Anna Fiori

Regulation by cannabinoid receptors of anandamide transport across the blood-brain barrier and through other endothelial cells

The endocannabinoid anandamide (AEA) has many neurovascular activities. However, it is not yet clear how AEA can be metabolized at the neurovascular interface, and how it can move through the vascular and the cerebral compartments. The results reported in this article show that isolated bovine brain microvessels, an ex vivo model of the blood-brain barrier, have detectable levels of endogenous AEA and possess the biochemical machinery to bind and metabolize it, i.e. type-1 and type-2 cannabinoid receptors (CB1R and CB2R), a selective AEA membrane transporter (AMT), an AEA-degrading fatty acid amide hydrolase, and the AEA-synthesizing enzymes N-acyltransferase and N-acyl-phosphatidylethanolamines-specific phospholipase D. We also show that activation of CB1R enhances AMT activity through increased nitric oxide synthase (NOS) activity and subsequent increase of NO production. AMT activity is instead reduced by activation of CB2R, which inhibits NOS and NO release. In addition, binding experiments and immunoelectronmicroscopy demonstrate that different endothelial cells vary in the expression of CB1R and CB2R on the luminal and/or abluminal sides. The different localization of CBRs can lead to a diverse effect on AMT activity on the luminal and abluminal membranes, suggesting that the distribution of these receptors may drive AEA directional transport through the blood-brain barrier and other endothelial cells.

Isolated Brain Microvessels as In Vitro Equivalents of the Blood‐Brain Barrier: Selective Removal by Collagenase of the A‐System of Neutral Amino Acid Transport

On treatment with collagenase, brain microvessels, together with several protein components, lose some enzymatic activities such as alkaline phosphatase and γ‐glutamyltranspeptidase, whereas no change occurs in the activities of 5′‐nucleotidase and glutamine synthetase. The energy‐requiring „A‐system” of polar neutral amino acid transport is also severely inactivated, whereas the L‐system for the facilitated exchange of branched chain and aromatic amino acids is preserved. In the collagenase‐digested microvessels, this leads to loss of the transtimulation effect of glutamine on the transport of large neutral amino acids, because such transtimulation is due to a cooperation between the A‐ and L‐systems. By contrast, NH4+ maintains (and even enhances) its ability to stimulate the L‐system of amino acid transport, presumably through glutamine synthesis within the endothelial cells.

The binding of mycolic acids to galectin-3: A novel interaction between a host soluble lectin and trafficking mycobacterial lipids?

Understanding the molecular mechanism of host‐pathogen interactions is the basis for drug design and vaccine development. The fine composition of mycolic acids (MA), the major constituents of Mycobacterium tuberculosis (Mtb) cell envelope, as well as other cell wall‐associated lipids, contribute to determine the virulence of a given strain. However, endogenous receptors for mycolic acids on susceptible cells exposed to mycobacterial infections have not been fully identified. Here, we show that galectin‐3, a multifunctional β‐galactoside binding lectin present mainly in the cytoplasm of inflammatory cells and also present on the cell surface, can recognize mycobacterial mycolic acids. MA can inhibit the lectin self‐association but not its carbohydrate‐binding abilities and can selectively interfere in the interaction of the lectin with its receptors on temperature‐sensitive dendritic cell line, suggesting that galectin‐3 could be involved in the recognition of trafficking mycolic acids and participate in their interaction with host cells.

Uptake of amino acids by brain microvessels isolated from rats with experimental chronic renal failure

Abstract: The neurological disorders seen in patients with chronic renal failure and liver cirrhosis are analogous. Previous in vivo studies have shown that the impaired blood‐brain amino acid transport seen in rats with chronic renal failure is similar to that of rats with portocaval anastomosis. To elucidate whether a comparable underlying pathogenic mechanism plays a role in both pathological conditions, blood and brain amino acid levels together with amino acid transport by isolated brain microvessels have been studied in rats with chronic renal failure and in sham‐operated rats. Brain microvessels isolated from rats with experimental chronic renal failure showed that the uptake of labeled large neutral amino acid, i.e., leucine or phenylalanine, but not of lysine or a‐methylaminoisobutyric acid, was significantly increased with respect to sham‐operated rats; conversely, the uptake of glutamic acid in rats with chronic renal failure was significantly lower compared with values in controls. Kinetic analysis indicated that this was mainly due to increased exchange transport activity (Vmax) of the L‐system, rather than to changes in the affinity (Km) of the carrier system for the relative substrate. These data, together with the significant rise of brain glutamine levels and an increased brain‐to‐plasma ratio of the sum of large neutral amino acids, are analogous to what was previously observed in rats with portocaval anastomosis. Because increased brain influx of large neutral amino acids, especially the neurotransmitter precursor amino acids, is thought to be responsible for the altered brain neurotransmission observed in chronic liver failure, these data support the hypothesis that the neurological disorders seen in chronic renal failure may result from functional modifications to the L‐systems transport activity at the blood‐brain barrier, similar to those observed in chronic liver failure.

