Early over-expression of low-affinity [3H]ryanodine receptor sites in heavy sarcoplasmic reticulum fraction from dystrophic chicken pectoralis major.
Heavy sarcoplasmic reticulum (SR) membranes enriched in [3H]ryanodine receptor have been isolated from pectoralis major (PM) of normal line 412 and dystrophic line 413 chickens paired at various stages during post-hatch development. Normal PM 2 days ex ovo yields 17% lower protein recovery in the heavy SR subfractions compared to preparations from paired dystrophic PM (0.80 vs. 0.96 mg/g PM, respectively). By 2 weeks ex ovo, protein recovery in normal SR subfractions decreases over 3-fold to 0.24 mg/g PM, whereas yields from dystrophic PM increase to 1.67 mg/g PM. Dystrophic preparations consistently give 7-9-fold higher recoveries of protein in heavy SR subfractions compared to normal PM when examined at 2, 4, and 5.5 weeks ex ovo. [3H]Ryanodine binding to normal SR from PM 2 days ex ovo exhibits nonlinear Scatchard plots which resolve into high-(Kd,app = 18 nM; Bmax = 1.7 pmol/mg protein) and low-(Kd,app = 532 nM; Bmax = 2.6 pmol/mg protein) affinity binding sites, whereas dystrophic preparations exhibit only high-affinity [3H]ryanodine binding sites (Kd,app = 31 nM; Bmax = 2.1 pmol/mg protein). Both normal and dystrophic PM have similar capacities to bind [3H]ryanodine (2.6 vs. 2.0 pmol/g PM, respectively) at 2 days ex ovo. However, at 2, 4, and 5.5 weeks ex ovo the density of high-affinity [3H]ryanodine binding sites in normal SR drops dramatically to 3.5, 1.2, and 0.4 pmol/mg protein, respectively, and corresponds with disappearance of the low-affinity binding sites. In marked contrast, heavy SR membranes from dystrophic PM 2, 4, and 5.5 weeks ex ovo, maintain their high-affinity binding sites for [3H]ryanodine and exhibit high densities of low-affinity binding sites (Kd,app = 725-4500 nM; Bmax = 15.4-25.1 pmol/mg protein). Early developmental over-expression of [3H]ryanodine binding capacity in dystrophic PM ranges from 34-fold to 388-fold that of normal PM at 2 weeks and 5.5 weeks, respectively, and correspond to the intensity with which high molecular weight doublets of Mr 340,000 and 320,000 stain on SDS-PAGE. Low-affinity [3H]ryanodine binding sites of dystrophic SR exhibit 4-6-fold higher sensitivity to activation by Ca2+ and altered sensitivity to activation by caffeine and adenine nucleotides. These results demonstrate that over-expression of junctional SR and [3H]ryanodine receptors having altered radioligand binding properties is a very early event in the post-hatch development of dystrophic PM.(ABSTRACT TRUNCATED AT 400 WORDS)
Pessah
I N
IN
Department of Veterinary Pharmacology and Toxicology, University of California, Davis 95616.
Schiedt
M J
MJ
D000818:Animals; D002110:Caffeine / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation; D007700:Kinetics; D008024:Ligands; D009137:Muscular Dystrophy, Animal / Q000378:metabolism*; D010369:Pectoralis Muscles / Q000378:metabolism*; D011201:Poultry Diseases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D008024:Ligands; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D000242:Cyclic AMP; D002118:Calcium