Cyclic AMP stabilizes the degradation of original junctional acetylcholine receptors in denervated muscle.

We used mouse diaphragm muscle in organ culture to study the stabilization of acetylcholine receptor (AChR) degradation at denervated neuromuscular junctions. After denervation, the degradation rate of the AChRs present prior to denervation (slowly degrading, or Rs, AChRs) accelerates from the predenervation degradation half-life (t1/2) of approximately 8-10 days to a t1/2 of approximately 2-3 days. We report that addition to the organ culture medium of pharmacological agents that elevate cytoplasmic cAMP levels (forskolin, dibutyryl cAMP, and 8-bromo-cAMP) reversed the change in t1/2 caused by denervation, whereas addition of 1,9-dideoxyforskolin, a forskolin analog that does not elevate cytoplasmic cAMP levels, did not reverse the effect of denervation. The degradation rate of AChRs in primary myotube cultures and that of the newly synthesized AChRs in denervated muscle were little affected by forskolin or dibutyryl cAMP. The possibility is raised that the modulation of Rs AChR degradation by innervation may be mediated by cAMP.     

Increased cyclic GMP in the end-plate region of denervated frog muscle

Denervated frog sartorius muscles showed an approximately 2–3 fold increase of cyclic GMP in their end-plate rich regions which did not appear up to 5 weeks after denervation in the normally end-plate-free pelvic region. No increase in cyclic AMP was seen in these preparations. The results suggest that the increase of cyclic GMP is related to processes specific to the region in which end plates are normally present.     

Increased cyclic GMP in the end-plate region of denervated frog muscle.

Denervated frog sartorius muscles showed an approximately 2--3 fold increase of cyclic GMP in their end-plate rich regions which did not appear up to 5 weeks after denervation in the normally end-plate-free pelvic region. No increase in cyclic AMP was seen in these preparations. The results suggest that the increase of cyclic GMP is related to processes specific to the region in which end plates are normally present.     

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."     

Acetylcholine receptors in normal and denervated rat diaphragm muscle. II. Comparison of junctional and extrajunctional receptors.

Acetylcholine (ACh) receptors have been purified separately from normal rat diaphragm muscle (junctional receptors) and from extrajunctional regions of denervated diaphragm (extrajunctional receptors) in order to compare their properties. The toxin-receptor complexes of the two receptors were indistinguishable by gel filtration and by zone sedimentation in sucrose gradients, and showed identical precipitation curves with rabbit antiserum to the eel ACh receptor. Both toxin-receptor complexes bind concanavalin A and are therefore probably glycoproteins. Low concentrations of d-tubocuratine (dTC) were more effective in decreasing the rate of toxin binding to junctional than to extrajunctional receptors. The apparent dissociation constant for dTC binding to the junctional receptor was 4.5 X 10 minus 8 M, whereas the value for the extrajunctional receptor was 5.5 X 10 minus 7 M. When the complexes were analyzed by isoelectric focusing, the junctional complex focused at approximately 0.15 pH unit lower than the extrajunctional complex. This result was also found with crude preparations of receptor. We conclude that junctional and extrajunctional receptors are similar but distinct molecules. The properties of receptors present in neonatal diaphragm muscle were also examined and found to be similar to those of receptors in denervated muscle, as shown by dTC inhibition and isoelectric focusing.     

Mechanical properties of denervated amphibian muscle

Denervated amphibian muscle does not show the prolongation of action potential found in mammalian denervated muscle. It was, therefore, predicted that denervated amphibian muscle would not show prolongation of the mechanical twitch. The sartorius muscles in one leg of toads--Xenopus borealis--were denervated for 140-268 days. Isometric twitch time to peak, time to half relaxation and twitch/tetanus ratio were not changed following denervation, confirming our prediction. Twitch tension decreased to 68% and tetanic tension decreased to 75% of control values. The maximum velocity of unloaded shortening (muscle length/s) was also unchanged.     

Analysis of changes in the membrane potential of denervated muscle

Quantitative analysis of the experimental data presented in the previous paper has shown that the electrogenic pump component of the membrane potential of muscle fibres on the third day after denervation is in the average 8.7 mV, and the diffusion component--12.9 mV lower than that in the normal fibres. It is due to a decrease of the stechiometric coefficient of Na+,K+-pump at denervation from 2.15 to 1.3 and to a change of the passive ionic permeability: at denervation permeability for Na+ increases from 0.52.10(-7) to 0.67.10(-7) cm.sec-1, and for K+ decreases from 0.75.10(-5) to 0.53.10(-5) cm.sec-1.