Cloning and function of the rat colonic epithelial K+ channel K(V)LQT1

K(V)LQT1 (K(V)LQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K+ channel in the colonic epithelium. We now report cloning and expression of K(V)LQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K(V)LQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K(V)LQT1 in crypt cells and surface epithelium. Expression of rK(V)LQT1 in Xenopus oocytes induced a typical delayed activated K+ current. that was further activated by increase of intracellular cAMP but not Ca2+ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K+ conductance in the colonic mucosal epithelium and inhibited whole cell K+ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K(V)QT1 is forming an important component of the basolateral cAMP-activated K+ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.

Electrolyte transport in the mouse trachea: No evidence for a contribution of luminal K+ conductance

Recent studies on frog skin acini have challenged the question whether Cl- secretion or Na+ absorption in the airways is driven by luminal K+ channels in series to a basolateral K+ conductance. We examined the possible role of luminal K+ channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl- secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+)2Cl(-)K(+) cotransporter azosemide. Similarly, the compound 29313, a blocker of basolateral KCNQ1/KCNE3 K+ channels effectively blocked Cl- secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K+ channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K+ channels in mouse airways, using luminal 29313, clotrimazole and Ba2+ or different K+ channel toxins such as charybdotoxin, apamin and alpha-dendrotoxin. Thus, the present study demonstrates Cl- secretion in mouse airways, which depends on basolateral Na(+)2Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl- channels. Cl- secretion is maintained by the activity of basolateral K+ channels, while no clear evidence was found for the presence of a luminal K+ conductance.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

GFP-tagged CFTR transgene is functional in the G551D cystic fibrosis mouse colon

Trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) is central to its function, with the most common mutation, DeltaF508, resulting in abnormal processing and trafficking. Therefore, there is a significant need to develop tools, which enable the trafficking of CFTR to be studied in vitro and in vivo. In previous studies it has been demonstrated that fusion of the green fluorescent protein (GFP) to the N-terminus of CFTR does lead to functional expression of CFTR chloride channels in epithelial cell lines. The aim of the present study was to examine whether it is possible to express GFP-tagged CFTR as a transgene in colonic and airway epithelial cells of cystic fibrosis (CF) mice and to correct the CF defect. Using the epithelial-specific human cytokeratin promoter K18, we generated bitransgenic mice cftr(G551D/G551D) K18-GFP-CFTR+/-, designated GFP mice. Transcripts for GFP-CFTR could be detected in bitransgenic mice by use of RT-PCR techniques. Expression of GFP-CFTR protein was detected specifically in the colonic epithelium by both direct GFP fluorescence and the use of an anti-GFP antibody. Ussing chamber studies showed that the ion transport defect in colon and airways observed in cftr(G551D/G551D) mice was partially corrected in the bitransgenic animals. Thus, K18-GFP-CFTR is functionally expressed in transgenic mice, which will be a valuable tool in studies on CFTR synthesis, processing and ion transport in native epithelial tissues.

Transient response of diffusion cell containing a porous solid

Transient response of an adsorbing or non-adsorbing tracer injected as step or square pulse input in a diffusion cell with two flowing streams across the pellet is theoretically investigated in this paper. Exact solutions and the asymptotic solutions in the time domain and in three different limits are obtained by using an integral transform technique and a singular perturbation technique, respectively. Parametric dependence of the concentrations in the top and bottom chambers can be revealed by investigating the asymptotic solutions, which are far simpler than their exact counterpart. In the time domain investigation, it is found that the bottom-chamber concentration is very sensitive to the value of the macropore effective diffusivity. Therefore this concentration could be used to extract diffusivity by fitting in the time domain. The bottom-chamber concentration is also sensitive to flow rate, pellet length chamber volume and the type of input (step and square input).

Anatomy of the mouthparts and digestive tract during feeding in larvae of the parasitoid wasp Trichogramma australicum Girault (Hymenoptera : Trichogrammatidae)

To aid in the development of artificial diets for mass rearing parasitioids, we investigated the anatomical changes in the digestive tract during feeding behaviour of larval Trichogramma australicum (Hymenoptera: Trichogrammatidae). Larvae begin to feed immediately upon eclosion and feed continuously for 4 h until replete. Feeding is characterised by rhythmic muscle contractions (ca 1 per s) of the pharynx. Contractions of the pharyngeal dilator muscles lift the roof of the lobe-shaped pharynx away from the floor of the chamber, opening the mouth and pumping food into the pharyngeal cavity. Another muscle contraction occurs about 0.5 s later, forcing the bolus of food through the oesophagus and into the midgut. The junction of fore- and midgut is marked by a cardiac valve. The midgut occupies most of the body cavity and is lined with highly vacuolated, flattened cells and a dispersed layer of muscle cells. In the centre of the midgut, food has the appearance of host egg contents. Food near the midgut epithelial cells has a finer, more homogeneous appearance. This change in the physical properties of the gut contents is indicative of the digestion process. In the prepupa, where digestion is complete, the entire gut contents have this appearance. After eclosion, the vitelline membrane remains attached to the posterior end of the larva. We believe this attachment to be adaptive in two ways: (1) to anchor the larva against the movements of its anterior portion, thereby increasing the efficiency of foraging within the egg, and (2) to prevent a free-floating membrane from clogging the mouthparts during ingestion. 1998 Elsevier Science Ltd. All rights reserved.

