Luminal Localization of Blood-Brain Barrier Sodium, Potassium Adenosine Triphosphatase Is Dependent on Fixation

Abstract

Cytochemical data in the literature reporting localization of sodium, potassium adenosine triphosphatase (Na(+), K(+)-ATPase) in the blood-brain barrier (BBB) have been contradictory. Whereas some studies showed the enzyme to be located exclusively on the abluminal endothelial plasma membrane, others demonstrated it on both the luminal and abluminal membranes. The influence of fixation on localization of the enzyme was not considered a critical factor, but our preliminary studies showed data to the contrary. We therefore quantitatively investigated the effect of commonly used fixatives on the localization pattern of the enzyme in adult rat cerebral microvessels. Fixation with 1%, 2%, and 4% formaldehyde allowed deposition of reaction product on both the luminal and abluminal plasma membranes. The luminal reaction was reduced with increasing concentration of formaldehyde. Glutaraldehyde at 0.1%, 0.25%, 0.5%, in combination with 2% formaldehyde, drastically inhibited the luminal reaction. The abluminal reaction was not significantly altered in all groups. These results show that luminal localization of BBB Na(+), K(+)-ATPase is strongly dependent on fixation. The lack of luminal localization, as reported in the literature, may have been the result of fixation. The currently accepted abluminal polarity of the enzyme should be viewed with caution.

Fluid and ion transfer across the blood–brain and blood–cerebrospinal fluid barriers; a comparative account of mechanisms and roles

“…However, even in these studies, because the assays were conducted after tissue fixation there is the ever present risk that Na + -pumps were inactivated prior to the assay [304, 313, 314]. Furthermore, it is conceivable that fixation might have affected the pumps in one membrane more than the other [311]. It should be noted that, using the K-NPPase cytochemical assay, differences in localization of the Na + -pumps were observed in the choroid plexus that have never been adequately explained [315].…”

“…levamisole. However, even so it is necessary to demonstrate that the activity detected requires the presence of K + and is inhibited by ouabain, usually at 1 mM.Manoonkitiwongsa et al [311] investigated the effects of a range of concentrations of formaldehyde and glutaraldehyde. They found that fixation with 2% formaldehyde yielded 1.5 as the ratio of the luminal to abluminal product densities but with 2% formaldehyde plus glutaraldehyde at 0.1, 0.25 or 0.5% the ratio decreased to 0.7, 0.5 or 0.4 [311].…”

“…They found that fixation with 2% formaldehyde yielded 1.5 as the ratio of the luminal to abluminal product densities but with 2% formaldehyde plus glutaraldehyde at 0.1, 0.25 or 0.5% the ratio decreased to 0.7, 0.5 or 0.4 [311]. They concluded that glutaraldehyde had an effect to selectively decrease luminal activity.…”

Blood-brain barrier Ca2+-ATPase cytochemistry: incubation media and fixation methods for differentiating Ca2+-specific ATPase from ecto-ATPase

“…There were both similarities and differences compared to tissues fi xed with 4% formaldehyde. The similarities were that reaction product was present at the BBB for all of these controls and the amount of reaction product on the abluminal surface of the endothelia ultrastructure is poor, because 4% formaldehyde is a weak fi xative, particularly compared to fi xatives containing even small amounts of glutaraldehyde (Manoonkitiwongsa et al 2000). Fig.…”

“…The diffi culty with image analysis procedures is that it can be very tedious and time-consuming, which can discourage investigators from using quantitative measurements or even the incubation medium of Ando et al (1981) for the BBB or any other tissue. We have had this experience with previous quantitative studies of Na ϩ , K ϩ -ATPase reaction product in the BBB (Manoonkitiwongsa et al 2000).…”