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Biomedical subjects

A Parini

Publications and source records attributed to A Parini.

At least 37 records · Page 2Linked to original sources
PubMed1997

I2-imidazoline binding sites and monoamine oxidase activity in human postmortem brain from patients with Parkinson's disease.

I2-imidazoline binding site (I2BS) has been identified with a regulatory site located on a sub-population of monoamine oxidase (MAO)-A and -B. Previous studies showed a modification of MAO and I2BS in the elderly and in neurodegenerative processes such as Alzheimer's disease. In the present study, we studied the potential modification of I2 binding sites and monoamine oxidases in Parkinson's disease. Putamen and cerebral cortex were collected from 17 normal subjects (79 +/- 12 yr) and 16 patients (76 +/- 9 yr) affected by Parkinson's disease. In mitochondrial preparations, radioligand binding studies with [3H]idazoxan showed that putamen and frontal cortex express equivalent amount of I2BS. The density and affinity of I2BS were similar in normal subjects (putamen: Bmax = 207 +/- 58 fmol/mg of protein, Kd = 10.1 +/- 3.4 nM; cerebral cortex: Bmax = 193 +/- 54 fmol/mg of protein, Kd = 12.8 +/- 6.8 nM) and Parkinson's disease patients (putamen: Bmax = 193 +/- 60 fmol/mg of protein, Kd = 9.8 +/- 4.6 nM; cerebral cortex: Bmax = 199 +/- 49 fmol/mg of protein, Kd = 15.9 +/- 8.1 nM). The activity of total monoamine oxidase and monoamine oxidase B, measured by [14C]tyramine and [14C]phenylethylamine oxidation, respectively, were higher in putamen than in cerebral cortex. No differences have been detected in the enzyme activity between normal and pathological subjects. These data suggest that, although MAO and I2BS may play a role in the development of Parkinson's disease, they are not altered in the chronic phase of this disease.

Aged↗
PubMed1997

Predominant expression of monoamine oxidase B isoform in rabbit renal proximal tubule: regulation by I2 imidazoline ligands in intact cells.

Previous studies have shown that a subpopulation of the catecholamine-degrading enzymes monoamine oxidase (MAO) A and B holds a previously unknown regulatory site, the I2-imidazoline binding site (I2BS). In the present work, we characterized the isoforms of monoamine oxidases expressed in the rabbit renal proximal tubule, defined their relationship with I2BS, and investigated the ability of I2BS ligands to inhibit enzyme activity in intact cells. Two findings indicate that MAO-B is the predominant isoform expressed in the renal proximal tubule cells: 1) Western blot performed with an anti-MAO-A/MAO-B polyclonal antiserum revealed a single 55-kDa band corresponding to MAO-B; 2) enzyme assays showed an elevated MAO-B activity ([14C]beta-phenylethylamine oxidation: Vmax = 1.31 +/- 0.41 nmol/min/mg protein), whereas MAO-A activity was only detectable ([14C]5-HT oxidation: Vmax = 80.3 +/- 19 pmol/min/mg protein). Photoaffinity labeling with the I2BS ligand [125I]2-(3-azido-4-iodophenoxy)-methylimidazoline revealed a single 55-kDa band, which indicates that MAO-B of the renal proximal tubule cells holds the I2 imidazoline binding site. [3H]Idazoxan binding studies and enzyme assays showed that, in intact cells, I2BS ligands bind to and inhibit MAO-B. Indeed, the increase in the accessibility of intracellular compartment by cell permeabilization did not enhance [3H]idazoxan binding, which indicates that, in intact cells, intracellular I2BS are fully occupied by imidazoline ligands. In addition, enzyme assays showed that incubation of proximal tubule cells with imidazoline ligands leads to a complete, dose-dependent inhibition of MAO activity. These data show the predominant expression of MAO-B in rabbit renal proximal tubule and its regulation by imidazoline ligands in intact cells.

Animals↗
PubMed1997

Localization of the imidazoline binding domain on monoamine oxidase B.

Monoamine oxidase B (MAO-B) was recently identified as a member of the family of imidazoline binding proteins. To localize the imidazoline binding domain on MAO-B, we labeled the domain with the imidazoline photoaffinity adduct [125I]2-(3-azido-4-iodophenoxy)methylimidazoline in rat and human liver and visualized labeled peptides by autoradiography/sodium dodecyl sulfate-polyacrylamide gel electrophoresis after CNBr cleavage of the labeled protein. Based on species-specific fragmentation patterns and immunoprecipitation of labeled peptides, the imidazoline binding domain was localized to residues K149 to M222 of human MAO-B. The imidazoline binding domain is encompassed within a region that influences substrate processing but is distinct from primary sites of interaction for the enzyme inhibitors pargyline and lazabemide (Ro 19-6327). Radioligand binding assays and photoaffinity labeling also indicated that the various classes of compounds did not cross-compete at the different enzyme domains. Identification of an imidazoline binding domain on MAO-B provides a new opportunity for the potential pharmacological development of imidazoline/guanidinium compounds and also presents additional avenues for structure/function analysis of the monoamine oxidase enzymes.

