Biomedical subjects

Mirko Moroni

Publications and source records attributed to Mirko Moroni.

6 recordsLinked to original sources

PubMed2006

Experimentally induced aggressiveness in heroin-dependent patients treated with buprenorphine: comparison of patients receiving methadone and healthy subjects.

Objective measures of experimentally induced aggressiveness were evaluated in heroin-dependent patients (HDP), 15 receiving buprenorphine (BUP) and 15 receiving methadone (METH) treatment. HDP were randomly assigned to BUP and METH groups. Fifteen healthy subjects (CONT) were included in the study as controls. During a laboratory task, the Point Subtraction Aggression Paradigm, subjects earned monetary reinforcement and could respond by ostensibly subtracting money from a fictitious subject (the aggressive response). Money-earning (points maintained) responses did not differ in BUP patients and in controls. In contrast, point-maintained responses were significantly lower in the group of HDP treated with METH than in both the BUP and CONT groups. Aggressive responses were significantly higher in the HDP group than in the CONT group. No significant differences in aggressive responses were found between the BUP and METH groups. Baseline concentrations of plasma adrenocorticotropic hormone (ACTH) and cortisol (CORT) were higher in HDP than in CONT. During the experimental task, ACTH and CORT increased significantly less in METH patients than in BUP patients and CONT. Norepinephrine (NE) and epinephrine (EPI) levels increased significantly more in HDP than in CONT, without any difference between the METH and BUP patients. PSAP aggressive responses positively correlated with NE and EPI changes, as well as with Buss-Durkee Hostility Inventory (BDHI) scores in both METH and BUP patients and also in CONT subjects. No correlation was found between the extent of heroin exposure, drug doses and aggressiveness levels. BUP, similarly to METH, does not seem to affect outward-directed aggressiveness, as aggressive responses related more to monoamine levels and personality traits than to the action of opioid agonists. Money-earning responses seemed to be unimpaired in BUP patients.

Adrenocorticotropic Hormone↗

PubMed2006

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human alpha 4 beta 2 nicotinic acetylcholine receptors in Xenopus oocytes.

Alpha4 and beta2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)-induced current responses, and increased the relative proportion of alpha4beta2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the alpha4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of alpha4S467A mutant subunits decreased steady levels of alpha4 and the relative proportion of alpha4beta2 receptors with low agonist sensitivity, whilst expression of alpha4S467D increased steady levels of alpha4 and alpha4beta2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14-3-3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of alpha4beta2 with high sensitivity to activation by ACh. Thus, post-translational modification affecting steady-state levels of alpha4 subunits provides a possible means for physiologically relevant, chaperone-mediated variation in the relative proportion of high and low agonist sensitivity alpha4beta2 nACh receptors.

14-3-3 Proteins↗

PubMed2006

alpha4beta2 nicotinic receptors with high and low acetylcholine sensitivity: pharmacology, stoichiometry, and sensitivity to long-term exposure to nicotine.

alpha4 and beta2 nicotinic acetylcholine receptor (nAChR) subunits expressed heterologously assemble into receptors with high (HS) and low (LS) sensitivity to acetylcholine (ACh); their relative proportions depend on the alpha4to beta2 ratio. In this study, injection of oocytes with 1:10 alpha4/beta2 subunit cDNA ratios favored expression of HS alpha4beta2 nAChRs, as evidenced by monophasic ACh concentration-response curves, whereas injections with 10:1 cDNA ratios favored expression of LS alpha4beta2 receptors. The stoichiometry was inferred from the shifts in the ACh EC(50) values caused by Leu to Thr mutations at position 9' of the second transmembrane domain of alpha4 and beta2. The 1:10 injection ratio produced the (alpha4)(2)(beta2)(3) stoichiometry, whereas 10:1 injections produced the (alpha4)(3)(beta2)(2) stoichiometry. The agonists epibatidine, 3-[2(S)-azetidinylmethoxy]pyridine (A-85380), 5-ethoxy-metanicotine (TC-2559), cytisine, and 3-Br-cytisine and the antagonists dihydro-beta-erythroidine and d-tubocurarine were more potent at HS receptors. TC-2559 was more efficacious than ACh at HS receptors but was a partial agonist at LS receptors. Epibatidine was more efficacious than ACh at LS receptors and a partial agonist at HS receptors. Cytisine and 5-halogenated cytisines had moderate efficacy at LS receptors but had almost no efficacy at HS receptors. By exploiting the differential effects of ACh, TC-2559 and 5-I-cytisine we evaluated the effects of long-term exposure to nicotine on HS and LS receptors expressed in Xenopus laevis oocytes after cDNA injections or microtransplantation of alpha4beta2 receptors assembled in human embryonic kidney 293 cells. We conclude that nicotine up-regulates HS alpha4beta2 receptors, probably by influencing the assembly of receptors rather than by altering the functional state of LS alpha4beta2 nAChRs.