Role of chemical cues from cotton in mediating host selection and oviposition behaviour in Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

We investigated the role of chemoreception in the host selection and oviposition behaviour of Helicoverpa armigera in the laboratory using five cotton genotypes and synthetic volatile terpenes. Female moths oviposited on substrates treated with methanol, ethanol, acetone and pentane extracts of leaves, squares and flowers of the cotton genotypes. Phytochemicals soluble in pentane were the most efficient in eliciting oviposition behaviour. In a two-way bioassay, pentane extracts of leaves or squares of a Multiple Host-plant Resistance genotype (MHR11), Deltapine commercial (DP90), and Smith Red Leaf (SRL) received significantly more eggs than solvent-treated controls. Extracts of squares of the native genotype Gossypium nelsonii did not receive more eggs. Females preferred DP90 and MHR11 to SRL and G. nelsonii. Female moths also laid more eggs on pentane extracts of MHR11 flowers than MHR11 leaves from preflowering, early flowering and peak-flowering plants. In a flight chamber, female moths used olfactory cues at short range to mediate oviposition and discrimination between host plants. Egg-laying, mated females were attracted at a distance (1.5 m) to volatile compounds released by whole plants and odours emanating from filter papers treated with synthetic volatile terpenes. Individually, the terpenes did not stimulate any significant oviposition response. However, there was a significant oviposition response to a mixture of equal volumes of the terpenes (trans-beta-caryophyllene, alpha-pinene, beta-pinene, myrcene, beta-bisabolol, and alpha-humulene). Conversely, antennectomised (moths with transected antennae), egg-laying, mated females did not stimulate any significant oviposition response. The significance of these findings in relation to H. armigera hostplant selection are discussed.

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neo-goniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H, curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in H. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

Implications of specimen preparation and of surface contamination for the measurement of the grain boundary carbon concentration of steels using x-ray microanalysis in an UHVFESTEM

The purpose of the present investigation was to gain an understanding of the nature of the carbon contamination on the surface of standard steel transmission electron spectroscopy (TEM) specimens, the effect of exposure of a clean specimen to normal laboratory air, and the efficacy of plasma-cleaning treatments. This knowledge is a necessary prerequisite to the development of appropriate specimen preparation and/or specimen cleaning methods. X-ray photoelectron spectroscopy in combination with argon ion beam profiling was used to characterize the specimen surfaces of X65 steel and 316 stainless steel. The only clean carbon-free surface obtained was that during argon etching of the sample in the surface analysis chamber. Any exposure of a previously cleaned sample to laboratory air resulted in a rapid carbon (hydrocarbon) contamination of the sample surface and the development of surface oxidation, Plasma cleaning with subsequent exposure of the specimen to the laboratory air also resulted in a carbon-contaminated surface. This suggests that procedures of preparation of TEM specimens of steels outside an ultrahigh vacuum chamber are unlikely to result in the lowering of contamination rates on specimens to levels where measurements for carbon in the grain boundaries are possible. What is needed is a cleaning system as an integral part of the specimen insertion system into the field-emission scanning transmission electron microscope. This cleaning could be carried out by argon ion etching. Copyright (C) 2000 John Wiley & Sons, Ltd.

Neurons in the hilus region of the rat hippocampus are depleted in number by exposure to alcohol during early postnatal life

We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life resulted in a deficit in spatial learning ability. This ability is controlled, at least in part, by the hippocampal formation. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life affected the number of specific neurons in the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol between postnatal days 10 and 15 by placing them for 3 h each day in a chamber containing ethanol vapor. The blood ethanol concentration was about 430 mg/dl at the end of the exposure period. Groups of ethanol-treated (ET) rats, separation controls (SC), and mother-reared controls (MRC) were anesthetized and killed at 16 days of age by perfusion with phosphate-buffered glutaraldehyde (2.5%). The Cavalieri principle was used to determine the volume of various subdivisions of the hippocampal formation (CA1, CA2+CA3, hilus, and granule cell layer), and the physical disector method was used to estimate the numerical densities of neurons within each subdivision. The total number of neurons was calculated by multiplying estimates of the numerical density with the volume. There were, on average, about 441,000 granule cells in the granule cell layer and 153,000 to 177,000 pyramidal cells in both the CA1 and CA2+CA3 regions in all three treatment groups. In the hilus region, ET rats had about 27,000 neuronal cells. This was significantly fewer than the average of 38,000 such neurons estimated to be present in both MRC and SC animals. Thus, neurons in the hilus region may be particularly vulnerable to the effects of a high dose of ethanol exposure during early postnatal life. (C) 2000 Wiley-Liss, Inc.

Morphology of the cloaca in the estuarine crocodile, Crocodylus porosus, and its plastic response to salinity

The cloacal complex of Crocodylus porosus is composed of three chambers (proctodaeum, urodaeum, and coprodaeum) separated by tight, muscular sphincters. The proctodaeum is proximal to the cloacal vent and houses the genitalia. The urodaeum is the largest chamber, is capable of storing large quantities of urine, and is lined with an epithelium with the capacity for transepithelial water and ion exchange. The coprodaeum, the most orad cloacal chamber, is a small, only marginally expandable chamber that has an epithelium composed almost entirely of mucus-secreting cells. The coprodaeum and lower intestine are reported to be the site(s) for urine modification in birds and bladderless lizards. A radiographic trace of urine storage in C. porosus kept for 2 months under hyperosmotic conditions showed no signs of retrograde movement of urine into the coprodaeum or rectum. Instead, urine was stored in the urodaeum of C. porosus. Examination of the mucosal surface of the urodaeum by SEM showed a plastic response to environmental salinity, with a possible increase in surface area in animals kept in hyperosmotic water compared with animals from fresh water. We propose the urodaeum as the primary site for postrenal modification of urine in C, porosus. (C) 2000 Wiley-Liss, Inc.