6. On the basis of these findings it is concluded that ACh receptors aggregate within the sarcolemma, spontaneously as well as in response to innervation. In the latter case extrajunctional receptors accumulate at the site of nerve contact thereby contributing to the development of high receptor density in the subneural muscle membrane. This process of receptor redistribution occurs in the absence of synaptic or contractile activity.7. Possible mechanisms involved in the redistribution of ACh receptors are discussed in relation to those which appear to modulate ligandinduced changes in the distribution of lectin and immunoglobulin receptors.

5. Fluorescent stain on innervated cells was restricted to the path of nerve-muscle contact and sometimes extended for greater lengths than the largest patches seen on non-contacted muscle cells.6. Similar long bands of stain associated with nerve-muscle contacts were observed when cultures were grown in high concentrations of curare and carbachol which prevented spontaneous twitching. They were also seen in cultures in which the addition of neural tube cells was delayed for 2-3 days.7. It is concluded that innervation caused receptors to accumulate at sites of nerve-muscle contact and that this process can operate independently of muscle activity.

SUMMARY1. a-Bungarotoxin was labelled with fluorescent dyes and used as a stain for visualizing the distribution of acetylcholine receptors in vertebrate skeletal muscle fibres.2. Dye-toxin conjugates had the same pharmacological properties as native toxin, but their potencies were lower.3. Fluorescent staining was examined in teased muscle fibres. The stain was found to be confined to the neuromuscular junction and associated with the subsynaptic membrane.4. Staining intensity was reduced by curare and even more so by carbachol, but not by atropine or neostigmine. Pre-treatment of muscles with unlabelled x-bungarotoxin entirely prevented staining.5. The staining at amphibian neuromuscular junctions was characterized by a pattern of intense transverse bands occurring at intervals of approximately 0-5-1 um, with fluorescence of lower intensity between them. Fluorescent staining was not detected on adjacent, extrasynaptic, muscle membrane. In side views the staining appeared as a fine line with small protuberances occurring at the same intervals as the intense bands seen face-on. These results indicate that acetylcholine receptors are associated with the entire subsynaptic membrane, including the membrane of the junctional folds and that their density changes abruptly at the border between synaptic and extrasynaptic muscle membrane.

Hybridoma techniques have been used to generate monoclonal antibodies to an antigen concentrated in the basal lamina at the Xenopus laevis neuromuscular junction. The antibodies selectively precipitate a high molecular weight heparan sulfate proteoglycan from conditioned medium of muscle cultures grown in the presence of [3~S]methionine or [35S]-sulfate. Electron microscope autoradiography of adult X. laevis muscle fibers exposed to 12Sl-labeled antibody confirms that the antigen is localized within the basal lamina of skeletal muscle fibers and is concentrated at least fivefold within the specialized basal lamina at the neuromuscular junction. Fluorescence immunocytochemical experiments suggest that a similar proteoglycan is also present in other basement membranes, including those associated with blood vessels, myelinated axons, nerve sheath, and notochord.During development in culture, the surface of embryonic muscle cells displays a conspicuously non-uniform distribution of this basal lamina proteoglycan, consisting of large areas with a low antigen site-density and a variety of discrete plaques and fibrils. Clusters of acetylcholine receptors that form on muscle cells cultured without nerve are invariably associated with adjacent, congruent plaques containing basal lamina proteoglycan. This is also true for clusters of junctional receptors formed during synaptogenesis in vitro. This correlation indicates that the spatial organization of receptor and proteoglycan is coordinately regulated, and suggests that interactions between these two species may contribute to the localization of acetylcholine receptors at the neuromuscular junction.The vertebrate neuromuscular junction is a region of elaborate morphological specialization for both the skeletal muscle fiber and motor neuron. This morphological complexity reflects a corresponding chemical specialization. It is well established, for example, that the postsynaptic membrane of the muscle fiber contains a high concentration of acetylcholine receptors (AChR), ~ and that the packing density of this integral membrane protein drops at least 50-fold within a few micrometers of the nerve terminal (1). It is also known that acetylcholinesterase (ACHE) is concentrated within the synaptic cleft (2, J Abbreviations used in this paper: ACHE, acetylcholinesterase; AChR, acetylcholine receptor; aBGT, a-bungarotoxin; FITC, fluorescein isothiocyanate; and TRITC, tetramethylrhodamine isothiocyanate.3), where it appears to be associated with the basal lamina (4,5). Immunocytochemical experiments indicate the existence of further chemical specialization in the muscle cytoskeleton (6-8), basal lamina (9), and the nerve terminal (10, 11). Undoubtedly the list of components concentrated at the neuromuscular junction will continue to increase as improved immunological techniques permit the identification and study of new molecular entities.In contrast to such evidence for chemical specialization, little is known about the cellular mechanisms responsible for the elaboration...

This study examined the effect of glycerol ingestion on fluid homeostasis, thermoregulation, and metabolism during rest and exercise. Six endurance-trained men ingested either 1 g glycerol in 20 ml H2O x kg(-1) body weight (bw) (GLY) or 20 ml H2O x kg(-1) bw (CON) in a randomized double-blind fashion, 120 min prior to undertaking 90 min of steady state cycle exercise (SS) at 98% of lactate threshold in dry heat (35 degrees C, 30% RH), with ingestion of CHO-electrolyte beverage (6% CHO) at 15-min intervals. A 15-min cycle, where performance was quantified in kJ, followed (PC). Pre-exercise urine volume was lower in GLY than CON (1119 +/- 97 vs. 1503 +/- 146 ml x 120 min(-1); p < .05). Heart rate was lower (p < .05) throughout SS in GLY, while forearm blood flow was higher (17.1 +/- 1.5 vs. 13.7 +/- 3.0 ml x 100 g tissue x min(-1); p < .05) and rectal temperature lower (38.7 +/- 0.1 vs. 39.1 +/- 0.1 degrees C; p < .05) in GLY late in SS. Despite these changes, skin and muscle temperatures and circulating catecholamines were not different between trials. Accordingly, no differences were observed in muscle glycogenolysis, lactate accumulation, adenine nucleotide, and phosphocreatine degradation or inosine 5'-monophosphate accumulation when comparing GLY with CON. Of note, the work performed during PC was 5% greater in GLY (252 +/- 10 vs. 240 +/- 9 kJ; p < .05). These results demonstrate that glycerol, when ingested with a bolus of water 2 hours prior to exercise, results in fluid retention, which is capable of reducing cardiovascular strain and enhancing thermoregulation. Furthermore, this practice increases exercise performance in the heat by mechanisms other than alterations in muscle metabolism.