The expression of native and oxidized LDL receptors in brain microvessels is specifically enhanced by astrocytes-derived soluble factor(s)

Ex vivo rat brain microvessels express receptors for native as well as for oxidized low‐density lipoproteins. In brain microvessels‐derived endothelial cells, the expression levels of both receptors were enhanced by co‐cultivation with rat astrocytes, even in the absence of actual contact between the two cell types, suggesting a soluble factor(s)‐based mechanism of induction. No modulation effect could be evidenced in a heterologous cellular system. Since both receptors were found to be expressed also in astrocytes, these cells are likely to contribute substantially to the lipoprotein management at the blood–brain barrier and in the brain compartment.

Interaction of L-DOPA decarboxylase with substrates: A spectrophotometric study

Mammalian L-DOPA decarboxylase (EC 4.1.1.26) is an enzyme with a broad specificity, which acts on the natural aromatic amino acids. It has been purified from kidney by Christenson et al. [l] , by Borri Voltattorni et al. [2] and by Lancaster and Sourkes [3] and its spectral properties have been described [4]. While the interactions between enzyme and substrates have been studied with the bacterial amino acid decarboxylases [ 51, no data of this sort are available with the mammalian enzymes, which differ from the others in the value of pH optimum; furthermore the mechanism of action of DOPA decarboxylase, notwithstanding the biochemical and pharmacological importance of the enzyme, has received little attention. We have studied the interaction of LDOPA decarboxylase from hog kidney with its substrates by means of spectrophotometry, and we have been able to provide evidence for at least three enzymesubstrate or enzyme-product complexes; a quite unusual absorbance spectrum has been observed for two of them.

Inhibitors binding to L-aromatic amino acid decarboxylase

The effect of a number of inhibitors of L-aromatic amino acid decarboxylase activity on the absorption spectrum of the enzyme-bound coenzyme has been studied. It has been observed that the compounds tested, even if devoid of the amino function and therefore unable to form the Schiff base with the coenzyme, modify significantly the enzyme spectrum, indicating their binding to the coenzyme active site. Spectral modifications suggest that at least two kinds of binding of inhibitors to L-aromatic amino acid decarboxylase may occur, depending on their structural features. Moreover, from the spectra obtained at different concentrations of the inhibitors their affinity constants have been determined: data indicate that the cathecol ring gives the largest contribution to the binding, while the presence of the carboxyl group, the aminic group and the aliphatic chain are responsible for a decrease in the binding, which could be relevant for the efficiency of the catalysis.

Inhibitory effect of somatostatin on neutral amino acid transport in isolated brain microvessels

In the presence of somatostatin‐14 or some of its receptorial agonists, the uptake of large neutral amino acids by isolated brain microvessels was found to be inhibited up to 50%, no other transport system being affected. Although the luminal and abluminal sides of brain endothelial cells are both capable of taking up large neutral amino acids, only uptake from the abluminal side appears to be inhibited by somatostatin. The involvement of a type‐2 somatostatin receptor was suggested by assays with a series of receptor‐specific somatostatin agonists, and was confirmed by the release of inhibition caused by a specific type‐2 receptor antagonist. A type‐2‐specific mRNA was indeed shown to be present in both bovine brain microvessels ex vivo and primary cultures of endothelial cells from rat brain microvessels.

Synergism between different transport systems stimulates the uptake of neutral amino acids by isolated brain microvessels

SummaryThe polar long-chain amino acids glutamine and methionine can be transported across the endothelial cells of brain microvessels either by an L-system which operates by a facilitated diffusion, exchanging mechanism, or by a concentrating, energy-dependent A-system. The presence of glutamine and/or of methionine can induce a synergism between the two transport systems which results, by a transstimulation mechanism, in a net increased uptake of neutral hydrophobic aminoacids. The methionine analog S-methylthiocysteine, which is the mixed disulfide resulting from the combination of methanethiol with cysteine, behaves similarly to methionine in stimulating the uptake of neutral hydrophobic amino acids. The same transstimulating effect can even be obtained in collagenase-treated, A-system-deprived microvessels by inducing the direct formation of S-methylthiocysteine within the cytoplasmic compartment of the endothelial cells.

Effect of the cationic polypeptide polylysine on neutral amino acid transport in isolated brain microvessels.

SummaryThe functionality of isolated brain microvessels — used as anin vitro model of the blood-brain barrier — can be influenced by interaction with cationic proteins. The various polylysines (Mr ranging from 0.9 to 180 kDa) tested affected the activity of both the Na+-dependent (“A”) and the Na+-independent (“L”) systems for neutral amino acid transport. Exposure to the 180 kDa polylysine caused a conspicuous inhibition of both transport systems, associated to an increased passive permeability. There was a constant, Mr-dependent, inhibition of the the L-system-mediated uptake of hydrophobic neutral amino acids. The activity of the A-system was enhanced, upon exposure to polymers larger than 22 kDa reaching its peak at 68 kDa and and declining at higher Mr values. The effect which was Na+-ions dependent and abolished by phloretine, could be essentially ascribed to an increased affinity of the MeAIB for its carrier (Km value decreasing from 265 to 169µM in presence of 68 kDa polylysine).