Animals↗
PubMed1996

Clotrimazole and efaroxan inhibit red cell Gardos channel independently of imidazoline I1 and I2 binding sites.

In the present report, we investigated the potential involvement of imidazoline I1 and I2 binding sites in the inhibition of the Ca(2+)-activated K+ channel (Gardos channel) by clotrimazole in human red cells. Ca(2+)-activated 86Rb influx was inhibited by clotrimazole and efaroxan but not by the imidazoline binding site ligands clonidine, moxonidine, cirazoline and idazoxan (100 microM). Binding studies with [3H]idazoxan and [3H]p-aminoclonidine did not reveal the expression of I1 and I2 binding sites in erythrocytes. These data indicate that the effects of clotrimazole and efaroxan on the erythrocyte Ca(2+)-activated K+ channel may be mediated by a 'non-I1/non-I2' binding site.

Adrenergic alpha-Antagonists↗
PubMed1996

The elusive family of imidazoline binding sites.

Various imidazoline and guanidinium derivatives elicit diverse cellular responses in peripheral and nervous tissues that are often difficult to attribute to known receptor signalling systems. Biochemical, functional and clinical evidence suggests that some activities of these compounds may be related to their action on defined imidazoline binding sites, which have been recently characterized. Unexpectedly, and of particular significance, recent data indicate that two members of the family of imidazoline binding sites are identical to the A and B isoforms of monoamine oxidase. In this article, Angelo Parini and colleagues summarize the evidence for the characterization and location of imidazoline binding sites, and speculate on the clinical implications of compounds acting on these sites.

Animals↗
PubMed1996

I2-imidazoline binding sites: relationship with different monoamine oxidase domains and identification of histidine residues mediating ligand binding regulation by H+1.

We have shown that I2-imidazoline binding sites (I2BSs) are located on both monoamine oxidases A (MAO-A) and B (MAO-B) and are selectively regulated by H+ and K+ in vitro. In the present study we used chemical modifying agents to investigate the localization of I2BSs with respect to different MAO domains and the mechanisms of ligand binding regulation by K+ and H+. In mitochondrial or solubilized preparations from rabbit kidney and liver, modification of cysteine residues, which are critical for MAO activity, did not affect [3H]idazoxan binding, indicating that I2BS is not associated to the cysteine-containing flavin adenine dinucleotide (FAD) prosthetic group or to the catalytic site of MAOs. Among various chemical modifying agents, only diethylpyrocarbonate and 4-bromophenacyl bromide, two histidine modifying agents, inhibited [3H]idazoxan binding to I2BS. The pH profile of diethylpyrocarbonate effect was consistent with the specific modification of histidine residues. In protection experiments, the effect of diethylpyrocarbonate was not prevented in the presence of saturating concentrations of amiloride, guanabenz or KCl, suggesting that these residues are not located within the ligand or K+ binding sites. In contrast, histidine residues appear to be within a MAO domain involved in regulation of [3H]idazoxan binding by H+. Indeed, the pH-dependent increase in [3H]idazoxan binding was fully abolished after treatment of solubilized material with diethylpyrocarbonate. In conclusion, our results show that MAO I2BSs are not located within the flavin adenine dinucleotide prosthetic group or the catalytic site. Histidine(s) residue(s) involved in the regulation of ligand binding to I2BS by H+ also has been identified.

Amino Acid Sequence↗
PubMed1995

Imidazoline/guanidinium binding domains on monoamine oxidases. Relationship to subtypes of imidazoline-binding proteins and tissue-specific interaction of imidazoline ligands with monoamine oxidase B.

