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.
Department of Pathology and Human Anatomy, Loma Linda University, CA 92350, USA.
P S
PS
Manoonkitiwongsa
E F
EF
Whitter
W
W
Wareesangtip
P J
PJ
McMillan
P B
PB
Nava
R L
RL
Schultz
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