The effects of mutated skeletal ryanodine receptors on calreticulin and calsequestrin expression in the brain and pituitary gland of boars.
Mutations in skeletal ryanodine receptors (sRyR) result in malignant hyperthermia in humans and Porcine Stress Syndrome (PSS) in pigs. Whether the sRyR is expressed in neuronal tissue and what impact it has on neuronal function is relatively unexplored. We have hypothesized that the presence of mutated sRyR may be accompanied by compensatory changes in Ca(2+)-binding protein expression. We were interested in whether pigs heterozygous for mutated sRyR would show changes in the expression of Ca(2+)-binding proteins, in specific regions of the brain, and whether changes in this expression would be accompanied by the presence of sRyR within that region. The objectives of the current experiments were to determine (1) whether calreticulin (CR) and calsequestrin (CS) are expressed in the pituitary gland and brain of the pig, (2) if boars heterozygous for mutated sRyR differed from wild-type boars in the expression of CR or CS, and (3) if altered Ca(2+)-binding protein expression would be accompanied by the presence of sRyR mRNA. Boars either heterozygous or wild-type (n=6) for the mutation in sRyR known to cause PSS, were euthanized and the pituitary gland and brains were collected for western blotting for CR and CS. An additional four wild-type boars were sacrificed and brains were collected for in situ hybridization for sRyR mRNA. Immunoreactive CR was expressed in porcine tissues with highest (p<0.0001) expression in the pituitary gland and lower but equivalent expression in the hypothalamus, frontal cortex, and hippocampus. Immunoreactive CS was not detectable in the pituitary gland while low levels were observed in the hypothalamus and frontal cortex. Dramatically higher (p<0.0001) levels of CS were found in the hippocampus. Genotype did not affect CR expression in the pituitary gland or any brain region examined. Immunoreactive CS levels were lower (p<0.002) in the hippocampus of heterozygous compared to wild-type boars. In situ hybridization experiments revealed the presence of sRyR mRNA in the hippocampus equally distributed across all cell subfields. In conclusion, both CR and CS were expressed in the porcine brain with specific patterns of expression across the brain regions examined. Boars heterozygous for mutated sRyR had lower CS in the hippocampus, which was accompanied by the expression of mRNA for sRyR.
Weaver
S A
SA
Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada. sweaver@www.ansc.purdue.edu
Schaefer
A L
AL
Dixon
W T
WT
D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D037282:Calreticulin; D002155:Calsequestrin / Q000235:genetics*; D005625:Frontal Lobe / Q000378:metabolism; D005786:Gene Expression Regulation; D006579:Heterozygote; D006624:Hippocampus / Q000378:metabolism; D007031:Hypothalamus / Q000378:metabolism; D008297:Male; D009154:Mutation; D010902:Pituitary Gland / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics* / Q000502:physiology; D013552:Swine
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002155:Calsequestrin; D020033:Protein Isoforms; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D019837:Ryanodine Receptor Calcium Release Channel