Enzymatic analysis of Blomia tropicalis and Blomia kulagini (Acari: Echimyopodidae) allergenic extracts obtained from different phases of culture growth

The majority of important allergenic extracts from arthropods present enzymatic activity. This activity has been studied particularly in Dermatophagoides house dust mites because of its implication in the stability and immunogenicity of extracts used as tools for the diagnosis and specific treatment of allergic diseases. Extracts from cultures of Blomia tropicalis [van Bronswijk (1973a, b). Acarologia 15:477–489, 490–505] and Blomia kulagini (Zakhvatkin 1936) were used to study enzymatic profiles during three growth periods of the mite population: latency phase, maximum mite concentration during exponential growth, and drop stage. The activities of 19 enzymes were analyzed using the Api Zym system. The results show a large variety of enzymes. Some enzymatic activity was found to be (almost) exclusively attributable to mites. The activity levels of proteases, glycosidases and lipases overlapped with the growth curve. Only phosphatase activity showed no significant change during mite growth when compared with the culture medium. We suggest that the glycosidases (β-galactosidase, β-glucuronidase, β-N-acetylglucosaminidase, α-mannosidase and α-fucosidase) and proteases (leucine aminopeptidase and trypsin) may constitute suitable parameters for inclusion in the quality control process for the production of allergenic mite extracts, and may help define a new index for conducting environmental controls.     

GLYCOSIDASES IN NORMAL AND SCRAPIE MOUSE BRAIN

Abstract— The pH optima of ten glycosidases have been determined in normal and scrapie-affected mouse brain. The enzymes α-mannosidase, α-glucosidase and β-glucosidase displayed two peaks of enzyme activity over the pH range examined.
There is a significant increase in the activity of the enzymes α-mannosidase, β-glucuronidase, N -acetyl β-D-glucosaminidase, N -acetyl β-galactosaminidase, β-glucosidase (pH 4.1), α-fucosidase and β-xylosidase in the brains of mice clinically affected with scrapie, whilst only α-mannosidase (pH 4.1), β-glucuronidase, N -acetyl-β-D-glucosinidase and N- acetyl-β-D-gaiactosaminidase are elevated before mice exhibit signs of the disease.     

Modulatory Action of α-Tocopherol on Erythrocyte Membrane Adenosine Triphosphatase against Radiation Damage in Oral Cancer

To investigate the possible effects of α-tocopherol on erythrocyte membrane adenosine triphosphatases against radiation damage in oral cancer patients. Adenosine triphosphatase activities were analysed in oral cancer patients before and after radiotherapy (at a dosage of 6000 cGY in five fractions per week for a period of six weeks) and after supplemented with α-tocopherol (400 IU per day for entire period of radiotherapy). The membrane bound enzymes such as Na⁺/K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and some trace elements were altered in oral cancer patients before and after radiotherapy. Supplemented with α-tocopherol modulates the erythrocyte membrane which is damaged by radiotherapy which suggests that α-tocopherol protects the erythrocyte membrane from radiation damage in oral cancer patients.     

Raman spectroscopic study of space structure of membrane proteins and membrane lipids in photodamaged human erythrocyte sensitized by hypocrellin B

Ｈｙｐｏｃｒｅｌｌｉｎａｎｄｉｔｓｄｅｒｉｖａｔｉｖｅｓａｒｅｗｅｌｌｋｎｏｗｎｐｈｏｔｏｓｅｎｓｉｔｉｚｅｒｓ[1,2].ＡｍｏｎｇｔｈｅｍｂｏｔｈｈｙｐｏｃｒｅｌｌｉｎＡ(ＨＡ)ａｎｄｈｙｐｏｃｒｅｌｌｉｎＢ(ＨＢ)ｓｈｏｗｐｒｏｍｉｓｉｎｇａｎｔｉｃａｎｃｅｒａｎｄａｎｔｉｖｉｒａｌａｂｉｌｉｔｙ[1—3].Ｉｎｏｒｄｅｒｔｏｉｎｖｅｓｔｉｇａｔｅｉｔｓｍｅｃｈａｎｉｓｍｓ,ｍａｎｙｍｅｔｈｏｄｓｈａｖｅｂｅｅｎｕｓｅｄｔｏｄｅｔｅｃｔｔｈｅａｃｔｉｖｅｏｘｙｇｅｎｓｕｃｈａｓ1Ｏ2,Ｏ2.-,.ＯＨａｎｄｓｅｍ…     

Biochemical Changes Associated with Brassica juncea Seed Development, II: Glycosidases

