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A Parini

Publications and source records attributed to A Parini.

At least 19 recordsLinked to original sources
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↗
PubMed1992

Characterization of mitochondrial imidazoline-guanidinium receptive sites (IGRS) in liver.

Some imidazoline and guanidinium antihypertensive drugs display high affinity for a nonadrenergic membrane protein, the imidazoline-guanidinium receptive site (IGRS), which is insensitive to catecholamine and physically distinct from alpha 2-adrenoceptor. In the present report, we characterized IGRS in human and rabbit liver using [3H]idazoxan as radioligand. By performing subcellular fractionation, we showed a significant increase in [3H]idazoxan binding sites on membrane fractions enriched in cytochrome oxidase activity, a mitochondrial marker. A further enrichment in [3H]idazoxan binding (53-fold with respect to the homogenate) was found in a purified preparation of mitochondrial outer membranes. This localization of IGRS will facilitate the characterization of its functional activity in liver.

Animals↗
PubMed1992

[Imidazoline-guanidine site: a subtype of imidazoline receptors].

Since the demonstration that imidazoline and guanidinium alpha-2 adrenergic agonists induce some of their functional effects by a "nonadrenergic" mechanism, many efforts have been done to identify an imidazoline receptor. Binding studies have allowed to characterize two classes of potential imidazoline receptors: the "(p-amino)clonidine" and the "idazoxan" binding sites. These last, that we named "imidazoline-guanidinium receptive sites" (IGRS) on the basis of their ligand-recognition properties, have been identified, for the first time, in the proximal tubule from rabbit and human kidney. In the present report we will summarize the studies that led us to the characterization of IGRS.

Animals↗
PubMed1991

Identification of an imidazoline-guanidinium receptive site in mitochondria from rabbit cerebral cortex.

In the present report, we used [3H]idazoxan to characterize imidazoline-guanidinium receptive sites (IGRS) in mitochondria from rabbit cerebral cortex. When compared to the starting homogenate, [3H]idazoxan binding was higher (1.161 +/- 0.159 vs. 0.102 +/- 0.024 pmol/mg of protein) in a membrane fraction 6-fold enriched in cytochrome oxidase activity, a specific marker for mitochondria. In addition, the enrichment of [3H]idazoxan binding sites positively correlates with cytochrome oxidase activity in different membrane preparations (r = 0.977, P less than 0.001). In competition studies, [3H]idazoxan binding was completely inhibited by imidazoline and guanidinium derivatives but not affected by 10 microM epinephrine. Taken together, these data show the localization of IGRS in the mitochondria from rabbit cerebral cortex.

Animals↗
PubMed1991

Imidazoline-guanidinium and alpha 2-adrenergic binding sites in basolateral membranes from human kidney.

In the present study, we used [3H]idazoxan and [3H]rauwolscine to characterize the imidazoline-guanidinium receptive site (IGRS) and alpha 2-adrenoceptors in the human renal proximal tubule, respectively. In purified basolateral membranes, 11-fold enriched in Na(+)-K+ ATPase. [3H]idazoxan and [3H]rauwolscine binding was twofold higher than in homogenates ([3H]idazoxan: 87 +/- 19 vs. 45 +/- 23.3 fmol/mg protein, P less than 0.05; [3H]rauwolscine: 56.4 +/- 21.4 vs. 25.2 +/- 7.3 fmol/mg protein, P less than 0.01). In competition studies performed at saturating concentration of [3H]idazoxan (15 NM), specific binding was competed for by epinephrine and rauwolscine only by 10-15% but was completely inhibited by imidazoline and guanidinium compounds. Thus, in human renal proximal tubule. [3H]idazoxan mainly binds to an IGRS. The highest density of alpha 2-adrenoceptors in basolateral membranes and of IGRS in partially purified membrane preparations, suggests that these two binding sites have a different subcellular localization. When compared to the rabbit renal IGRS, the human [3H]idazoxan binding site displays different affinities for guanabenz, rilmenidine, clonidine, amiloride and its derivatives that persist after membrane solubilization. In contrast, the human and rabbit renal IGRS share similar regulatory properties such as the sensitivity to K+ and the insensitivity to Na+, divalent cations and 5'-guanylylimidodiphosphate (Gpp(NH)p). In conclusion, we demonstrated that, in the human renal proximal tubule, alpha 2-adrenoceptors are mainly located in basolateral membranes while IGRS appear to be associated with another cell compartment. As indicated by their common interaction with imidazoline and guanidinium derivatives and by similar regulatory properties, human and rabbit IGRS belong to the same family of membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Subcellular distribution of imidazoline-guanidinium-receptive sites in human and rabbit liver. Major localization to the mitochondrial outer membrane.

