7-Nitrobenzofurazan (NBD) derivatives of 5'-N-ethylcarboxamidoadenosine (NECA) as new fluorescent probes for human A(3) adenosine receptors.

New fluorescent ligands for adenosine receptors (ARs), obtained by the insertion, in the N(6) position of NECA, of NBD-moieties with linear alkyl spacers of increasing length, proved to possess a high affinity and selectivity for the A(3) subtype expressed in CHO cells. In fluorescence microscopy assays, compound 2d, the most active and selective for human A(3)-AR, permitted visualization and localization of this human receptor subtype, showing its potential suitability for internalization and trafficking studies in living cells.

Peripheral-type benzodiazepine receptor ligands: mitochondrial permeability transition induction in rat cardiac tissue.

Strong evidence is emerging that mitochondrial permeability transition (MPT) may be important in certain physiological conditions and, above all, in the processes of cell damage and death. Reversible MPT, triggered by inducing agents in the presence of calcium ions, has resulted in the opening of a dynamic multiprotein complex formed in the inner mitochondrial membrane and has caused large-amplitude mitochondrial swelling. In the present work, the exposure of de-energized rat cardiac mitochondria to peripheral benzodiazepine receptor (PBR) ligands (1-(2-chlorophenyl-N-methyl-1-methylpropyl)-3-isoquinolinecarboxamide (PK 11195), 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864), and diazepam) produced a dose-dependent and cyclosporin A (CSP)-sensitive loss of absorbance, which was indicative of mitochondrial swelling. By contrast, the addition of a high-affinity central benzodiazepine receptor ligand (clonazepam) was ineffective, even at the highest concentration tested. The ultrastructural changes associated with swelling were similar in mitochondria exposed either to PK 11195 or to calcium. Supporting the apoptotic role of PK 11195-induced swelling, supernatants from mitochondria that had undergone permeability transition caused apoptotic changes in isolated cardiac nuclei. In addition, ultrastructural abnormalities were observed in rat cardiac tissue following in vivo PK 11195 administration, with these abnormalities being prevented by CSP co-administration. These data indicate that PBR ligands induce mitochondrial permeability transition and ultrastructural alterations in isolated cardiac mitochondria as well as in myocardiocytes, suggesting a novel strategy for studying the implication of PBR ligands as apoptosis inducers, through a probable effect on the MPT pore.

Effects of newly-developed benzodiazepine ligands on noise-induced mitochondrial damage in the rat.

In this study we measured the ability of three newly-synthesized N-arylalkylindol-3-ylglyoxylylamide derivatives, which have recently been characterized as partial agonists at central benzodiazepine binding sites, to prevent the rat cardiac mitochondrial alterations resulting from acute loud noise exposure. In particular, we evaluated the effects of these new compounds on the ultrastructural damage induced by noise stress on the right atrium and ventricle after 6 and 12 hr of loud noise exposure. In parallel experiments, we measured the affinity of these compounds for peripheral benzodiazepine binding sites. Following a single injection of the test products, we observed a cardioprotective effect which was more marked after 6 hr compared with 12 hr of noise exposure. Confirming our recent data showing that full agonists at benzodiazepine receptors produce cardioprotection, we demonstrate in this study that partial agonists, like indolylglyoxylylamides, can also produce a cardioprotective effect. Based on their greater affinity in binding studies, the protective activity seems to be related more to their action at central than at peripheral benzodiazepine receptors.

Effect of noise exposure on rat cardiac peripheral benzodiazepine receptors.

Noise is an environmental physical agent, which is regarded as a stressful stimulus: impairment and modifications in biological functions are reported, after loud noise exposure, at several levels in human and animal organs and apparatuses, as well as in the endocrine, cardiovascular and nervous system. In the present study equilibrium binding parameters of peripheral benzodiazepine receptors (PBRs) labelled by the specific radioligand [3H]PK 11195, were evaluated in cardiac tissue of rats submitted to 6 or 12 h noise exposure and of rats treated "in vivo" with PBR ligands such as PK 11195, Ro54864, diazepam and then noise-exposed. Results revealed a statistically significant decrease in the maximum number of binding sites (Bmax) of [3H]PK 11195 in atrial membranes of 6 or 12 h noise exposed rats, compared with sham-exposed animals, without any change in the dissociation constant (Kd). The "in vivo" PBR ligand pre-treatment counteracted the noise-induced modifications of PBR density. As PBRs are mainly located on mitochondria we also investigated whether noise exposure can affect the [3H]PK 11195 binding parameters in isolated cardiac mitochondrial fractions. Results indicated a significant Bmax value decrease in right atrial mitochondrial fractions of rats 6 or 12 h noise-exposed. Furthermore, as PBR has been suggested to be a supramolecular complex that might coincide with the not-yet-established structure of the mitochondrial permeability transition (MPT)-pore, the status of the MPT-pore in isolated heart mitochondria was investigated in noise- and sham-exposed rats. The loss of absorbance associated with the calcium-induced MPT-pore opening was greater in mitochondria isolated from hearts of 6 h noise- than those of sham-exposed rats. In conclusion, these findings represent a further instance for PBR density decrease in response to a stressful stimulus, like noise; in addition they revealed that "in vivo" administration of PBR ligands significantly prevents this decrease. Finally, our data also suggest the involvement of MPT in the response of an organism to noise stress.

A(1) adenosine receptors in human neutrophils: direct binding and electron microscope visualization.

By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A(1) adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A(1) receptors in human neutrophils have not been investigated. In the present study, we used the agonist [(3)H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A(1) binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [(3)H] CHA bound A(1) adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 +/- 0.08 nM and a maximum density of binding sites of 7.1 +/- 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A(1) adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A(1) binding sites and to follow the intracellular trafficking of these receptors.

Fluorescent probes for adenosine receptors: synthesis and biology of N6-dansylaminoalkyl-substituted NECA derivatives.

New fluorescent ligands for adenosine receptors are described; these compounds were obtained by the insertion, in the N6 position of NECA (a potent adenosine agonist), of dansylaminoalkyl moieties with alkyl spacers of increasing carbon chain length (from 3 to 12). Among them, the compound with a C6 alkyl spacer proved to be the most interesting one, showing a marked selectivity for the A1 receptor subtype; furthermore, in fluorescence microscopy assays it proved to be able to visualize and localize this receptor subtype at the level of the molecular layer of the rate cerebellar cortex.