Molecular weight estimates of insect cholinergic receptors by radiation inactivation.

Molecular weight (MW) estimates for sites to which the radiolabelled cholinergic receptor probes alpha-bungarotoxin and N-methylscopolamine bind in CNS extracts of the cockroach Periplaneta americana have been made by radiation inactivation analysis. The MW of 77,600 determined for [3H]N-methylscopolamine binding sites agrees well with published values for vertebrate muscarinic acetylcholine receptor sites. In contrast, N-[propionyl-3H]propionylated alpha-bungarotoxin binds to a separate membrane component of lower MW (108,000) than previously reported values for vertebrate and insect nicotinic acetylcholine receptors. This appears to represent one or more subunits of the receptor complex, containing the recognition site.

[Radioprotectors of receptor action]. / Radioprotektory retseptornogo deistviia.

Based on analysis of own and literature data the separate class of radioprotectors, namely protectors of receptor action, has been distinguished. The main subclassed and representatives, common peculiarities and advantages (especially, low values of ED50 and high therapeutic indexes) of the protectors have been characterized. Possible participation of second messengers and alternative (non-receptor) hypothesis of hormone radioprotective effect are discussed.

Protective effect of laser phototherapy on acetylcholine receptors and creatine kinase activity in denervated muscle.

OBJECTIVE: This study was designed to assess the status of skeletal muscles after laser treatment during long-term denervation processes, by investigating changes in the level of acetylcholine receptors (AChR) and creatine kinase (CK) activity in the denervated gastrocnemius muscle of the rat. BACKGROUND DATA: Progressive muscle atrophy is common in patients with severe peripheral nerve injury. Denervated muscles can account for significant differences in the extent of AChR and CK activity during the denervation period. MATERIAL AND METHODS: The study was conducted on 96 rats: 48 that received laser treatment and 48 untreated controls. The gastrocnemius muscle was denervated by removing a 10 mm segment of the sciatic nerve. Low power laser irradiation was delivered transcutaneously to the right gastrocnemius muscle (HeNe continuous wave [CW] laser, 632.8 nm, 35 mW, 30 min) for 14 consecutive days. Under general anesthesia, the rats were euthanized at seven time points: day 7 (n=10), day 14 (n=10), day 21 (n=10), day 30 (n=5), day 60 (n=4), day 120 (n=5), and day 210 (n=4), with and without laser treatment, respectively. AChR was quantified by the (125)I-α-bungarotoxin. CK activity was measured by a specific spectrophotometric method. RESULTS: Laser treatment had a significant therapeutic effect on the denervated muscle during the first 21 days for AChR and the first 30 days for CK activity. CONCLUSIONS: In the early stages of muscle atrophy, laser phototherapy may preserve the denervated muscle by maintaining CK activity and the amount of AChR.

Degeneration and recovery of the neuromuscular junction after application of extracorporeal shock wave therapy.

It is known that free nerve endings are degenerated after application of shock waves. We therefore hypothesized that the application of shock waves to muscle induces dysfunction of neuromuscular transmission at neuromuscular junctions. We investigated changes in neuromuscular transmission in response to shock wave application. Sprague-Dawley rats were used in this study. Two thousand shock waves at an energy flux density of 0.18 mJ/mm(2) were applied to their right calf muscles. Neuromuscular junctions of gastrocnemius muscles were evaluated using rhodamine-α-bungarotoxin on the day of treatment (n = 5). Amplitude and latency of compound muscle action potentials were measured on the day of treatment and 1, 2, 4, 6, and 8 weeks after treatment (n = 10, each group). Degenerated acetylcholine receptors existed in all treated muscles. Although the action potential amplitude on the treated side was significantly less than on the control side from the day of treatment (25.1 ± 7.8 vs. 34.5 ± 9.1, p = 0.012) to 6 weeks (27.9 ± 7.2 vs. 34.5 ± 7.2, p = 0.037), there was no significant difference at 8 weeks. There was no significant difference in transmission latency between the groups. The application of shock waves to muscle induced a transient dysfunction of nerve conduction at neuromuscular junctions.

Low-level microwave irradiation and central cholinergic activity: a dose-response study.

Rats were irradiated with circularly polarized, 2,450-MHz pulsed microwaves (2-microseconds pulses, 500 pulses per second [pps]) for 45 min in the cylindrical waveguide system of Guy et al:(Radio Sci 14:63-74, 1979). Immediately after exposure, sodium-dependent high-affinity choline uptake, an indicator of cholinergic activity in neural tissue, was measured in the striatum, frontal cortex, hippocampus, and hypothalamus. The power density was set to give average whole-body specific absorption rates (SAR) of 0.3, 0.45, 0.6, 0.75, 0.9, or 1.2 W/kg to study the dose-response relationship between the rate of microwave energy absorption and cholinergic activity in the different areas of the brain. Decrease in choline uptake was observed in the striatum at a SAR of 0.75 W/kg and above, whereas for the frontal cortex and hippocampus, decreases in choline uptake were observed at a SAR of 0.45 W/kg and above. No significant effect was observed in the hypothalamus at the irradiation power densities studied. The probit analysis was used to determine the SAR50 in each brain area, i.e., the SAR at which 50% of maximum response was elicited. SAR50 values for the striatum, frontal cortex, and hippocampus were 0.65, 0.38, and 0.44 W/kg, respectively.

