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PubMed1995

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.

Animals↗
PubMed1976

Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers.

We have made direct, quantitative measurements of the lateral motion and age-dependent distribution of acetylcholine receptors (AChR) on the surface of rat myotubes in primary culture. AChR were fluorescently marked with tetramethylrhodamine-labeled alpha-bungarotoxin and AChR lateral motion was measured by the fluoresence photobleaching recovery technique. We found two coexisting distinct classes of AChR: (i) mobile, uniformly distributed AChR that appear on all myotubes shortly after fusion from myoblasts; and (ii) immobile, dense, highly granular AChR in patches of 10-60 mum size that appear shortly after fusion and disappear after myotubes have become extensively interconnected. In addition, evidence of turnover of AChR labeled with tetramethylrhodamine-alpha-bungarotoxin is seen in the gradual internalization of surface fluorescence within 36 hr after labeling. The relevance of these results to an understanding of the membrane dynamics and localization of muscle AChR is discussed.

Age Factors↗
PubMed1993

Neurotrophin 3 supports the survival of developing muscle sensory neurons in culture.

Target-dependent cell death of different sub-populations of sensory neurons may be regulated by different trophic factors. To investigate this possibility, we have taken advantage of the fact that the fractions of muscle sensory and cutaneous sensory neurons in chicken dorsal root ganglia (DRG) are probably different at different segmental levels, and we have compared the responses of chicken DRG from levels that do and do not innervate limb tissue to various growth factors in vitro. Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) both supported neurite outgrowth from DRG explanted from all segmental levels. In contrast, neurotrophin 3 (NT-3) supported robust neurite growth only from DRG explanted from the cervical or lumbar levels, which innervate limb muscles. Similarly, NGF and BDNF both promoted survival of dissociated neurons from limb and nonlimb segmental levels, whereas NT-3 promoted survival of more neurons from limb compared to nonlimb levels. This suggests that muscle sensory neurons, which are probably more prevalent at the cervical and lumbar levels, may be specifically affected by NT-3. To evaluate this possibility directly, we compared the survival of retrogradely labeled muscle and cutaneous neurons in NGF, BDNF, and NT-3. Identified muscle sensory neurons survived best in vitro in the presence of NT-3, while the survival of identified cutaneous sensory neurons was greatest in NGF. This work provides direct evidence for a potential role of NT-3 versus NGF in the survival of a specific subpopulation of DRG neurons.

Animals↗
PubMed1977

Effects of innervation on acetylcholine sensitivity of developing muscle in vitro.

1. Chick embryo skeletal muscle fibres were grown in culture. The acetylcholine (AACh) sensitivity of non-innervated fibres was compared with that of fibres innervated in vitro by chick embryo ciliary ganglion neurones. 2. The general pattern of ACh sensitivity was unchanged by innervation: ACh hot spots were superimposed on a background of uniform ACh sensitivity. 3. Quantitative comparisons revealed two differences between non-innervated and innervated fibres. First, hot spots were encountered about one third more often on innervated fibres. Secondly, about one-third of the hot spots on innervated fibres had significantly higher ACh sensitivities than the remainder, which were similar to those on control fibres. 4. Apossible explanation of these results is that nerves which form synapses induce the appearanceof end-plates which have higher ACh sensitivities than the pre-existing ACh hot spots.

Acetylcholine↗
PubMed1990

Stretch-sensitive channels in developing muscle cells from a mouse cell line.

1. Recordings of single-channel activity were made from cell-attached patches on mouse C2 muscle cells at morphologically identifiable stages of myogenesis in vitro. We have identified Ca2(+)-permeable, cation-selective channels that are gated by applying suction to the patch electrode and by changes in membrane potential and have analysed single-channel properties as well as channel expression during myogenesis. 2. Single-channel activity could be detected when the membrane was held at steady negative potentials. With monovalent cations in the electrode, the single-channel current-voltage (i-V) relations were linear. The channel is permeable to Li+, Na+, K+, Rb+ and Cs+, but is not strongly selective among the monovalent cations as judged by measurements of single-channel conductance and reversal potential. 3. With 110 mM of either CaCl2 or BaCl2 as the only inward change carrier, slope conductances were approximately 13 and 24 pS and currents reversed at approximately +22 and +17 mV, respectively. The relative permeability of Ca2+ to K+ calculated from the constant-field equation was PCa/PK = approximately 2. 4. Channel openings occurred as bursts of brief openings and closings separated by much longer closed periods. Closed-time histograms were best fitted with three exponential components, while histograms of burst duration were best fitted with two exponential components, reflecting the short and long bursts in the single-channel records. 5. Applying suction to the patch electrode while recording at steady negative membrane potentials produced channel openings to discrete current levels. Mean channel open probability depended linearly on the square of the applied pressure and was greater at positive membrane potentials. The permeability of the channel to monovalent and divalent cations was indistinguishable from the spontaneous activity recorded at steady negative potentials. 6. Channel activity recorded from cell-attached patches in the absence of applied pressure depended on membrane potential increasing approximately e-fold per 38 mV with depolarization. Analysis of the kinetics of the response to membrane potential showed that the depolarization reduced the duration of the slowest component of the closed-time distribution. 7. The lanthanide cation gadolinium (Gd) reduced the amplitude of the unitary currents in a concentration-dependent manner. The amplitudes of both inward and outward currents were reduced to the same extent suggesting block is voltage-independent. Gd produced half-maximal inhibition of the unitary current at approximately 6 microM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2003

The potential value and toxicity of chromium picolinate as a nutritional supplement, weight loss agent and muscle development agent.

The element chromium apparently has a role in maintaining proper carbohydrate and lipid metabolism in mammals. As this role probably involves potentiation of insulin signalling, chromium dietary supplementation has been postulated to potentially have effects on body composition, including reducing fat mass and increasing lean body mass. Because the supplement is absorbed better than dietary chromium, most studies have focused on the use of chromium picolinate [Cr(pic)(3)]. Cr(pic)(3) has been amazingly popular with the general public, especially with athletes who may have exercise-induced increased urinary chromium loss; however, its effectiveness in manifesting body composition changes has been an area of intense debate in the last decade. Additionally, claims have appeared that the supplement might give rise to deleterious effects. However, over a decade of human studies with Cr(pic)(3) indicate that the supplement has not demonstrated effects on the body composition of healthy individuals, even when taken in combination with an exercise training programme. Recent cell culture and in vivo rat studies have indicated that Cr(pic)(3) probably generates oxidative damage of DNA and lipids and is mutagenic, although the significance of these results on humans taking the supplement for prolonged periods of time is unknown and should be a focus for future investigations. Given that in vitro studies suggest that other forms of chromium used as nutritional supplements, such as chromium chloride, are unlikely to be susceptible to generating this type of oxidative damage, the use of these compounds, rather than Cr(pic)(3), would appear warranted. Potential neurological effects (both beneficial and deleterious) from Cr(pic)(3) supplementation require further study.

Animals↗