Differential Effects of Hypoxia on Ligand Binding Properties of Nicotinic and Muscarinic Acetylcholine Receptors on Cultured Bovine Adrenal Chromaffin Cells

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l -[³H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and ²²Na⁺ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O₂/79% N₂ (control) or 100% N₂ (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the K_D under hypoxic conditions was twice as large as that under control conditions, whereas the B _max was unchanged. When the specific [³H]nicotine binding was kinetically analyzed, the association constant ( k ₁) but not the dissociation constant ( k ₋₁) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine-evoked ²²Na⁺ flux into the cells was suppressed by hypoxia. In contrast, specific [³H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.     

Block by 5-hydroxytryptamine of neuronal acetylcholine receptor channels expressed inXenopus oocytes

Summary 1. Effects of 5-hydroxytryptamine (5-HT) on neuronal nicotinic acetylcholine (ACh) receptor channels were investigated by expressing cloned channel subunits inXenopus oocytes.2. When channels were expressed with a combination of ₃ and ₄ subunits, 5-HT (10 to 300 µM) reversibly inhibited an inward current activated by 100 µM ACh in a concentration-dependent manner. The inhibition was also observed when ₃ subunit was combined with ₂ subunit instead of ₄ subunit, or ₄ subunit was combined with ₂ or _4–1 subunit instead of ₃ subunit to express channels.3. Compounds known to antagonize at 5-HT receptors (LY53857, metoclopramide and propranolol) exhibited an agonistic effect: they inhibited the ACh-activated current.4. The results suggest that 5-HT inhibits recombinant neuronal nicotinic receptor channels through a binding-site distinct from conventional 5-HT receptors. The binding-site may not be attributed to a unique type of channel subunits.     

Effects of surfactant proteins SP-B and SP-C on dynamic and static mechanics of immature lungs

Kobayashi, Tsutomu, Katsumi Tashiro, Ken Yamamoto, ShunichiNitta, Shigeo Ohmura, and Yasuhiro Suzuki. Effects of surfactant proteins SP-B and SP-C on dynamic and static mechanics of immature lungs. J. Appl. Physiol. 83(6):1849-1856, 1997.To investigate the effects of surfactantproteins B (SP-B) and C (SP-C) on lung mechanics, we compared tidal andstatic lung volumes of immature rabbits anesthetized with pentobarbitalsodium and given reconstituted test surfactants (RTS).With a series of RTS having various SP-B concentrations (0-0.7%)but a fixed SP-C concentration (1.4%), both the tidal volume with25-cmH₂O insufflation pressure and the static volume deflated to5-cmH₂O airway pressure increased, significantly correlating with the SP-B concentration: the former increased from 6.5 to 26.0 ml/kg (mean), and the latter increased from6.4 to 31.8 ml/kg. With another series of RTS having afixed SP-B concentration (0.7%) but various SP-C concentrations(0-1.4%), the tidal volume increased from 5.1 to 24.8 ml/kg,significantly correlating with the SP-C concentration, whereas thestatic volume increased from 3.4 to 32.0 ml/kg, the ceiling value, inthe presence of a minimal concentration of SP-C (0.18%). Inconclusion, certain doses of SP-B and SP-C were indispensable foroptimizing dynamic lung mechanics; the static mechanics, however,required significantly less SP-C.

     

Localization of flagellin genes on the physical map of Methanococcus voltae

Methanococcus voltae DNA, digested individually with the restriction enzymes ApaI, SacII, BamHI, or EagI, was resolved by pulsed-field gel electrophoresis reproducing the previously published digestion patterns. Hybridization of a flagellin gene-specific probe to such gels dried down (unblots) resulted in the identification of one band per enzyme harboring the flagellin genes. These bands all overlapped, revealing that an approximately 15-kb BamHI/EagI DNA fragment should harbor the flagellin genes. Double digestion with BamHI and EagI resulted in the resolution of two bands in the 15-kb region of the gel. Separation of these two fragments prior to blotting and probing with a flagellar gene-specific probe revealed that one of these fragments possessed the flagellar sequences. The presence of an EagI restriction site in flaB3 localized the flagellin genes precisely at the junction of EagI fragments Ea2 and Ea5 at approximately the 1800-kb position of the physical map.     

