An in situ cytochemical evaluation of blood–brain barrier sodium, potassium-activated adenosine triphosphatase polarity

Abstract

It is presently believed that sodium, potassium-activated adenosine triphosphatase (Na +, K +-ATPase) is localized on the abluminal plasma membrane of brain endothelial cells. But there have been contrary reports from some cytochemical studies. We examined the localization of the enzyme in rat cerebral microvessel endothelium using the in situ model originally employed to establish the abluminal polarity concept. Alterations in fixation and incubation media from the original reports were conducted to determine the effect on localization pattern. With the Ernst indirect incubation method as originally used, three types of localization patterns were obtained: abluminal only, luminal only, and on both surfaces of endothelial cells. With the direct incubation method of Mayahara, reaction product was seen on both surfaces. Reduction in fixation time followed by the use of the indirect incubation method resulted in a complete loss of the reaction product. The same reduction in fixation time followed by the use of the direct method did not alter the localization pattern of the enzyme. Our results demonstrated that Na +, K +-ATPase is localized on both surfaces of brain endothelial cells. The localization pattern of Na +, K +-ATPase is significantly dependent upon fixation and the incubation medium used in the in situ model. Data discrepancies for the enzyme as reported in the literature appear to be caused by differences in cytochemical protocols, rather than the biological reasons advocated by other investigators. We conclude that past cytochemical reports of blood–brain barrier (BBB) Na +, K +-ATPase abluminal localization were incomplete. The currently held abluminal polarity theory of the enzyme needs to be reexamined. Past basic and clinical cytochemical studies of BBB Na +, K +-ATPase should be viewed and interpreted with caution.