simulant. The full hyphenated AF(4)-MALS-ICP-MS system showed, however, that none of the characteristic clay minerals was detectable, and it is concluded that clay nanoparticles were absent in the migrate. Finally, by means of centrifugation experiments, a platelet aspect ratio of 320 was calculated for montmorillonite clay using AF(4)-MALS for platelet size measurements.

The lateral intercellular space as osmotic coupling compartment in isotonic transport.

Solute-coupled water transport and isotonic transport are basic functions of low- and high-resistance epithelia. These functions are studied with the epithelium bathed on the two sides with physiological saline of similar composition. Hence, at transepithelial equilibrium water enters the epithelial cells from both sides, and with the reflection coefficient of tight junction being larger than that of the interspace basement membrane, all of the water leaves the epithelium through the interspace basement membrane. The common design of transporting epithelia leads to the theory that an osmotic coupling of water absorption to ion flow is energized by lateral Na(+)/K(+) pumps. We show that the theory accounts quantitatively for steady- and time dependent states of solute-coupled fluid uptake by toad skin epithelium. Our experimental results exclude definitively three alternative theories of epithelial solute-water coupling: stoichiometric coupling at the molecular level by transport proteins like SGLT1, electro-osmosis and a 'junctional fluid transfer mechanism'. Convection-diffusion out of the lateral space constitutes the fundamental problem of isotonic transport by making the emerging fluid hypertonic relative to the fluid in the lateral intercellular space. In the Na(+) recirculation theory the 'surplus of solutes' is returned to the lateral space via the cells energized by the lateral Na(+)/K(+) pumps. We show that this theory accounts quantitatively for isotonic and hypotonic transport at transepithelial osmotic equilibrium as observed in toad skin epithelium in vitro. Our conclusions are further developed for discussing their application to solute-solvent coupling in other vertebrate epithelia such as small intestine, proximal tubule of glomerular kidney and gallbladder. Evidence is discussed that the Na(+) recirculation theory is not irreconcilable with the wide range of metabolic cost of Na(+) transport observed in fluid-transporting epithelia.

Intake of household salt in a Danish population.

OBJECTIVE: To quantify the intake of household salt and its contribution to the total salt intake in a Danish population. METHODS: Eighty-seven healthy individuals (37 men and 50 women), aged 20-55 years, recruited from the area of Copenhagen, completed the study. Total salt intake was estimated from the mean urinary excretion of sodium in four 24-h collections. Household salt, added to the food by the volunteers, was assessed using a lithium-marker technique. RESULTS: Total salt intake was 10.6+/-3.3 g day(-1) (mean+/-s.d.) in men and 7.1+/-2.3 g day(-1) in women. Median intake of household salt was 1.0 g day(-1) in men and 0.5 g day(-1) in women, corresponding to 10.2 and 8.7% of total salt intake in men and women, respectively. A significant difference between sexes was found regarding total salt intake (P<0.0001), but no difference was found if total salt intake was measured per energy intake. No significant difference was found between sexes regarding intake of household salt, and neither the educational level nor the age was associated to either total salt intake or intake of household salt. CONCLUSION: These findings support the assertion that the total salt intake in the Danish population is above the recommended intake and that the salt intake cannot be sufficiently lowered simply by lowering the use of household salt. Focus needs to be addressed to salt added during the processing or manufacture of foods, as this is the greatest source of salt intake at least in this group of healthy volunteers.

Fluid transport and ion fluxes in mammalian kidney proximal tubule: a model analysis of isotonic transport.

AIM: By mathematical modelling, we analyse conditions for near-isotonic and isotonic transport by mammalian kidney proximal tubule. METHODS: The model comprises compliant lateral intercellular space (lis) and cells, and infinitely large luminal and peritubular compartments with diffusible species: Na+, K+, Cl- and an intracellular non-diffusible anion. Unknown model variables are solute concentrations, electrical potentials, volumes and hydrostatic pressures in cell and lis, and transepithelial potential. We used data mainly from rat proximal tubule to model epithelial cells and interspace with luminal and peritubular baths of identical composition. RESULTS: The model of the tubular epithelium with physiological water permeability and paracellular electrical resistance generates solute coupled water uptake with an approx. 3% hypertonic absorbate. This function remains unperturbed following 'blocking' of apical water channels and in 'aquaporin-null' simulation. Reduced rate of volume reabsorption in AQP(-/-) mice would also require decreased apical sodium permeability. Paracellular convection accounts for approx. 36% of the net Na+ absorption, and the model epithelium accomplishes uphill water transport similar to rat proximal tubule. Na+ recirculation is required for truly isotonic transport. The tonicity of the absorbate and the recirculation flux depend critically on ion permeabilities of interspace basement membrane. CONCLUSION: Our model based on solute-solvent coupling in lateral space simulates major physiological features of proximal tubule, including significantly lower water permeability of the AQP1-null preparation, and a ratio of net sodium uptake and oxygen consumption exceeding that predicted from stoichiometry of the Na+/K+-pump. Physical properties of interspace basement membrane are critical for obtaining near-isotonic and truly isotonic transport.

