Incorporation of highly purified melittin into phosphatidylcholine bilayer vesicles.

Melittin free of phospholipase A2 was prepared. In the absence of salt this highly pure protein starts to aggregate in solution at a protein concentration of Cp greater than 10(-3) M. In high salt solution (2 M) aggregation starts at Cp greater than 10(-6) M. This was determined from the blue shift of the intrinsic fluorescence of the protein. Reinvestigation of the quenching behaviour clearly shows that self-aggregation cannot be deduced from quenching experiments using nitrate or 2,2,6,6-tetramethylpiperidine-1-oxyl as quencher. The incorporation of melittin into phosphatidylcholine bilayer vesicles was studied by fluorescence quenching and by energy-transfer experiments using 2- and 6-anthroyloxypalmitic acid as acceptor and peptide tryptophan as donor. Incorporation of melittin into small unilamellar vesicles was found to be reduced below the lipid phase transition temperature, Tt, whereas it incorporates and distributes more randomly above Tt. Cooling the temperature below Tt after incubation at T greater than Tt leads to a deeper incorporation of the peptide into the lipid bilayer due to electrostatic interaction between the lipid phosphate groups and the positively charged amino acids. This stabilizing effect is lost above Tt and melittin is extruded to the polar phase. Quenching experiments support this finding. EPR measurements clearly demonstrate that even in the presence of high amounts of melittin up to 10 mol% with respect to the lipid broadening of the phase transition curves was only observed with fatty acid spin labels, where the doxyl group is localized near the bilayer surface. The order degree of the inner part of the bilayer remains almost unchanged even in the presence of high melittin content.

Blood-brain barrier characteristic enzymatic properties in cultured brain capillary endothelial cells.

Isolated brain capillary endothelial cells contain high activity levels of the blood-brain barrier (BBB) marker enzymes gamma-glutamyltranspeptidase (gamma-GT) and alkaline phosphatase (ALP). In primary culture the activities of these specific enzymes decrease with increasing cell proliferation to a constant low value characteristic for a confluent monolayer. However, activities are retained in non-proliferating cells. After passage of cells from a confluent cell monolayer a further reduction of enzyme activity was observed which corresponds to the newly triggered cell proliferation. Culture of cerebral endothelial cells on structural components of the native vascular basement membrane-like type IV collagen, fibronectin, laminin or a commercially available basement membrane cannot prevent the activity decrease of both gamma-GT and ALP. Antiserum raised against a native renal dog gamma-GT binds to the cerebral endothelial gamma-GT and suppresses its activity. The relative activity decrease induced by a given amount of anti-gamma-GT-antiserum is constant at all times in culture. This result clearly shows that the observed decrease in gamma-GT activity in proliferating cells in culture correlates to a decreased number of enzyme molecules per cell and not to an inhibition of expressed enzymes. Possibly the de novo synthesis of this enzyme is prevented in vitro. In contrast to the loss of the activity of the BBB marker enzymes gamma-GT and ALP, the activity of angiotensin-converting enzyme (ACE), a marker for all vascular endothelial cells, is highly preserved in cultured cerebral endothelial cells.

Correlation of zonula occludens ZO-1 antigen expression and transendothelial resistance in porcine and rat cultured cerebral endothelial cells.

Cerebral endothelial cell cultures (CECC) have been utilized as an in vitro system for simulating the blood-brain barrier. We have studied the expression of a tight junction associated protein (ZO-1) concomitantly with measurements of transendothelial electrical resistance and freeze-fracturing to test the barrier properties of CECC monolayers and, thereby, their capability in serving as an in vitro blood-brain barrier model. Freshly isolated CECC aggregates and 7 day old CECCs in primary culture express the ZO-1 protein. Measurements of the transcellular resistance of CECC monolayers yielded an electrical resistance of about 90 omega cm2. The low electrical resistance of cerebral endothelial monolayers together with the reduced complexity of the tight junction complement in vitro are indicative of reduced barrier properties of the in vitro system.

Characterization of gamma-glutamyl transpeptidase activity of cultured endothelial cells from porcine brain capillaries.

Endothelial cells were isolated with high viability (greater than 93%) from porcine brain capillaries by Percoll gradient centrifugation after purely enzymatic digestion. Primary cultures were grown to confluent cell monolayers and quantitated for the activity of gamma-glutamyl transpeptidase. The gamma-glutamyl transpeptidase activity starts from a high enzymatic level, decreases with time in culture to about 15% of the initial value, and remains constant at this level after day 10 in culture. The activity progression depends on surface conditions. In the presence of collagen, an exponential decrease starts immediately after seeding, with a time constant of 70 +/- 10 h. In the absence of collagen, gamma-glutamyl transpeptidase activity first decreases on day 1 after plating, recovers to the initial value on day 2 and 3 and afterwards declines exponentially to a low and constant activity level. Ethanol added to the cell culture at a time when low constant activity is reached, reactivates the gamma-glutamyl transpeptidase to 30% of the initial value.