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A Parini

Publications and source records attributed to A Parini.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1989

Selective inhibition of adrenaline-induced human platelet aggregation by the structurally related Paf antagonist Ro 19-3704.

1. Two non-lipid antagonists of platelet-activating factor acether (Paf), BN 52021 and WEB 2086, at concentrations which completely blocked Paf-induced platelet aggregation, failed to interfere with aggregation by adrenaline. In contrast, Ro 19-3704, a structurally related antagonist of Paf, inhibited concentration-dependently aggregation induced by adrenaline or by the simultaneous addition of submaximal concentrations of adrenaline and Paf. Reversal of aggregation was obtained when Ro 19-3704 was added to the platelet suspension after adrenaline. 2. Ro 19-3704 was selective for Paf and adrenaline since it failed to interfere with platelet aggregation induced by arachidonic acid or ADP. CV-3988, an antagonist of Paf structurally similar to Ro 19-3704, also inhibited adrenaline-induced aggregation. However, a morpholine analogue (MA) of Paf, which has no anti-Paf activity, failed to interfere with the aggregation induced by adrenaline. This suggests that the effect of Ro 19-3704 and CV-3988 on adrenaline is not simply due to their lipid structure. 3. Experiments on plasma membrane preparations showed that Ro 19-3704 inhibited [3H]-yohimbine binding with an inhibition constant (Ki) of 7 +/- 3 microM. In contrast, BN 52021 and MA did not interfere with [3H]-yohimbine binding. Equilibrium binding experiments showed that Ro 19-3704 increased the apparent KD of [3H]-yohimbine binding from 2.02 +/- 0.15 to 7.3 +/- 0.4 nM. The Paf antagonist Ro 19-3704 interacts specifically with the alpha 2-adrenoceptor and may thus prevent the early steps involved in the mechanism of adrenaline-induced platelet activation.

5-Hydroxytryptophan↗
PubMed1989

Adrenergic agonists and the Na+-K+-adenosine triphosphatase from rabbit proximal tubules and their basolateral membranes.

Several studies suggested that catecholamines modulate renal sodium and water excretion by direct stimulation of adrenergic receptors located on the renal proximal tubule. However, neither the mechanism nor the class of adrenoceptor involved in this effect have yet been established definitively. In the present study, we examined the effects of L-norepinephrine (NE) and selective alpha-1, alpha-2 and beta adrenergic agonists on monovalent cation transport and on Na+-K+-adenosine triphosphatase (ATPase) activity from homogenates, intact tubules and highly purified basolateral membranes prepared from superficial rabbit kidney cortex. Our results showed that neither NE nor specific alpha-1, alpha-2 and beta adrenergic agonists (10 microM) modified ouabain-sensitive uptake of 86Rb+ (a K+ analog) in intact proximal tubules. Similarly, it is demonstrated that NE and alpha and beta adrenergic agonists did not affect Na+-K+-ATPase activity from homogenates, intact tubules and basolateral membranes. The integrity of the alpha-2 adrenergic receptor system, the predominant adrenergic subtype in rabbit proximal tubule, was supported by the following findings: 1) maximal binding of [3H] rauwolscine was about 4-fold higher in basolateral membranes than in homogenates; 2) 5'-guanylimidodiphosphate induced a 27-fold increase in the Ki of NE for alpha-2 receptor in basolateral membranes; 3) NE (5 microM) inhibited by 35% parathyroid hormone-stimulated cyclic AMP production in intact tubules. In conclusion, these data fail to demonstrate that NE, as well as other adrenergic agonists, directly increases Na+-K+-ATPase in the rabbit proximal tubule. Further investigations are needed to clarify the interaction of catecholamines with the renal Na+K+ pump.

Animals↗
PubMed1989

[Selectivity of alpha 2-adrenergic agonists for the imidazoline-guanidine and alpha 2-adrenergic receptors].

Imidazolines have been proposed as highly selective drugs for alpha 2-adrenergic receptors. However, we have recently showed that the imidazoline ligand 3H-RX 781094 (idazoxan) binds to both alpha 2-receptors and imidazoline guanidinium receptive substance (IGRS) in rabbit renal proximal tubule. Binding of 3H-RX 781094 to the purified basolateral membranes (15-fold enriched in Na-KATPase activity) was rapid (t 1/2 = 5 mn.) reversible (t 1/2) = 4 mn.), saturable and of high affinity. Scatchard analysis of equilibrium binding data showed that 3H-RX 781094 labels 566 +/- 118 fmol/mg of proteins of binding sites with an apparent dissociation constant (Kd) of 1.45 +/- 0.14 nM. On the other hand, the non imidazoline ligand 3H-rauwolscine binds only to the alpha 2-adrenergic receptors with a maximal density of 155 +/- 28 fmol/mg of protein and a Kd of 11.5 +/- 1.5 nM. In order to define the relative affinity of the alpha-2-agonists, clonidine, rilmenidine and guanfacine for the two classes of receptors, we performed competition studies of the alpha 2-antagonists 3H-RX 781094 (imidazoline) and 3H-rauwolscine (non imidazoline) binding to basolateral membranes from rabbit proximal tubule. The order of potency for inhibition of the two radioligand binding was rilmenidine greater than clonidine greater than guanfacine for 3H-RX 781094 and clonidine greater than guanfacine greater than rilmenidine for 3H-rauwolscine. Therefore, rilmenidine displayed a higher affinity for IGRS than for alpha 2 adrenergic receptors; on the other hand, clonidine and guanfacine preferentially interact with alpha 2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