Ontogeny and the effects of corticosteroid pretreatment on aquaporin water channels in the ovine cerebral cortex.
The aim of the present study was to determine the ontogeny and effects of corticosteroid pretreatment on aquaporin 4 (AQP4) channel mRNA and protein expression in the cerebral cortex of sheep during development. A portion of the cerebral cortex was snap-frozen from fetuses of dexamethasone- and placebo-treated ewes at 60%, 80% and 90% of gestation, dexamethasone- and placebo-treated newborn lambs and adult sheep. Cerebral cortical samples were obtained 18 h after the last of four 6 mg dexamethasone or placebo injections were given over 48 h to the ewes and adult sheep. Lambs were treated with 0.01 mg kg(-1) dexamethasone or placebo in the same schedule as the ewes and adult sheep. Amplification of an ovine AQP4 cDNA fragment was accomplished by reverse transcription-polymerase chain reaction using primers based on a homologous bovine sequence. The resulting cDNA was used to determine AQP4 channel mRNA expression by Northern hybridisation using phosphorimaging. The relative abundance of AQP4 mRNA was normalised to the ovine ribosomal gene L32. A portion of the frontal cortex was also analysed for AQP4 protein expression by Western immunoblot. Densitometry was performed and the results expressed as a ratio to an adult brain pool. Aquaporin 4 channel mRNA and protein were detectable as early as at 60% gestation. There were no changes in AQP4 mRNA expression among the fetal, newborn and adult groups or after dexamethasone pretreatment in any age group. The expression of the AQP4 protein was higher (P < 0.05) in fetuses at 80% and 90% of gestation (2.9- and 3.3-fold, respectively), in lambs (3.2-fold) and in adult sheep (3.8-fold) compared with fetuses at 60% of gestation, as well as in adult sheep (1.3-fold) compared with fetuses at 80% of gestation. Dexamethasone pretreatment resulted in decreases (P < 0.05) in AQP4 protein expression in the lambs and adult sheep, but not in the fetal groups. We conclude that: (1) AQP4 mRNA and protein were expressed early in fetal and throughout ovine development; (2) protein, but not mRNA, expression increased between 60% and 80% of gestation and did not differ from adult levels by 90% of gestation; and (3) dexamethasone pretreatment resulted in decreases in AQP4 protein expression in lambs and adult sheep, but not in fetuses. The maturational increases in AQP4 protein expression and dexamethasone-related decreases in expression were post-transcriptional, because changes in AQP4 mRNA expression were not observed.
Ron
Nitin P
NP
Department of Pediatrics, Brown University School of Medicine and Women and Infants' Hospital of Rhode Island, Providence, 02905, USA.
Kazianis
John A
JA
Padbury
James F
JF
Brown
Courtney M
CM
McGonnigal
Bethany G
BG
Sysyn
Gregory D
GD
Sadowska
Grazyna B
GB
Stonestreet
Barbara S
BS
D000305:Adrenal Cortex Hormones / Q000008:administration & dosage*; D000818:Animals; D051401:Aquaporin 4 / Q000032:analysis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D002540:Cerebral Cortex / Q000737:chemistry* / Q000196:embryology*; D003907:Dexamethasone / Q000008:administration & dosage; D047109:Fetal Development; D005865:Gestational Age; D006854:Hydrocortisone / Q000097:blood; D012333:RNA, Messenger / Q000032:analysis; D012756:Sheep / Q000196:embryology*
D000305:Adrenal Cortex Hormones; D051401:Aquaporin 4; D012333:RNA, Messenger; D003907:Dexamethasone; D006854:Hydrocortisone