Changes in α- and β-galactosidase, glucosidase, and α-mannosidase and β-xylosidase activities were analyzed in developing mustard (Brassica juncea) seed. A cubic polynomial described the seed dry weight data adequately. A close parallelism between the water content and dry matter accumulation was shown (r= 0.991). Glycosidase activities were detected in both cytoplasmic and wall fractions. The trend was similar in both of the fractions, but the activity of α-mannosidase and α-galactosidase was considerably greater in the wall-bound fraction. The water content and the activity of glycosidic enzymes showed a significant linear correlation (p < 0.001). The results suggest that glycosidases have an important role in cell wall loosening during mustard seed development. Received July 10, 1997; accepted January 28, 1998     

Comparative Characterization of Laminarinases from the Filamentous Marine Fungi Chaetomium Indicum Corda and Trichoderma Aureviride Rifai

Marine filamentous fungi (103 strains) isolated from various marine habitats were studied for their ability to produce extracellular O-glycosylhydrolases. Cultural filtrates of these strains were shown to contain a series of glycanases (laminarinases, amylases, cellulases, pustulanases) and glycosidases (β-glucosidases, N-acetyl-β-glucosaminidases, β-galactosidases, α-mannosidases).Two species of marine fungi from different habitats were chosen for isolation of laminarinases and detailed study on enzyme properties. The fungus Chaetomium indicum associated with the alga Fucus evanescens C. Agardh was collected near the Kuril Islands, and T. aureviride was sampled from bottom deposits of South China Sea. Properties of extracellular laminarinases were similar: temperature optimums (40–45 ^∘C), molecular masses (54–56 kDa), K _m (0.1–0.3 mg ml⁻¹). Temperature stability of laminarinase of C. indicum was significantly higher than those from T. aureveride. It is shown that these enzymes are specific to β-1,3-bonds in glucans, release predominantly glucose from laminaran and do not catalyze reaction of transglycosylation. Accoding to these data enzymes are exo-1,3-β-D-glucan-glucanohydrolases (EC 3.2.1.58). Inhibitor analysis demonstrated the significant role of tryptophan and tyrosine residues in the catalytic activity of enzymes. Molecules of T. aureviride laminarinase contained the functionally important thiol group.     

Biosynthesis,processing, and extracellular release of α-l-fucosidase in lymphoid cell lines of different genetic origins

In humans, the quantity of α-l-fucosidase in serum is determined by heredity. The mechanism controlling levels of the enzyme in serum is unknown. Lymphoid cell lines derived from individuals with either low, intermediate, or high α-l-fucosidase in serum were established. Steady-state levels of intracellular and extracellular α-l-fucosidase as well as rates of synthesis and secretion of enzyme overlapped among the cell lines. Thus,_vivo} serum phenotypes were not expressed in this system. No appreciable differences in the qualitative processing of newly made α-l-fucosidase were observed among these lymphoid cell lines. Cells pulse-labeled with³⁵S-methionine from 0.25 to 2 hr had an intracellular form of enzyme with aM _r=58,000. Cells pulsed for 1.5 hr and chased for 21 hr with unlabeled methionine had an intracellular form ofM _r=60,000 and an extracellular form ofM _r=62,000. All three enzyme forms were glycoproteins with a common polypeptide chain ofM _r=52,000 but with different carbohydrate moieties. No evidence for a high molecular mass precursor form of α-l-fucosidase was found. Fucosidosis is a rare, inherited disease in which α-l-fucosidase activity in tissues and body fluids is low or absent. The mutations for fucosidosis and the serum polymorphism map separately. Lymphoid cells from two siblings with fucosidosis had 8-fold to 341-fold less intracellular α-l-fucosidase protein with 11-fold to 56-fold lower specific activities than control cells. Residual mutant enzyme was a glycoprotein with a polypeptide chain virtually the same size (M _r=52,000) as control enzyme. However, residual mutant enzyme was hypoglycosylated and hypersecreted as compared to control enzyme. This research was supported by National Institutes of Health Grant DK 32161.     