Imidazoline-guanidinium-receptive site (IGRS) is a membrane protein that, even if recognized by a series of imidazoline and guanidinium alpha 2-adrenergic compounds, is insensitive to catecholamine and physically distinct from alpha 2 receptors (Parini, A., Coupry, I., Graham, R. M., Uzielli, I., Atlas, D., and Lanier, S. M. (1989) J. Biol. Chem. 264, 11874-11878). In the present report, we defined the subcellular localization of IGRS by performing binding studies with the imidazoline radioligand [3H]idazoxan. Binding studies on subcellular fractions of homogenates from human and rabbit liver showed a significant increase in [3H]idazoxan binding in a membrane fraction enriched in cytochrome oxidase activity, a specific marker for mitochondria. The enrichment in [3H]idazoxan binding sites correlates closely with cytochrome oxidase activity in the nuclear, mitochondrial, plasma membrane, microsomal, and soluble fractions (r = 0.966, p less than 0.002) but not with the specific markers for other cell compartments, suggesting a major localization of IGRS in mitochondria. Separation of inner and outer mitochondrial membranes by digitonin treatment showed that [3H]idazoxan binding correlates positively with monoamine oxidase (r = 0.960) and negatively with cytochrome oxidase (r = -0.950) activities. In addition, in highly purified preparations of outer mitochondrial membranes obtained by hypotonic shock, [3H]idazoxan binding activity was 12.5-fold enriched with respect to intact mitochondria. Taken together, these data show, for the first time, that IGRS in human and rabbit liver are mainly associated with the outer mitochondrial membranes. This demonstration of the major mitochondrial localization of IGRS will facilitate the characterization of its functional activity in liver.

Animals↗
PubMed1991

Contribution of alpha 2-adrenoceptors to the central cardiovascular effects of clonidine and S 8350 in anaesthetized rats.

1. The alpha 2-adrenoceptor agonist clonidine elicits centrally mediated effects through an interaction with both alpha 2-adrenoceptors and imidazoline binding sites. 2. We selected a new oxazoline derivative, S 8350, which competes with [3H]-yohimbine for binding to cerebral alpha 2-adrenoceptors (IC50, 67 +/= 17 nmol/L) and displays a higher affinity (35-fold) for alpha 2- than for alpha 1-adrenoceptors. 3. As observed for clonidine, intravenous (i.v.) administration of S 8350 resulted in a brief pressor effect followed by a prolonged hypotension. When S 8350 was administered i.v. to spinally pithed rats, only a rise in blood pressure was observed. 4. In order to discriminate the cardiovascular effects related to the central imidazoline receptor or alpha 2-adrenoceptor activation, the effects of intracisternal (i.c.) administration of clonidine and S 8350 were investigated in the rat. 5. In the anaesthetized rat, both clonidine and S 8350 displayed a profound central (i.c. route) hypotensive effect associated with a bradycardia. 6. The cardiovascular effects of S 8350 were abolished by the central administration of the selective alpha 2-adrenoceptor antagonist rauwolscine. Conversely, rauwolscine completely prevented bradycardia but it induced only a partial reversion of the hypotension elicited by clonidine. 7. These results suggest that central alpha 2-adrenoceptors are responsible for hypotension and bradycardia while imidazoline binding sites do not apparently contribute to heart rate control.

Adrenergic alpha-Agonists↗
PubMed1990

Effect of imidazolines on Na+ transport and intracellular pH in renal proximal tubule cells.

Recently, we characterized an imidazoline-guanidinium receptive site (IGRS) in the renal proximal tubule of rabbit kidney. Although recognized by a series of imidazoline and guanidinium alpha-2 adrenergic compounds, IGRS is insensitive to catecholamines and can be physically separated from alpha-2 adrenergic receptors after solubilization. In the present study, we investigated the effect of imidazoline derivatives on 22Na+ uptake and intracellular pH in isolated cells from rabbit renal proximal tubule. After 5 min of preincubation, idazoxan inhibited the total 22Na+ influx (-30%) in a dose-dependent manner, with a maximum effect at 10(-5) M. The effect of idazoxan was not competitive as shown by the decrease of the maximal velocity of 22Na+ entry (control: 3.80 +/- 0.42; idazoxan 10(-5) M: 3.23 +/- 0.33 nmol/30 s per mg protein, P less than 0.01). A series of imidazoline derivatives inhibited 22Na+ entry with an order of potency similar to that previously found for inhibition of [3H]idazoxan binding to IGRS (cirazoline greater than idazoxan greater than UK 14304 greater than rilmenidine much greater than cimetidine). The inhibition of 22Na+ uptake by these compounds does not appear to be related to interaction with alpha-adrenergic receptors since it was observed in the presence of saturating concentrations of the adrenergic antagonists rauwolscine (alpha-2) or prazosin (alpha-1). When tested on the regulation of intracellular pH by fluorimetric techniques, 10(-5) M cirazoline or idazoxan inhibited by 20% the velocity of the sodium-dependent H+ efflux in acidified cells (P less than 0.02). The concomitant inhibition of 22Na+ entry and of cell realkalinization suggests that imidazoline derivatives inhibit Na+/H(+)-exchanger. This effect could be mediated via the renal IGRS and intracellular second messengers that are not yet known.