Pharmacologically active compounds with an imidazoline and/or guanidinium moiety are recognized with high affinity by a family of membrane-bound proteins collectively known as imidazoline binding sites or imidazoline/guanidinium receptive sites. Two such receptive sites may correspond to imidazoline binding domains identified on the A and B isoforms of monoamine oxidase (MAO), but the detection of monoamine oxidase isoforms in multiple tissues contrasts with the restricted expression of imidazoline-binding proteins. To address these issues, we determined the relationship between monoamine oxidase isoforms and subtypes of imidazoline-binding proteins in human tissues known to express one or both isoforms of MAO. 2-(3-Azido-4-[125I]iodophenoxy)methylimidazoline ([125I]A-ZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an M(r) = approximately 59,000 peptide in liver and an M(r) = approximately 63,000 peptide in placenta, consistent with the M(r) of the MAO isoforms identified by immunoblots in these tissues. The photolabeled species in liver was immunoprecipitated with MAO-B selective antibodies, whereas the photolabeled species in placenta was immunoprecipitated by MAO-A selective antibodies consistent with the isoform of MAO predominantly expressed in these tissues. The imidazoline/guanidinium ligands interact with the enzyme at a site distinct from the substrate recognition domain, and the immunoprecipitated peptides in liver and placenta display distinct ligand recognition properties consistent with those reported for subtypes of imidazoline binding sites. However, the imidazoline binding domain was not detected in platelet membrane preparations containing amounts of MAO-B equivalent to those in the photolabeled liver membranes indicating that recognition of this domain is tissue-restricted. Restricted access to the imidazoline binding domain on platelet MAO-B was not altered by membrane washing with 500 mM KCl or by solubilization and partial purification of the enzyme suggesting that there are distinct subpopulations of MAO. Identification of a binding domain on MAO that recognizes this class of pharmacologically active compounds suggests a novel mechanism for regulation of substrate oxidation/selectivity or that the enzyme may subserve an as yet undefined function.

Affinity Labels↗
PubMed1995

Localization of I2-imidazoline binding sites on monoamine oxidases.

Imidazoline binding sites (IBS) were proposed to be responsible for some of the pharmacological and therapeutic activities of imidazoline and related compounds and have been classified into two subtypes, I1BS and I2BS. Convergent studies attribute a role in central blood pressure regulation to the I1BS. In contrast, the function of I2BS remains unknown. In the present study, by combining biochemical and molecular biology approaches, we show that 1) microsequencing of I2BS purified from rabbit kidney mitochondria allowed the recovery of four peptide sequence stretches displaying up to 85.7% similarity with human, rat, and bovine monoamine oxidases (MAO)-A and -B; 2) I2BS and MAO displayed identical biophysical characteristics as their activities, measured by [3H]idazoxan binding and [14C]tyramine oxidation, respectively, could not be separated using various chromatographic procedures; and 3) heterologous expression of human placenta MAO-A and human liver MAO-B in yeast, inherently devoid of I2BS and MAO activities, led to the coexpression of [3H]idazoxan binding sites displaying ligand-recognition properties typical of I2BS. These results show definitely that I2BS is located on both MAO-A and -B. The fact that I2BS ligands inhibited MAO activity independently of the interaction with the catalytic region suggests that I2BS might be a previously unknown MAO regulatory site.

Amino Acid Sequence↗
PubMed1995

Selectivity of rilmenidine for I1-imidazoline-binding sites in rabbit proximal tubule cells.

Imidazoline and imidazoline-like compounds may elicit their pharmacological activities through the interaction with three membrane proteins: alpha 2-adrenergic receptors (alpha 2-AR), I1-binding sites (I1BS), and I2-binding sites (I2BS). We have recently shown that these three proteins are co-expressed in the renal proximal tubule cells, where they could mediate the renal effects of imidazoline and structurally related antihypertensive drugs such as clonidine and rilmenidine. To identify the receptor involved in regulation of the tubular effects of clonidine and rilmenidine, we performed binding studies on isolated cells from rabbit kidney proximal tubule using [3H]idazoxan, an I1/I2 ligand, and [3H]rauwolscine, a selective alpha 2-adrenergic antagonist. Competition studies of [3H]idazoxan binding showed that rilmenidine and clonidine interact with both I1BS and I2BS. The comparison of inhibition constants (rilmenidine: Ki for I1BS. 7.1 +/- 3.5 nM; Ki for I2BS, 5,189 +/- 1,816 nM; clonidine: Ki for I1BS, 58.2 +/- 17.3 nM; Ki for I2BS, 4,179 +/- 2,633 nM) demonstrated that rilmenidine and clonidine are 731-and 72-fold, respectively, more selective for I1BS than for I2BS. In addition, in competition experiments with [3H]-rauwolscine, rilmenidine poorly interacted with alpha 2-AR (Ki 2,440 +/- 322 nM), whereas clonidine displayed an affinity (Ki, 32 +/- 12 nM) close to that observed for I1BS. Taken together, these data show that although clonidine is not able to discriminate alpha 2-AR from I1BS, rilmenidine selectively binds only to I1BS. This suggests that the renal effects of rilmenidine are related to its selective interaction with this class of binding site.

Adrenergic alpha-Agonists↗
PubMed1994

Development of a high-affinity radioiodinated ligand for identification of imidazoline/guanidinium receptive sites (IGRS): intratissue distribution of IGRS in liver, forebrain, and kidney.