Target size of the ryanodine receptor from junctional terminal cisternae of sarcoplasmic reticulum.

Target inactivation analysis was carried out on the ryanodine receptor. This receptor recently has been implicated as the channel involved in the calcium release process in excitation-contraction coupling and was localized to the junctional terminal cisternae of sarcoplasmic reticulum from skeletal muscle [Fleischer, S., Ogunbunmi, E. M., Dixon, M. C., & Fleer, E.A.M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7256-7259]. Irradiation of the junctional terminal cisternae resulted in an exponential decrease in ryanodine binding with radiation dose, thereby consistent with target theory. The target molecular weight was found to be 138,000 +/- 21,000, i.e., smaller than the polypeptide that binds ryanodine. The calcium pump protein in the same membrane preparation served as an internal control to validate the methodology.

[The effect of ultrahigh-frequency electromagnetic radiation on learning and memory processes]. / Vliianie élektromagnitnogo izlucheniia sverkhvysokoi chastoty na protsessy obucheniia i pamiati.

Low-intensity electromagnetic field (12.6 cm, 2375 MHz, power density 1 mW/cm2) produced retrograde amnesia in the rat passive avoidance test. No effect was registered of microwave irradiation on the open field behavior and the pain sensitivity. Functional activity of the m-cholinergic receptors decreased, but their number increased in the brain cortex. It is suggested that cholinergic system plays an important role in the effects of electromagnetic field on memory processes.

Subnanosecond polarized fluorescence photobleaching: rotational diffusion of acetylcholine receptors on developing muscle cells.

Polarized fluorescence recovery after photobleaching (PFRAP) is a technique for measuring the rate of rotational motion of biomolecules on living, nondeoxygenated cells with characteristic times previously ranging from milliseconds to many seconds. Although very broad, that time range excludes the possibility of quantitatively observing freely rotating membrane protein monomers that typically should have a characteristic decay time of only several microseconds. This report describes an extension of the PFRAP technique to a much shorter time scale. With this new system, PFRAP experiments can be conducted with sample time as short as 0.4 microseconds and detection of possible characteristic times of less than 2 microseconds. The system is tested on rhodamine-alpha-bungarotoxin-labeled acetylcholine receptors (AChRs) on myotubes grown in primary cultures of embryonic rat muscle, in both endogenously clustered and nonclustered regions of AChR distribution. It is found that approximately 40% of the AChRs in nonclustered regions undergoes rotational diffusion fast enough to possibly arise from unrestricted monomer Brownian motion. The AChRs in clusters, on the other hand, are almost immobile. The effects of rat embryonic brain extract (which contains AChR aggregating factors) on the myotube AChR were also examined by the fast PFRAP system. Brain extract is known to abolish the presence of endogenous clusters and to induce the formation of new clusters. It is found here that rotational diffusion of AChR in the extract-induced clusters is as slow as that in endogenous clusters on untreated cells but that rotational diffusion in the nonclustered regions of extract-treated myotubes remains rapid.

[Action of a UHF field on GABA-ergic and acetylcholinergic systems in synaptic transmission]. / Deistvie SVCh polia na GAMKergicheskie i atsetilkholinergicheskie sistemy sinapticheskoi peredachi.

It was shown that exposure of rats to microwaves (800 MHz, 16 Hz modulation, 1-3 mW/cm2) decreased muscimol binding with synaptic membranes and reduced acetycholinesterase activity in the rat brain.

Size of acetylcholine receptors in the membrane. An improved version of the radiation inactivation method.

The radiation inactivation method was used to study the size of acetylcholine receptors in the intact membrane-bound state. This technique was reinvestigated, and modifications were made which remove substantial difficulties affecting previous applications of it to such proteins. The molecular size was deduced here by reference to a set of protein standards: an inactivation ratio was defined relative to a given internal enzyme molecular weight standard, and a linear calibration plot for the inactivation ratios of the protein standards was constructed and applied. The acetylcholine receptor in Torpedo electric organ, cat denervated muscle, and chick embryonic muscle was found by this method to exist in the membrane as a homogeneous population of the same size in each case. This receptor, when identified thus by the alpha-neurotoxin-binding target structure, has an apparent molecular weight of 300000 or a molecular volume of about 350 nm3. In comparison, the molecular weight of the cat muscle receptor when solubilized, as analyzed by gel electrophoresis after extensive cross-linking, was found to be 270000 +/- 20000. These two values are thought to be equivalent by virtue of the situation and structure of the receptor protein in the cell membrane. If a disulfide-bridge dimeric receptor exists in the membrane (as other evidence has indicated for Torpedo), each monomer acts independently there in binding alpha-neurotoxin, since the monomers can be inactivated independently by irradiation in the Torpedo membrane. In the muscle membrane no evidence for the existence of receptor dimers, of any kind, has been found.