Establishment of an abalone digestive gland cell line secreting various glycosidases in protein-free culture

A cell line designated as ADG was established from an abalone digestive gland using ERDF medium supplemented with 8% fetal bovine serum (FBS), 8% abalone hemolymph, and high concentrations of NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂. ADG cells proliferated better in protein-free medium than in FBS-supplemented medium. Among 9 kinds of media examined, ERDF medium was shown to be optimal for cell growth. ADG cells secreted 13 different kinds of glycosidases in protein-free medium: α-L-fucosidase, β-L-fucosidase, α-D-galactosidase, β-D-galactosidase, N-acetyl-α-D-galactosaminidase, N-acetyl-β-D-galactosaminidase, α-D-glucosidase, β-D-glucosidase, N-acetyl-α-D-glucosaminidase, N-acetyl-β-D-glucosaminidase, α-D-mannosidase, β-D-mannosidase, β-D-xylosidase, and 1-3 xylanase. When ADG cells were cultured in Grace’s insect cell medium, the activity of some secreted glycosidases increased 25-fold to 65-fold per cell as compared with control cells cultured in ERDF medium. ADG - abalone digestive gland; ERDF - enriched RDF; FBS - fetal bovine serum; L-15 - Leibovitz’s L-15 media; DME - Dulbecco’s modified Eagle medium; F-12 - nutrient mixture (Ham); LDF - L-15; DME: F-12 = 10 : 7 : 3; MEM - minimum essential medium; RPMI - RPMI medium 1640; 199 - media 199; GIC - Grace’s insect cell medium; pNP -p -nitrophenol. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhancement of α- and β-Galactosidase Activity in <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> by Different Metal Ions

The hydrolysis of oligosaccharides and lactose is of great importance to the food industry. Normally, oligosaccharides like raffinose, stachyose, and verbascose which are rich in different plants like soy bean are considered indigestible by the human gut. Moreover, many humans suffer from lactose intolerance due to the absence of effective enzyme that can digest lactose. α-Galactosidase can digest oligosaccharides like raffinose, while β-galactosidases can hydrolyze lactose. Therefore, selection of microorganisms safe for human use and capable of producing high levels of enzymes becomes an attractive task. The objective of this study was to investigate the enhancement of α- and β-galactosidase activity in Lactobacillus reuteri by different metal ions. Ten millimolar of Na⁺, K⁺, Fe²⁺, and Mg²⁺ and 1 mM of Mn²⁺ were added separately to the growth culture of six strains of L. reuteri (CF2-7F, DSM20016, MF14-C, MM2-3, MM7, and SD2112). Results showed that L. reuteri CF2-7F had the highest α- and β-galactosidase activity when grown in the medium with added Mn²⁺ ions (22.7 and 19.3 Gal U/ml, respectively). 0.0274% of Mn²⁺ ions lead to 27, 18% enhancement of α- and β-galactosidase activity over the control group, and therefore, it could be added to the growth culture of CF2-7F to produce enhanced levels of α- and β-galactosidase activity. The addition of Fe²⁺ led to a significant (P < 0.01) decrease in the activity of both enzymes for most strains. This study shows that modified culture medium with that 0.0274% Mn²⁺ can be used to promote the production for α- and β-galactosidase in L. reuteri CF2-7F, which may lead to enhancement of α- and β-galactosidase activity and have a good potential to be used in the food industry.     

Glycosidases of apple fruit: A multi-functional β-galactosidase

Extraction of Spartan apple ( Malus domestica ) fruit acetone powder and fractionation of the extract on DEAE-agarose allowed detection and quantification of 10 glycosidases active toward 4-methylumbelliferyl glycosides. Hydrolysis was measured fluorimetrically. The predominant activity, a β- d -galactosidase (EC 3.2.1.23), labile upon purification, was stabilized by soluble PVP. Molecular weights, measured by gel permeation HPLC, pH optima and K_m values were obtained for most glycosidase activities. Multiple forms of several activities were found. The major α- d - and β- d -galactosidases were resolved on phosphocellulose. The β- d -galactosidase so obtained had associated α- l -arabinopyranosidase and β- d -fucosidase activities which were retained upon GP-HPLC. Mixed substrate kinetic analysis and inhibition analysis of this fraction indicated that the enzyme has 3 catalytic sites, 1 for each substrate, whose substrates mutually influence each other's activity positively.     

Membrane destabilization induced by the human immunodeficiency virus type-1 fusion peptide

Summary The human immunodeficiency virus type-1 (HIV-1) fusion peptide, corresponding to a sequence of 23 amino acid residues at the N-terminus of the spike transmembrane subunit gp41, has the capacity to destabilize negatively charged and neutral large unilamellar vesicles, representing, respectively, the acidic and the neutral fraction of the plasma membrane lipids of viral target cells. As revealed by infrared spectroscopy, the peptide associated with the vesicles may exist in different conformations. In negatively charged membranes the structure is mainly an α-helix, while in Ca²⁺-neutralized negatively charged membranes the conformation switches to a predominantly extended conformation. In membranes composed of zwitterionic phospholipids and cholesterol, the peptide also adopts a predominant extended structure. The α-helical structure permeabilizes negatively charged vesicles but does not induce membrane fusion. The peptide in β-type conformation, on the other hand, permeabilizes neutral membranes and triggers fusion. As seen by³¹P NMR, the latter structure also exhibits the capacity to alter the lamellar organization of the membrane.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Estradiol inhibits the effects of extracellular ATP in human sperm by a non genomic mechanism of action

