Effect of trypsinization in cell culture on bradykinin receptors in vascular endothelial cells

Two types of receptors (B₁R, B₂R) for kinins are defined in mammalian species. Comparative experiments involving recombinant fusion proteins consisting of rabbit B₁R or B₂R fused to GFP-related proteins are exploited to study the regulation of the response to kinins at the receptor level. The following points will be briefly reviewed and supported by some novel data. (1) The constitutive B₂Rs are internalized upon agonist stimulation, but completely recycled to the cell surface; however, B₂R destruction can be achieved following limited proteolysis (extracellular trypsin, neutrophil proteases), a plausible down-regulation mechanism in pathology. (2) The inducible B₁Rs, stimulated by des-Arg⁹-kinins, are not phosphorylated nor internalized upon agonist stimulation, but rather undergo a reversible redistribution to caveolae-related rafts. B₂Rs are also subjected to this translocation, but only transiently (before endocytosis). (3) Based on the analysis of rabbit aortic smooth muscle cells, B₁R induction by cytokines is dependent on nuclear factor κB in rabbit vascular tissue, but exogenous kinins acting on either receptor type do not induce B₁R expression.

In this paper the anatomy of the blood-brain barrier and the properties of peripheral versus brain-microvessel-endothelial cells (BMEC) are outlined and the isolation and application of microvessels are discussed. In addition, the isolation and culture of BMEC are described and discussed and the application of this system in drug transport-and metabolism-studies, visualization of drug transport routes, and in the study of changes in BBB functionality during inflammation, is illustrated.

In the present work we studied the effect of adenosine and various analogues on cAMP level in guinea pig coronary endothelial cells of microvascular origin. The tested adenosine agonists mediate a concentration-dependent increase in cAMP level. The rank order of potency was 5′-N-ethylcarboxamidoadenosine (NECA) > CGS 21680 > N⁶-phenylisopropyladenosine (R-PIA) > 2-chloro-N⁶-cyclopentyladenosine (CCPA) which is typical for an adenosine A₂ receptor. Their respective concentrations for half maximal stimulation of cAMP formation were 0.36 μM, 0.82 μM, 4.7 μM and 9.8 μM. The tested agonist showed differences in efficacy, NECA being the most efficacious. R-PIA, CCPA and adenosine were less efficacious, suggesting partial agonism. The efficacy of adenosine was unchanged by the addition of the nucleoside transport inhibitor S(4-nitrobenzyl)-6-thioinosine (NBTI, 10 μM) suggesting that inhibition of adenylyl cyclase through P-site activation is not responsible for the observed low efficacy of adenosine. We could demonstrate CGS 21680 activation of adenylyl cyclase in a peripheral receptor. We therefore suggest that the endothelial adenosine receptor resembles the striatal adenosine A_2a receptor.

The cloned pulp-cell line RDP4-1 increases cAMP production, hydrolyses phosphoinositide (PI) and mobilizes calcium in response to prostaglandin E₂ (PGE₂) and PGF₂-α. The effect of bradykinin (BK) on intracellular signalling systems and DNA synthesis was studied in these cells. BK (10 μM) transiently increased cytoplasmic free calcium ([Ca²⁺]i) both in the presence and absence of external calcium. After stimulation with BK (10 μM), cells did not respond significantly to PGE₂ (0.5 μg/ml). Pretreatment with indomethacin (30 μM) inhibited the [Ca²⁺]i increment by BK (10 μM), but not by the subsequent addition of PGE₂ (0.5 μg/ml). Also, pretreatment with PGE₂ (0.5 μg/ml) blocked the action of BK (10 μM). BK (0.1–100 μM) stimulated PI hydrolysis and cAMP production in a dose-dependent manner. Both the PI and the cAMP responses were inhibited by indomethacin (30 μM), as was the calcium response. BK (0.01–10 μM) also stimulated release of arachidonic acid and its metabolites dose-dependently. However, prolonged exposure to BK in serum-deficient medium did not exert any effect on DNA synthesis. RDP 4-1 cells, therefore, appear to respond to BK with increased cAMP production, PI hydrolysis and calcium mobilization. The inhibition of these effects of BK by indomethacin raises the possibility that cyclo-oxygenase product(s), especially PGE₂ or PGE₂-like compounds, may be responsible for evoking these effects. These results indicate that BK may stimulate or modulate cell metabolism in the dental pulp.

The contractile responses of cultured rat and calf endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts (FB) to vasoactive mediators (thrombin, serotonin, bradykinin, and histamine), forskolin, and cytochalasin B were compared. Cells were grown on a pliable silicone membrane, and contraction was assessed, using time-lapse video microscopy, by recording changes in the wrinkling of the silicone as the cells exerted tension on the surface. We found that all cells contracted in the presence of serum or thrombin and that VSMC and FB also contracted with serotonin stimulation. Bradykinin and histamine were not contractants in this system. Discrepancies between these results and reports of changes in permeability of endothelial layers in vitro and in vivo may be due to (1) the vascular segment from which EC were studied or (2) the possibility that certain mediators may provoke a noncontractile response that results in gap formation. Thus changes in vascular permeability, which occur during inflammation, may have both contractile and noncontractile components. Forskolin, known to indirectly inhibit myosin light-chain kinase activity, and cytochalasin B were potent relaxants, suggesting a similar smooth muscle-like contractile mechanism for all three cell types.

The high-affinity uptake of the acidic amino acidd-aspartate was inhibited in a dose- and time-dependent manner, when C6 cells were exposed to trypsin. The protease decreased the maximal velocity for uptake but not itsK_m, consistent with a reduction in the number of competent carriers at the plasma membrane. Cellular energy production and [K⁺]_i were unaffected, indicating that the transporter itself was the site of trypsin action. Maximum inhibition of uptake was 50%, which siggests the presence of a heterogeneous population of transporters, only half of which is sensitive to trypsin. These results support our earlier postulate that in glial cells, the high-affinity transporter for acidic amino acids is a transmembrane protein, part of which extends into the external environment.