Calcium-dependent ATPase unlike ecto-ATPase is located primarily on the luminal surface of brain endothelial cells.
Numerous cytochemical studies have reported that calcium-activated adenosine triphosphatase (Ca2+-ATPase) is localized on the abluminal plasma membrane of mature brain endothelial cells. Since the effects of fixation and co-localization of ecto-ATPase have never been properly addressed, we investigated the influence of these parameters on Ca2+-ATPase localization in rat cerebral microvessel endothelium. Formaldehyde at 2% resulted in only abluminal staining while both luminal and abluminal surfaces were equally stained following 4% formaldehyde. Fixation with 2% formaldehyde plus 0.25% glutaraldehyde revealed more abluminal staining than luminal while 2% formaldehyde plus 0.5% glutaraldehyde produced vessels with staining similar to 4% and 2% formaldehyde plus 0.25% glutaraldehyde. The abluminal reaction appeared unaltered when ATP was replaced by GTP, CTP, UTP, ADP or when Ca2+ was replaced by Mg2+ or Mn2+ or p-chloromercuribenzoate included as inhibitor. But the luminal reaction was diminished. Contrary to previous reports, our results showed that Ca2+-specific ATPase is located more on the luminal surface while the abluminal reaction is primarily due to ecto-ATPase. The strong Ca2+-specific-ATPase luminal localization explains the stable Ca2+ gradient between blood and brain, and is not necessarily indicative of immature or pathological vessels as interpreted in the past.
Manoonkitiwongsa
P S
PS
Department of Pathology and Human Anatomy, Loma Linda University, CA 92350, USA.
Whitter
E F
EF
Wareesangtip
W
W
McMillan
P J
PJ
Nava
P B
PB
Schultz
R L
RL
D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001812:Blood-Brain Barrier; D001921:Brain / Q000098:blood supply*; D001923:Brain Chemistry; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D004730:Endothelium, Vascular / Q000737:chemistry*; D005404:Fixatives; D005557:Formaldehyde; D005976:Glutaral; D008274:Magnesium; D008297:Male; D008345:Manganese; D011122:Polyphosphates / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016707:Tissue Fixation
D005404:Fixatives; D011122:Polyphosphates; D005557:Formaldehyde; D000249:Adenosine Monophosphate; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; C005692:triphosphoric acid; D005976:Glutaral