Steroid hormones, beside their classical genomic mechanism of action, exert rapid, non genomic effects in different cell types. These effects are mediated by still poorly characterized plasma membrane receptors that appear to be distinct from the classic intracellular receptors. In the present study we evaluated the non genomic effects of estradiol (17βE₂) in human sperm and its effects on sperm stimulation by extracellular ATP, a potent activator of sperm acrosome reaction. In human sperm 17βE₂ induced a rapid increase of intracellular calcium (Ca²⁺) concentrations dependent on an influx of Ca²⁺ from the extracellular medium. The monitoring of the plasma membrane potential variations induced by 17βE₂ showed that this steroid induces a rapid plasma membrane hyperpolarization that was dependent on the presence of Ca²⁺ in the extracellular medium since it was absent in Ca²⁺ free-medium. When sperm were pre-incubated in the presence of the K⁺ channel inhibitor tetra-ethylammonium, the 17βE₂ induced plasma membrane hyperpolarization was blunted suggesting the involvement of K⁺ channels in the hyperpolarizing effects of 17βE₂. Extracellular ATP induced a rapid plasma membrane depolarization followed by acrosome reaction. Sperm pre-incubation with 17βE₂ inhibited the effects of extracellular ATP on sperm plasma membrane potential variations and acrosome reaction. The effects of 17βE₂ were specific since its inactive steroisomer 17αE₂ was inactive. Furthermore the effects of 17βE₂ were not inhibited by tamoxifen, an antagonist of the classic 17βE₂ intracellular receptor.     

A study of the enzymatic profile of soil isolates of Nocardia asteroides

In this study, using the API-ZYM system, we have reported the enzyme profile of 42 soil strains and 2 clinical strains of Nocardia asteroides isolated locally. Of the 19 enzymes tested, only 7 were demonstrable in over 90% of the soil isolates. These included alkaline phosphatase, esterase lipase, leucine arylamidase, acid phosphatase, phosphohydrolase, α-glucosidase and β-glucosidase. In addition, β-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-β-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in >95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-glucoronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. With the exception of valine arylamidase, which was lacking in all but one isolate, the enzyme profiles of the soil isolates were comparable with the clinical isolates of N. asteroides reported in previous studies. The reasons for this difference in the two sets of isolates is not clear. The study reinforces the view that specific differences in the enzymatic profiles of Nocardia species could be used for their rapid identification. However, more extensive studies are needed to establish the reproducibility of this method. To the best of our knowledge, this is the first study of the enzymatic profile of soil isolates of N. asteroides originating from a single geographic region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Studies on cell wall loosening enzymes in hormone treated internodes of <Emphasis Type="Italic">Merremia emarginata</Emphasis>

The cell wall loosening enzymes viz. glycosidases, polygalacturonase and xylanase were analyzed in cytoplasmic and wall bound fractions extracted from control and hormone (GA₃ NAA, PAA) treated internodes, as they are known to play a key role in cell wall metabolism. Among the glycosidases, wall bound β-glucosidase and α-galactosidase activities were significantly correlated with age of control internodes. Cytoplasmic α-galactosidase showed significant correlation in hormone treated internodes. Maximum correlation was observed in GA₃, followed by PAA and NAA. Wall bound xylanase activity was well correlated with length only in NAA treated internodes and less after GA₃ treatment while cytoplasmic xylanase showed correlation with intrnode length only in control and after NAA treatment. Cytoplasmic polygalacturonase showed correlation with internode length only after GA₃ treatment while wall bound polygalacturonase showed correlation with internode length after NAA treatment. The possible role of these enzymes in internode development is discussed.     

Short-term stretch translocates the alpha-1-subunit of the Na pump to plasma membrane

Short-term (2–30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na⁺,K⁺-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na⁺,K⁺-ATPase α-1-subunit protein. Membrane marker enzyme, 5′ nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na⁺,K⁺-ATPase α-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na⁺,K⁺-ATPase α-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5′ nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the α-subunit of the enzyme from endosomes to the plasma membrane.     

Laminin chains in the basement membranes of human thymus

Summary In recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β₄ integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.     