Amiloride↗
PubMed1990

Imidazoline-guanidinium receptive site in renal proximal tubule: asymmetric distribution, regulation by cations and interaction with an endogenous clonidine displacing substance.

In the present report we have used [3H]idazoxan to characterize the rabbit renal imidazoline preferring site by defining its plasmalemma distribution, its regulation by cations and the type of interaction with the clonidine displacing substance (CDS), a putative endogenous ligand for the imidazoline receptor. The density of [3H]idazoxan binding sites was 12-fold higher in purified basolateral membranes than in brush-border membranes (maximal binding activity, 566 +/- 118 vs. 46 +/- 2 fmol/mg of protein). In basolateral membranes, [3H]idazoxan binding was inhibited not only by imidazoline compounds but also by guanidinium analogs such as guanabenz, amiloride, 5-(M-ethyl-N-isopropyl)amiloride and phenamylamiloride. Amiloride had no effect on the dissociation rate of [3H]idazoxan, suggesting a direct interaction of this molecule with the ligand binding site. [3H]Idazoxan binding was 80% inhibited by 150 mM K+ or Rb+. The effect of K+ appeared to occur through the interaction with an allosteric site in as much as both the apparent dissociation constant and the dissociation rate of [3H]idazoxan were increased in the presence of 75 mM K+. CDS inhibited [3H]idazoxan binding with a half-maximal effective concentration of 2 U/250 microliters. The competitive nature of CDS effect was indicated by the increase in the apparent dissociation constant of [3H]idazoxan (Kd from 3 +/- 0.3 to 8.5 +/- 0.2 nM, P less than .01) in the presence of CDS. In conclusion, our findings showed that the imidazoline-guanidinium receptive site is located mainly in the basolateral side of the tubular cell, recognizes CDS and is regulated by K+.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1989

Characterization of an imidazoline/guanidinium receptive site distinct from the alpha 2-adrenergic receptor.

alpha 2-Adrenergic receptors recognize a number of molecules with diverse chemical structures, including the yohimban diastereoisomers yohimbine and rauwolscine, catecholamines, guanidinium analogs, and imidazolines, such as clonidine. The affinity of the receptor protein for some of these ligands can vary by 10-100-fold among various tissues and species, suggesting a heterogeneous class of binding sites. Certain cellular effects elicited by the compounds possessing an imidazoline or guanidinium moiety may actually be mediated by a membrane receptor distinct from the alpha 2-adrenergic receptor. To determine whether this imidazoline/guanidinium receptive site (IGRS) and the alpha 2-adrenergic receptor represent distinct proteins, we solubilized and partially characterized the two binding sites in rabbit kidney. This tissue expresses both alpha 2-adrenergic receptors and high affinity imidazoline/guanidinium binding sites, the latter which are rauwolscine-insensitive but can be identified with the benzodioxan [3H]idazoxan. The IGRS and alpha 2-adrenergic receptor in rabbit kidney exhibit distinct ligand recognition properties, which are maintained after solubilization and partial purification. In addition, the two receptors can be physically separated by heparin-agarose or lectin affinity chromatography indicating that the two binding sites are distinct entities. [3H]Idazoxan binding is trypsin-sensitive, indicating that the IGRS is a protein rather than a lipid component of the plasma membrane. [3H]Idazoxan binding is not inhibited by endogenous agonists for known neurotransmitter receptors. However, the IGRS does recognize clonidine-displacing substance, a small non-catechol compound isolated from calf brain, suggesting the existence of a previously uncharacterized hormonal/neurotransmitter receptor system.

Animals↗
PubMed1989

Glycerol, sodium phosphate, and sodium chloride permit the solubilization and partial purification of rat hepatic alpha 1-receptors by 3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate.

