Gary W. Goldstein

Polarity of the blood-brain barrier: Distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells

The subcellular distribution in brain capillaries of alkaline phosphatase and Na+, K+-ATPase was investigated by two methods. Cytochemical studies using whole brain perfusion and electron microscopic examination indicated that alkaline phosphatase activity was located in both the luminal and antiluminal cytoplasmic membranes of the brain capillary endothelial cells. By contrast, the K+-dependent phosphatase activity associated with Na+, K+-ATPase was located in only the antiluminal membrane. Biochemical studies using membranes prepared by homogenization of isolated brain capillaries and density gradient centrifugation resulted in identification of two plasma membrane fractions. The light fraction contained alkaline phosphatase but very little Na+, K+-ATPase while the heavier fraction contained both enzyme activities. In addition, gamma-glutamyl transpeptidase showed a distribution similar to alkaline phosphatase while 5-nucleotidase activity was distributed with the Na+, K+-ATPase activity. We conclude that the luminal and antiluminal membranes of brain capillaries are biochemically and functionally different. This polarity should permit active solute transport across brain capillary endothelial cells which are the cells responsible for the blood-brain barrier.

Isolation of metabolically active capillaries from rat brain

MANY substances circulating in the blood do not enter the central nervous system or achieve only limited steadystate concentrations in the brain. To account for these observations, a functional barrier is considered to separate the central nervous system from the vascular elements. Recent studies suggest that this blood-brain barrier may be related to the unique ultra structural anatomy of endothelial cells in small blood vessels of the brain. These cells are circumferentially sealed together by tight junctions, lack fenestrations and are surrounded by astroglial processes (REESE & KARNOVSKY, 1967). Despite this anatomical arrangement, some metabolites appear to be transported across the blood-brain carrier by stereospecific carrier-mediated transport systems (OLDENDORF, 1971). This suggests that transcapillary movement of specific molecules might occur through the cells of the capillary wall. Thus, metabolic study of capillaries isolated from brain may shed light on some of the factors that regulate the permeability of the blood-brain barrier. Recently, several laboratories have reported methods for preparing brain capillary segments (SIAKOTOS et al., 1969; J o o & KARNUCHINA, 1973; ORLOWSKI et al., 1974; BRENDEL et at., 1974). These studies have been limited by a requirement for large quantities of bovine brain or have not presented sufficient criteria of purification and metabolic activity. In this report, we describe a novel method for isolating brain capillaries from rat brain which are suitable for study in uitro. They appear to be free of neuronal and glial contamination, contain characteristic marker enzymes in high concentration, and are capable of carrying out glucose transport and oxidation. Brain capillaries were isolated in physiologic buffer without the addition of digestive enzymes in order to minimize damage to cellular metabolism and membrane transport. The cerebral cortices from 20 one-month-old rats were minced in 200ml of oxygen saturated Ringer’s solution containing 1.2 mM-MgCl,, 15 mwHepes buffer (pH 7.4), 5mM-glucose and 1% fraction V bovine serum albumin. The mince was passed serially through 670, 335 and 116pm nylon meshes and then centrifuged at loo0 x g for 10min. In order to remove cellular debris and myelin the pellet was suspended in 100 ml of the Same buffer now

Relation of potassium transport to oxidative metabolism in isolated brain capillaries.

1. The uptake of K by a capillary suspension isolated from rat brain was studied with the radioactive analogue 86Rb.

Carrier mediated glucose transport in capillaries isolated from rat brain.

Abstract— Uptake of 2‐deoxy‐d‐glucose (2‐DG) was investigated in capillaries isolated from rat brain. A high affinity, carrier‐mediated transport system was defined with an apparent Km for 2‐DG of 93 μM. Uptake was temperature‐dependent and markedly inhibited by phloretin and selected hexose isomers. The apparent Ki for d‐glucose inhibition of 2‐DG uptake was 98 μM. Essentially all of the 2‐DG retained by the capillary preparation could be released by sonication and 95% was recovered as free unphosphorylated 2‐DG. Uptake was not sodium‐dependent and not altered by insulin. These results suggest that movement of glucose across the blood‐brain barrier through endothelial cells probably is not rate‐limiting unless blood glucose levels are extremely low.

Electron probe microanalysis of isolated brain capillaries poisoned with lead

The blood-brain barrier has been proposed as an important site for the toxic action of lead in the central nervous system. To investigate this, capillary endothelial cells were isolated from rat cortex and exposed to lead in vitro. Tissue suspensions were then prepared for electron microscopy and X-ray microprobe analysis. In cells exposed in vitro to lead, electron-dense deposits were observed within mitochondria. With X-ray analysis, it was determined that these intramitochondrial deposits contained lead in a non-crystalline matrix. Also, lead appeared to be accumulated in the same intramitochondrial areas as calcium. The results suggest that lead is preferentially sequestered in mitochondria of capillary endothelial cells. Further, this selective localization may be associated with lead-induced disruptions in intracellular calcium metabolism and transepithelial transport processes.

Synthesis and secretion of an albumin-like protein by cultured bovine tracheal gland serous cells

Bovine tracheal submucosal gland serous cells were incubated with (35S) methionine. The proteins synthesized and secreted into the culture medium during pulse and chase periods were analyzed. Three major protein bands with apparent molecular weight values of 67 kD, 47 kD and 32 kD were detected by fluorography following SDS-polyacrylamide gel electrophoresis. Based on the molecular weight, immuno-reactive cross reactivity and surface molecular change characteristics, the labeled Mr 67 kD protein was identified as an albumin-like protein. Our results suggest that albumin found in airway secretions can originate, at least in part, from tracheal gland serous cells.

Culture of endothelial cells from neural capillaries

In brain and retinal capillaries, the endothelial cells are sealed together by continuous tight junctions (1, 2), contain few pinocytotic vesicles (1, 2), and have a polar distribution of transport carriers between their luminal and antiluminal plasma membranes (3, 4). These features distinguish neural from systemic capillaries and result in formation of the blood-brain and blood-retinal barriers. However, in addition to providing a permeability barrier to some polar solutes, the endothelial cells in neural capillaries must regulate the movement of many other solutes into and out of the nervous system. Since it is difficult to study the cellular mechanisms involved in control of neural capillary permeability in intact animals, methods were developed to isolate microvessels from brain and retina. Isolated microvessels are metabolically active and have been useful in characterizing the biochemical properties of this specialized endothelium as well as studying its response to toxic agents. There are, however, several limitations to the use of freshly isolated neural microvessels. Studies are restricted to short incubations lasting minutes to several hours so that it is not possible to study the long-term response of microvessels to injury. In addition, investigations of transport by isolated capillaries are limited to studies of solute movement into the cells rather than across a layer of the cells. Furthermore, the microvessels contain per-icytes and occasional smooth muscle cells as well as endothelial cells. This fact hinders interpretation of some of the metabolic responses of the isolated capillaries. Finally, while growth of new brain or retinal capillaries is a prominent reaction in several diseases, this proliferative response cannot be studied with isolated microvessels. Since these problems might be circumvented by growing purified capillary endothelial cells in monolayer culture, several laboratories have begun to develop methods for culturing endothelial cells from brain and retinal microvessels. This review will consider the early advances that have been made in culturing these cells.