The effect of the lipid-binding site of the ankyrin-binding domain of erythroid β-spectrin on the properties of natural membranes and skeletal structures

It was previously shown that the beta-spectrin ankyrin-binding domain binds lipid domains rich in PE in an ankyrin-dependent manner, and that its N-terminal sequence is crucial in interactions with phospholipids. In this study, the effect of the full-length ankyrin-binding domain of β-spectrin on natural erythrocyte and HeLa cell membranes was tested. It was found that, when encapsulated in resealed erythrocyte ghosts, the protein representing the full-length ankyrin-binding domain strongly affected the shape and barrier properties of the erythrocyte membrane, and induced partial spectrin release from the membrane, while truncated mutants had no effect. As found previously (Bok et al. Cell Biol. Int. 31 (2007) 1482–94), overexpression of the full-length GFP-tagged ankyrin-binding domain aggregated and induced aggregation of endogenous spectrin, but this was not the case with overexpression of proteins truncated at their N-terminus. Here, we show that the aggregation of spectrin was accompanied by the aggregation of integral membrane proteins that are known to be connected to spectrin via ankyrin, i.e. Na⁺K⁺ATP-ase, IP3 receptor protein and L1 CAM. By contrast, the morphology of the actin cytoskeleton remained unchanged and aggregation of cadherin E or N did not occur upon the overexpression of either full-length or truncated ankyrin-binding domain proteins. The obtained results indicate a substantial role of the lipid-binding part of the β-spectrin ankyrin-binding domain in the determination of the membrane and spectrin-based skeleton functional properties.     

Palytoxin Acts on Na+,K+-ATPase but not Nongastric H+,K+-ATPase

Palytoxin (PTX) opens a pathway for ions to pass through Na,K-ATPase. We investigate here whether PTX also acts on nongastric H,K-ATPases. The following combinations of cRNA were expressed in Xenopus laevis oocytes: Bufo marinus bladder H,K-ATPase α₂- and Na,K-ATPase β₂-subunits; Bufo Na,K-ATPase α₁- and Na,K-ATPase β₂-subunits; and Bufo Na,K-ATPase β₂-subunit alone. The response to PTX was measured after blocking endogenous Xenopus Na,K-ATPase with 10 μm ouabain. Functional expression was confirmed by measuring ⁸⁶Rb uptake. PTX (5 nm) produced a large increase of membrane conductance in oocytes expressing Bufo Na,K-ATPase, but no significant increase occurred in oocytes expressing Bufo H,K-ATPase or in those injected with Bufo β₂-subunit alone. Expression of the following combinations of cDNA was investigated in HeLa cells: rat colonic H,K-ATPase α₁-subunit and Na,K-ATPase β₁-subunit; rat Na,K-ATPase α₂-subunit and Na,K-ATPase β₂-subunit; and rat Na,K-ATPase β₁- or Na,K-ATPase β₂-subunit alone. Measurement of increases in ⁸⁶Rb uptake confirmed that both rat Na,K and H,K pumps were functional in HeLa cells expressing rat colonic HKα₁/NKβ₁ and NKα₂/NKβ₂. Whole-cell patch-clamp measurements in HeLa cells expressing rat colonic HKα₁/NKβ₁ exposed to 100 nm PTX showed no significant increase of membrane current, and there was no membrane conductance increase in HeLa cells transfected with rat NKβ₁- or rat NKβ₂-subunit alone. However, in HeLa cells expressing rat NKα₂/NKβ₂, outward current was observed after pump activation by 20 mm K⁺ and a large membrane conductance increase occurred after 100 nm PTX. We conclude that nongastric H,K-ATPases are not sensitive to PTX when expressed in these cells, whereas PTX does act on Na,K-ATPase.     

Characterization of neuronal zebra finch GABAA receptors: steroid effects

Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize γ-aminobutyric acid (GABA) type A receptors (GABA_A) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation by 17β-estradiol(E₂), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked currents were inhibited by picrotoxin (10 μmol l⁻¹) and bicuculline methiodide (10 μmol l⁻¹) and potentiated by pentobarbital (100 μmol l⁻¹) and propofol (3 μmol l⁻¹). Loreclezole (10 μmol l⁻¹) potentiated GABA-evoked currents, suggesting the presence of β₂, β₃ and/or β₄ subunits. Diazepam (1 μmol l⁻¹) potentiated currents, while Zn²⁺ (1 μmol l⁻¹) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l⁻¹) potentiated currents, whereas E₂ (1 μmol l⁻¹), 5α- and 5β-DHT (1 μmol l⁻¹), and corticosterone (10 μmol l⁻¹) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABA_A receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little or no acute modulation by sex steroids or corticosterone. Accepted: 12 November 1997