CHAPS [3-(3-cholamidylpropyl)-dimethylammonio-1-propanesulfonate], a zwitterionic detergent, has been used to solubilize the rat hepatic alpha 1-adrenergic receptor. Although the use of this detergent alone permitted a poor receptor solubilization, the inclusion of sodium phosphate, sodium chloride, and glycerol to the medium allowed 30% of the binding activity observed in plasma membranes to be recovered. Binding of the selective alpha 1-adrenergic antagonist, [3H]prazosin, by the solubilized preparation was saturable and of high affinity. In addition, binding of the radioligand was inhibited by a variety of adrenergic agents with affinity, specificity, and stereoselectivity comparable to that observed in plasma membranes. The use of glycerol in the solubilization medium permitted recovery of the solubilized receptor in a stable form (T1/2 = 72 h at 4 degrees C). Sequential affinity and size-exclusion gel chromatography allowed a 1000-fold purification of the solubilized receptor. The Stokes' radius and the apparent molecular mass of the purified receptor-Chaps complex (48.4 A and 160,000 Da, respectively), determined by gel filtration chromatography, were similar to those previously obtained for the rat hepatic alpha 1-receptor purified after solubilization with the nonionic detergent digitonin. These data indicate that the combination of Chaps, sodium phosphate, sodium chloride, and glycerol permitted the solubilization and partial purification of hepatic alpha 1-receptor in an active and stable form. The use of this technique might be useful for the solubilization of other membrane-bound proteins by Chaps whose biophysical characteristics make it an ideal detergent for reconstitution experiments.

Animals↗
PubMed1989

Different affinities of alpha 2-agonists for imidazoline and alpha 2-adrenergic receptors.

It has recently been shown that imidazoline alpha 2-adrenergic agonists, such as clonidine and UK 14,304, selectively bind to both alpha 2- and imidazoline receptors in basolateral membranes from rabbit renal proximal tubule. In order to define the relative affinity of three antihypertensive alpha 2-agonists for the two classes of receptors, we performed competition studies of imidazoline alpha 2-antagonist 3H-RX 781094 and nonimidazoline antagonist 3H-rauwolscine binding to basolateral membranes from rabbit proximal tubule. The order of potency for inhibition of radioligand binding to basolateral membranes was rilmenidine greater than clonidine greater than guanfacine and clonidine greater than guanfacine greater than rilmenidine for 3H-RX 781094 and 3H-rauwolscine binding, respectively. These data show that not only clonidine, but also rilmenidine and guanfacine, drugs usually used as specific alpha 2-agonists, bind to both alpha 2- and imidazoline receptors. The higher affinity of these molecules for one or the other class of receptors could explain their different capacity to induce hypotension and side effects.

Adrenergic alpha-Agonists↗
PubMed1989

Alpha-adrenoceptor properties in rat strains sensitive or resistant to salt-induced hypertension.

Cerebral and renal alpha 2-adrenoceptors are implicated in the control of sympathetic activity and of sodium reabsorption respectively. In addition, sodium ions play an important role in the regulation of either alpha 2-adrenoceptor densities and affinities for adrenergic agonists. In the present study, alpha-adrenoceptor properties were investigated in genetically predetermined salt-sensitive and salt-resistant Dahl and Sabra rats. Cerebral alpha 2-adrenoceptor densities were higher in salt-resistant than in salt-sensitive Dahl and Sabra rats. In contrast, renal alpha 2-adrenoceptor density was higher in salt-sensitive than in salt-resistant rats. No difference in cerebral and renal alpha 1-adrenoceptor densities was observed between Dahl and Sabra substrains. Noradrenaline content in cerebral and renal cortex were also similar in both these rat substrains. Sodium ions markedly increased cerebral and renal high-affinity alpha 2-adrenoceptor densities in salt-sensitive but not in salt-resistant rats. Cerebral and renal alpha 1-adrenoceptor densities were unchanged in salt-sensitive and salt-resistant substrains of Dahl and Sabra rats. In addition, sodium ions reduced the affinity of adrenaline for renal alpha 2-adrenoceptors in salt-sensitive rats but not in salt-resistant rats. We can conclude that there exist genetically determined differences in the densities and properties of cerebral and renal alpha 2-adrenoceptors between salt-sensitive and salt-resistant rat strains. Abnormal densities of alpha 2-adrenoceptors may play a primary role in the role in the development of hypertension in salt-sensitive animals. These results also suggest an association between absence of sodium regulation of alpha 2-adrenoceptors and resistance to salt-induced hypertension. The absence of sodium regulation in salt-resistant rats may be linked either to a particular receptor conformation or to an abnormal structure of the receptor system. This property may represent a genetically-mediated change responsible for the resistance to the development of salt-induced hypertension.

Animals↗