An improved method for cell and spore counts during aerobic cultivation of crystal toxin producingBacillus sphaericus

Summary A simple improved method including sonication treatment is proposed to determine accurate cell and spore counts forBacillus sphaericus, a microorganism which can form cell aggregates. Sonication (10 watt power output) for 40 seconds after dilution of culture broth was effective in dispersing clumps of cells and spores without disruption. This improved method gave approximately 2 times higher cell and spore counts compared with the conventional counting methods (without sonication).     

A comparative dynamic study of the effectiveness of gastric cytoprotection by vitamin A, De-Nol, sucralfate and ulcer healing by pirenzepine in patients with chronic gastric ulcer (a multiclinical and randomized study)

The gastric cytoprotective effects of vitamin A, De-Nol and sucralfate were compared with the effectiveness of pirenzepine in healing ulcer in patients with chronic gastric ulcer. A total of 100 patients was randomized into different groups: the patients were treated with antacids, vitamin A (3 X 50.000 IU), De-Nol liquid (4 X 5 ml), sucralfate (4 X 1 g) or pirenzepine (3 X 50 mg). The treatment was continued for 4 weeks. At the beginning, 2 and 4 weeks after starting treatment the patients were subjected to endoscopy and the size of the ulcer was measured planimetrically. The ulcer-healing effect of De-Nol liquid was significantly better than that of the antacids (p less than 0.01). Ulcer size was reduced significantly in all groups (p less than 0.01), however, at the end of the study the gastric ulcers were smallest in the De-Nol treated group (p less than 0.001). The dynamics of ulcer healing in the second week was most favourable in the patients receiving vitamin A (p less than 0.01). The present data point to the cytoprotective effects of De-Nol liquid, vitamin A and sucralfate and to their ability of healing chronic gastric ulcers.     

Formation and regeneration ofHalobacterium spheroplasts

Spheroplasts ofHalobacterium cutirubrum were formed upon suspension of cell pellets in 0.1M MES buffer, pH 7.0, containing 0.5M sucrose, 0.25M NaCl, and 0.01M MgCl₂. The spheroplasts regenerated into rod-shaped bacteria when plated on a complex medium containing 15% (wt/vol) sucrose, undergoing several divisions as spherical bodies before the rod shape developed. The frequency of regeneration was approximately 5% of the total spheroplasts plated. The yield of regenerants was increased significantly (to approximately 35%) when bovine serum albumin was present in the spheroplasting buffer and dilution media. The conditions for spheroplast formation and regeneration inH. cutirubrum were also found effective forHalobacterium salinarium but not forHalobacterium halobium.NRCC Paper no. 23080.     

13C-NMR of double and triple bond carbon atoms of unsaturated fatty acid methyl esters

The carbon magnetic resonance spectra of many fatty acid methyl esters with cis and trans double bonds and triple bonds at various positions and in many different combinations have been investigated.The influence of the ester group on double and triple bonds in the fatty acid chain depends strongly on the positions of these bonds. For a given position the influence is constant, even if one or more other double or triple bonds are present.Together with the evaluated chemical shift parameters for the effects of double and triple bonds on each other, complete assignments are possible and spectra of various types of unsaturated esters can be predicted with high accuracy (±0.1 ppm).     

An ultrastructural examination of everted rat jejunum

Male, albino, Sprague-Dawley rats were sacrificed by cervical separation. Segments of jejunum were excised, everted and examined with the electron microscope. Examination of tissue fixed immediately after eversion revealed the following changes as compared to non-everted segments fixed $\text{in}$$\text{situ}$ and $\text{in}$$\text{vitro}$: 1) an increase in the length of microvilli from (mean ± S. E.) 0.991 ± 0.011μ for normal tissue to 1.389 ± 0.023μ for everted tissue, 2) an increase in width of microvilli from (mean ± S. E.) 0.089 ± 0.001μ for normal tissue to 0.097 ± 0.001μ for everted tissue, 3) an increase in length and number of lateral membrane interdigitations, and 4) the appearance of intercellular “lakes” in the lateral spaces. The above changes are in those structures hypothesized to be involved with salt and water transport across epithelia and may reflect altered transport rates $\text{in}$$\text{vitro}$ as compared to $\text{in}$$\text{vivo}$.