Na+ recirculation and isosmotic transport.

The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.

Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater.

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.

Monitoring the content and intake of trace elements from food in Denmark.

The content of cadmium, lead, nickel, mercury and selenium in 83 foods was monitored from 1993 to 1997. In comparison with similar results from 1988 to 1992, a general decrease in lead levels had occurred, whereas the contents of cadmium, nickel, mercury and selenium were stable or declined only slightly. The distribution in dietary intake of the five trace elements was estimated by combining the mean trace element concentrations with food consumption data from 1837 Danes aged 15-80 years. The lead intake for 1993-97 showed a decrease in comparison with similar estimates from the previous monitoring cycles: 1983-87 and 1988-92. The intake of cadmium and mercury decreased to a lesser extent, whereas the intake of selenium and nickel remained unchanged in the same period. The dietary intake of trace elements was compared with the provisional tolerable weekly intake (PTWI). The 95th percentile of the distribution in cadmium intake amounts to 34% of PTWI, which is relatively high, and therefore calls for a more detailed future risk assessment. The intakes of lead and mercury were 11% of PTWI and, like the intake of nickel, did not cause any health concern in the adult population. The Danes ingest close to 100% of the Nordic Nutrition Recommendation for selenium at 50 microg day(-1), and no individuals had an intake less than the lower limit of 20 microg day(-1).

Lymph osmolality and rehydration from NaCl solutions by toads, Bufo marinus.

Toads, Bufo marinus, allowed to maintain an ad libitum state of hydration were dehydrated by 10 15% of their standard weight and allowed to rehydrate from either deionized water or from 10 or 50 mmol l(-1) NaCl solutions. Toads rehydrating from the dilute salt solutions recovered a larger fraction of their standard weight than did toads rehydrating from deionized water despite there being a reduced osmotic gradient. Amiloride did not reduce water gain from these solutions. Water uptake from 100 mmol l(-1) sucrose and 50 mmol l(-1) Na gluconate was reduced relative to deionized water by a fraction predicted from the osmotic gradient. Thus, the presence of both Na+ and Cl- are required for the augmentation of water gain from dilute salt solutions. Toads allowed to rehydrate from 120 mmol l(-1) NaCl for 180 min recovered nearly as much water as toads rehydrating from deionized water for 120 min and the lymph osmolality was not reduced relative to the dehydrated condition. The recovery of water from the salt solution was greater than that predicted from the reduced osmotic gradient and amiloride partially inhibited the rehydration from 120 mmol l(-1) NaCl. Solute coupled water transport can therefore be demonstrated in living animals but only from a NaCl solution that is nearly isoosmotic with the lymph. The mechanism for enhanced water gain from dilute salt solutions remains unresolved.

Absorption and retention of selenium from shrimps in man.

This study was undertaken to evaluate the bioavailability of selenium in shrimps, a possible good source of selenium, by measurements of the absorption and retention of selenium and the effects on plasma selenium concentration and glutathione peroxidase activity. Twelve healthy young subjects (9F and 3M) received 100 g of shrimps each day for six weeks in addition to their habitual diet. In the third week of the study a balance period was inserted in which the subjects received all food from the department and collected faeces and urine over 5 days. Blood samples were collected at commencement of the study, after 2, 4, and 6 weeks. The selenium intake increased from 39.4 +/- 15.3 microg/d to 127 +/- 5.5 microg/d with the addition of shrimps. The apparent absorption of selenium from shrimps was 83 +/- 4%. Faecal and urinary selenium excretion was 32.5 +/- 17.0 microg/d and 21.2 +/- 9.0 microg/d, respectively and the total retention of selenium was 3.1 +/- 1.1 mg. Plasma selenium concentrations were 95.2 +/- 9.7 microg/L and 101.5 +/- 9.7 microg/L before and after six weeks of shrimp intake, respectively (p<0.05). Plasma and erythrocyte glutathione peroxidase activities were not influenced by shrimp intake. Thus, despite the high absorption and retention, plasma selenium concentrations were only moderately affected by an increase in selenium intake of about 100 microg/d in the chemical forms found in shrimp. Whether the accumulation of selenium from shrimps in tissues may represent a potential hazard is to be further investigated.

