Induction of a fatigue-resistant phenotype in rabbit fast muscle by small daily amounts of stimulation.

We have shown that fatigue resistance can be induced in rabbit tibialis anterior (TA) muscles without excessive power loss by continuous stimulation at low frequencies, such as 5 Hz, and that the same result is obtained by delivering a 10-Hz pattern in equal on/off periods. Here we ask whether the same phenotype could be produced with daily amounts of stimulation that would be more appropriate for clinical use. We stimulated rabbit TA muscles for 6 wk, alternating fixed 30-min on periods of stimulation at 10 Hz with off periods of different duration. All patterns transformed fast-glycolytic fibers into fast-oxidative fibers. The muscles had fatigue-resistant properties but retained a higher contractile speed and power production than muscles transformed completely to the slow-oxidative type. We conclude that in the rabbit as little as one 30-min period of stimulation in 24 h can result in a substantial increase in the resistance of the muscle to fatigue.

Dynamics of stimulation-induced muscle adaptation: insights from varying the duty cycle.

We sought to gain insight into the dynamics of the signalling process that initiates adaptive change in mammalian skeletal muscles in response to chronic neuromuscular stimulation. Programmable miniature stimulators were implanted into rabbits and used to impose one of the following patterns on the dorsiflexors of one ankle: 10 Hz delivered in equal on/off periods of 30 s, 30 min, or 12 h (all equivalent in terms of aggregate impulse activity to continuous 5 Hz). Two further groups received continuous stimulation at 5 Hz or 10 Hz. In every case the stimulation pattern was maintained continuously for 6 weeks. Tibialis anterior muscles stimulated intermittently with equal on/off periods of 30 s, 30 min and 12 h had contractile characteristics that were significantly slower than the contralateral, unstimulated muscles but did not differ from those of muscles stimulated continuously at 5 Hz. Muscles stimulated continuously at 10 Hz were significantly slower than either contralateral muscles or muscles stimulated with any of the other patterns. Corresponding changes were seen in myosin heavy chain isoform composition. The fatigue index, defined as the fraction of tension remaining after 5 min of a standard fatigue test, was 0.4 for muscles in the contralateral group but equal to or greater than 0.85 for muscles of all the stimulated groups. These results were interpreted with the help of a simple model of the growth and decay of a putative signalling substance based on first order kinetics. The model suggests a rate constant for the accumulation of the signalling substance that is greater than 30 h(-1), and a rate constant for its removal that is greater than 50 h(-1).

Functional N-methyl-D-aspartate receptors in clonal rat phaeochromocytoma cells.

1. To characterize from a molecular and functional point of view the endogenous NMDA receptors expressed by phaeochromocytoma (PC12) cells, experiments involving polymerase chain reaction (PCR) amplification, Western blotting and patch-clamp analysis of undifferentiated and nerve growth factor (NGF)-differentiated PC12 cells were performed. 2. Analysis of PC12 mRNA demonstrated the presence of NMDAR1 and NMDAR2C transcripts. The NMDAR1 subunits lack the amino terminal insert of twenty-one amino acid residues, where as transcripts with and without deletions I and II at the 3' end of the coding region were detected. Thus, NMDA receptors of the PC12 cells might include NMDAR1A, NMDAR1E, NMDAR1C and NMDAR1D subunits. 3. Differentiation by NGF treatment of PC12 cells did not alter mRNA expression for NMDA receptor subunits significantly but induced an increase in both the NMDAR1 protein and the total amount of functional receptors that correlated well with a parallel increase in membrane area. 4. NMDA receptors in differentiated PC12 cells had a high affinity for both glutamate and glycine. These were estimated kinetically as 0.59 microM and 74 nM, respectively. Responses to glutamate or NMDA were non-desensitizing in the presence of saturating glycine, but slowly desensitized with low concentrations of glycine. Currents were completely blocked by D-aminophosphonovalerate (APV), 7-Cl-kynurenate and phencyclidine, and showed a voltage-dependent magnesium blockade. Spermine did not potentiate but inhibited NMDA receptor-mediated responses in a voltage-independent manner. 5. With 0.5 mM Ca2+, single-channel analysis revealed very brief openings (mean open time (t(o)) = 0.42 ms), with at least two conductive states, 55 and 33 pS, both having markedly low open probability. At 2 mM Ca2+, conductances were reduced to 39 and 19 pS, without an effect in open probability or mean open time. 6. The functional properties of NMDA receptors in PC12 cells were very similar to those described for NMDAR1A-NMDAR2C heteromers recombinantly expressed. The PC12 cell line provides a simple and reproducible system to analyse some specific NMDA receptor properties.