An acetylcholine receptor regulatory site in BC3H1 cells: characterized by laser-pulse photolysis in the microsecond-to-millisecond time region.

When a neurotransmitter binds to its specific receptor, the protein forms transmembrane channels through which ions flow, leading to changes in transmembrane voltage that trigger signal transmission between neurons. How do inhibitors affect this process? Interesting and extensive information comes from investigations of the acetylcholine receptor, the best known of these proteins. This receptor is inhibited by cationic inhibitors, including local anesthetics, and acetylcholine at high concentrations. The accepted mechanism, elegant in its simplicity, is that these compounds enter the receptor-channel after it opens and block inorganic ion flux. This mechanism requires that the inhibitors affect only the apparent rate constant for channel closing (k'cl). An alternative mechanism invokes a specific regulatory (inhibitory) site to which inhibitors bind before the channel opens and the signal is transmitted. This mechanism requires that the inhibitors affect the apparent rate constants for both channel opening (k'op) and closing. The effect of inhibitors on k'op has not been determined previously. This report describes the use of a newly developed laser-pulse photolysis technique with a dead time of approximately 120 microseconds to determine the effect of a local anesthetic, procaine, one of the best studied cationic inhibitors of the acetylcholine receptor, on both k'op and k'cl. Both k'op and k'cl were found to decrease with increasing procaine concentration. This effect of the inhibitor of k'op cannot be explained by the open-channel-blocking mechanism but is consistent with the existence of a regulatory (inhibitory) receptor site.

Rose bengal activates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

The photooxidizing xanthene dye rose bengal (10 nM to 1 microM) stimulates rapid Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles. Following fusion of sarcoplasmic reticulum (SR) vesicles to an artificial bilayer, reconstituted Ca2+ channel activity is stimulated by nanomolar concentrations of rose bengal in the presence of a broad-spectrum light source. Rose bengal does not appear to affect K+ channels present in the SR. Following reconstitution of the sulfhydryl-activated 106-kDa Ca2+ channel protein into a bilayer, rose bengal activates the isolated protein in a light-dependent manner. Ryanodine at a concentration of 10 nM is shown to lock the 106-kDa channel protein in a subconductance state which can be reversed by subsequent addition of 500 nM rose bengal. This apparent displacement of bound ryanodine by nanomolar concentrations of rose bengal is also directly observed upon measurement of [3H]ryanodine binding to JSR vesicles. These observations indicate that photooxidation of rose bengal causes a stimulation of the Ca2+ release protein from skeletal muscle sarcoplasmic reticulum by interacting with the ryanodine binding site. Furthermore, similar effects of rose bengal on isolated SR vesicles, on single channel measurements following fusion of SR vesicles, and following incorporation of the isolated 106-kDa protein strongly implicates the 106-kDa sulfhydryl-activated Ca2+ channel protein in the Ca2+ release process.

[The effect of the parenteral administration of alpha-ketoglutarate on the resistance of rats to ionizing radiation and on their cholinergic systems]. / Vplyv parenteral'noho vvedennia al'fa-ketohlutaratu na rezystentnist' shchuriv do ionizuiuchoho oprominennia ta kholinerhichnu systemu ïkh orhanizmu.

We have investigate the effect of sodium ketoglutarate intraperitoneal injection (20 mg/100 g body weight) made 0.5 hour before and 1, 2 and 3 hours after total X-ray treatment (259 mKl/kg) on the survival of rats. Simultaneously we have define changes in cholinesterase-acetylcholine system and content of adrenaline and noradrenaline in liver and pancreas tissues, small intestines mucous and in blood. All the data were taking at 1, 2, 5, 4, 24 and 72 hours after treatment and sodium ketoglutarate injection. We have found that sodium ketoglutarate injection made 0.5 hour before the treatment results in increasing of percentage death rate and injection made 1 and 2 hours after treatment results in increase in survival of rats. The effect of alpha-ketoglutarate injection made 1 hour after treatment in more pronounced. It is accompanied with increase of cholinergic status of organism on the catecholamine deficit background, decrease in the cholinesterase activity and increase of acetylcholine content in tissues of treated organism.