Maxi K+ channels co-localised with CFTR in the apical membrane of an exocrine gland acinus: possible involvement in secretion.

The primary secretion formed in various exocrine glands has a [K+] 2-5 times that of plasma. In this study we measured the transepithelial flux of 36Cl-, 22Na+ and 42K+ across the frog skin and applied the single-channel patch-clamp technique to the apical membrane of frog skin gland acini to investigate the pathway taken by K+ secreted by the glands. Transepithelial K+ secretion was active and was driven by a larger force than the secretion of Na+. When driving Na+ through the epithelium by clamping the transepithelial potential to 100 mV (apical solution reference), blockers of cellular secretion (apical 5-nitro-2-(3-phenylpropylamino)benzoate or basolateral quinine or furosemide) decreased K+ secretion but left Na+ secretion unaffected. We conclude that K+ follows a transcellular pathway across the epithelium. Patch-clamp analysis of the apical membrane of microdissected gland acini revealed a population of voltage- and calcium-activated K+ channels of the maxi K+ type. In cell-attached patches these channels were activated by membrane potential depolarisation or exposure to prostaglandin E2 and had a permeability of 3.6 +/- 0.3 x 10(-13) cm3 s-1, giving a calculated conductance of 170 pS with 125 mM K+ on both sides of the membrane. In inside-out patches the channels were activated by increasing intracellular [Ca2+] from 10(-7) to 10(-6) M and were blocked by Ba2+ added to the cytoplasmic side. Exposure of inside-out patches containing the maxi K+ channel to ATP on the inside activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, confirming that both channels are co-localised to the apical membrane. We interpret these findings in terms of a model where transepithelial NaCl secretion can be supported in part by an apical K+ conductance.

Role of mitochondria-rich cells for passive chloride transport, with a discussion of Ussing's contribution to our understanding of shunt pathways in epithelia.

A non-invasive method is applied for studying ion transport by single isolated epidermal mitochondria-rich (MR) cells. MR cells of toad skin (Bufo bufo) were prepared by trypsin (or pronase) treatment of the isolated epithelium bathed in Ca2+-free Ringer. Glass pipettes were pulled and heat- polished to obtain a tip of 2-4 mm with parallel walls and low tip resistances. The neck of an MR-cell was sucked into the tip of the pipette for being 'clamped mechanically' by the heat-polished glass wall. In this configuration the apical cell membrane faces the pipette solution while the major neck region and the cell body are in the electrically grounded bath. With Ringer in bath and pipette, transcellular voltage clamp currents were composed of an ohmic (I(leak)) and a dynamic (I(dynamic)) component. The dynamic component was studied by stepping the transcellular potential (Vp) from a holding value of +50 mV to the hyperpolarizing region (50 > Vp > or = -100 mV). The steady state I(dynamic)-Vp relationship was strongly outward rectified with I(dynamic) being practically zero for Vp > 0 mV. At Vp = -100 mV, MR cells isolated by trypsin or pronase generated a steady-state I(dynamic) of,-2.72 +/- 0.40 nA/cell (N = 21 MR cells). Continuous superfusion of the MR cell during recording increased the current to -7.99 +/- 1.48 nA/cell (N = 10 MR cells). The time course of the reversible activation of G(dynamic) varied among cells, but was usually sigmoid with T1/2 decreasing with Vp (-25 > or = Vp > or = -100 mV). T1/2 was in the order of 10 sec at Vp = -100 mV. The single-MR-cell currents recorded in this study are fully compatible with Cl- currents estimated by relating density of MR cells to transepithelial ICl or by measurements with the self-referencing ('vibrating') probe technique. In the discussion, Ussing's work on epithelial shunt pathways is considered. His thinking and experiments leading to his theory of isotonic transport in leaky epithelia is emphasized. It is our thesis that the understanding of the physiology of epithelia owes as much to Ussing's studies of shunt pathways as to his studies of the active sodium pathway.

Results and complications of tension-free vaginal tape (TVT) for surgical treatment of female stress urinary incontinence.

Thirty-one patients with stress urinary incontinence were operated on using tension-free vaginal tape (TVT). All were evaluated preoperatively with urodynamics, pad test and stress test. Conservative treatment was without significant effect. Three months after the operation no patients had stress incontinence but I with mixed incontinence experienced deterioration of her urge incontinence and 2 experienced de novo urge incontinence. The de novo urge incontinence was significantly improved and the urodynamic investigation normal after approximately 5 months. One patient with a previous operation with Kelly sutures under the urethra developed a urethrovaginal fistula. Fifteen patients were observed for 1 year. One patient who was continent after 3 months developed slight stress incontinence.