Imidazoline/guanidinium receptive sites (IGRS) are membrane proteins that exhibit high affinity for various compounds with an imidazoline or guanidinium moiety. The structure of these binding sites and their significance in the broad pharmacological action of such ligands are unclear. To address this issue, we developed selective high affinity compounds that could be radioiodinated and used as molecular probes for structural characterization of these proteins. This report describes the synthesis and characterization of such a molecule, 2-(3-amino-4-[125I]iodophenoxy)methylimidazoline ([125I]AMIPI). [125I]AMIPI is structurally related to cirazoline, an imidazoline exhibiting high affinity for IGRS and the family of related imidazoline binding sites. The phenyl-substituted analogue of cirazoline, 2-(3-aminophenoxy)methylimidazoline, was generated by alkylation of acetamidophenol with 2-chloromethylimidazoline. 2-(3-Aminophenoxy)methylimidazoline exhibited high affinity for IGRS in rabbit kidney membranes, as determined in competition binding studies with [3H]idazoxan (Ki = 12.5 +/- 7.5 nM), and was radioiodinated by chloramine-T oxidation to yield [125I]AMIPI. The binding properties of [125I]AMIPI were determined in membranes prepared from two representative tissues, rabbit kidney cortex and rat liver. Specific binding of [125I]AMIPI was saturable and of high affinity, as determined by Scatchard analysis of saturation binding isotherms (rabbit kidney, Kd = 2.0 +/- 0.9 nM, Bmax = 554 +/- 201 fmol/mg, five experiments; rat liver, Kd = 2.6 +/- 1.3 nM, Bmax = 73 +/- 10 fmol/mg, three experiments).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Purification and characterization of mitochondrial imidazoline-guanidinium receptive site from rabbit kidney.

The imidazoline-guanidinium receptive site (IGRS) is a membrane-bound protein that may mediate some of the pharmacological effects of imidazoline and guanidinium compounds. The structure and functionality of this protein are unknown but, in addition to its location at the plasma membrane, it is found in high density in the outer membrane of mitochondria (Tesson, F., Prip-Buus, C., Lemoine, A., Pegorier, J.-P., and Parini, A. (1991) J. Biol. Chem. 266, 155-160). Using a two-step procedure, we report the purification of mitochondrial IGRS from rabbit kidney to the apparent homogeneity. After solubilization of mitochondrial membranes with digitonin, an apparently homogeneous IGRS preparation was obtained by two sequential purification steps, chromatofocusing and hydroxylapatite-agarose chromatography. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified preparation after silver staining or radioiodination indicated that IGRS binding subunit was purified at the apparent homogeneity since a single band (M(r) approximately 60,000) was observed. IGRS behaves as an acidic protein (pI 5.5) whose binding activity is regulated by H+ concentration near a physiological pH of 7.4. The ability to achieve rapid purification of IGRS should facilitate efforts to define molecular properties and functionality of this protein.

Animals↗
PubMed1992

Tissue-specific localization of mitochondrial imidazoline-guanidinium receptive sites.

In the present report, we studied the distribution of the imidazoline-guanidinium receptive site in mitochondrial fractions from different rabbit and human tissues. Binding studies of the imidazoline-guanidinium receptive site ligand [3H]idazoxan, allowed to distinguish two groups of tissues: the first one, including kidney, brain and liver, displays a high density of imidazoline-guanidinium receptive site; the second one, consisting of striated and smooth muscle, enterocytes, lung, spleen and heart, is characterized by 4- to 16-fold lower binding site density. The demonstration that mitochondrial imidazoline-guanidinium receptive sites are not equally expressed in all tissues can be considered as a further progress towards the characterization of their functional activity.

Animals↗
PubMed1992

Characterization of imidazoline-guanidinium receptive sites in renal medulla from human kidney.

Previous studies showed that alpha 2-adrenergic receptors and imidazoline-guanidinium receptive sites (IGRS) are colocalized in rabbit and human renal proximal tubule. In the present study we investigated the localization of these two binding sites in the renal medulla from human kidney. Binding studies performed with [3H]idazoxan (IGRS ligand) and [3H]rauwolscine (alpha 2-adrenergic ligand) showed that, in membrane preparations from renal medulla, the density of IGRS was 3.6-fold higher than that of alpha 2-adrenergic receptors (134 +/- 7 v 37 +/- 5 fmol/mg protein, respectively). These data indicate that imidazoline, guanidinium, and oxazoline derivatives could induce their therapeutic effects through the interaction with IGRS and/or alpha 2-adrenergic receptors located not only in the renal proximal tubule but also in other segments of the nephron.

Adrenergic alpha-Antagonists↗