Acetylcholine↗

PubMed2006

5-I A-85380 and TC-2559 differentially activate heterologously expressed alpha4beta2 nicotinic receptors.

The neuronal nicotinic acetylcholine receptor alpha4 and beta2 subunits expressed in heterologous expression systems assemble into at least two distinct subunit stoichiometries of alpha4beta2 receptor. The (alpha4)2(beta2)3 stoichiometry is about 100-fold more sensitive to acetylcholine than the (alpha4)3(beta2)2 stoichiometry. In order to investigate if agonists in general distinguish high- and low-affinity alpha4beta2 nicotinic acetylcholine receptors, we have expressed human alpha4 and beta2 nicotinic acetylcholine receptor subunits in two different expression systems. The relative amounts of alpha4beta2 nicotinic acetylcholine receptors with high- and low-affinity for acetylcholine were manipulated by (a) injecting the subunit cDNAs at different alpha:beta ratios into Xenopus oocytes and (b) by culturing HEK-293 cells stably expressing alpha4beta2 nicotinic acetylcholine receptors overnight at different temperatures. The sensitivities of the alpha4beta2 nicotinic acetylcholine receptors to the agonists acetylcholine, 5-I A-85380, and TC-2559 were investigated using the voltage-clamp technique on Xenopus oocytes and using a fluorescent imaging plate reader to measure calcium responses from HEK-293 cells. Like acetylcholine, 5-I A-85380 produced biphasic concentration-response curves and the high-affinity component became larger when the cells were manipulated to produce a greater proportion of (alpha4)2(beta2)3 nicotinic acetylcholine receptors. Interestingly, under all circumstances, TC-2559 produced monophasic concentration-response curves. In oocytes injected with alpha4 and beta2 subunits in the 1:1 ratio the maximum effect of TC-2559 was 28% of that of acetylcholine. The EC50 for TC-2559 was not changed when oocytes were manipulated to express exclusively (alpha4)2(beta2)3 nicotinic acetylcholine receptors, however, the maximum effect of TC-2559 was dramatically enhanced. These results suggest that TC-2559 is a selective agonist of the (alpha4)2(beta2)3 nicotinic acetylcholine receptor stoichiometry.

Animals↗

PubMed2006

Stoichiometry and pharmacology of two human alpha4beta2 nicotinic receptor types.

The alpha4beta2 nicotinic acetylcholine receptor (nAChR) is the most abundant nAChR subtype in the brain, where it forms the high-affinity binding site for nicotine. The alpha4beta2 nAChR belongs to a gene family of ligand-gated ion channels that also includes muscle nAChRs, GABAA receptors, and glycine receptors and that assembles into pentameric structures. alpha4 and beta2 nAChR subunits expressed heterologously in Xenopus laevis oocytes assemble into a mixture of high- and low-affinity functional receptors, giving rise to biphasic ACh concentration-response curves (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001). High- and low-affinity alpha4beta2 nAChRs differ significantly in their functional and pharmacological properties (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003) and result from the assembly of alpha4 and beta2 subunits into two distinct stoichiometric arrangements: (alpha4)2(beta2)3(high-affinity subtype) and (alpha4)3(beta2)2 (low-affinity subtype) (Nelson et al., 2003). In this study we have examined the functional and pharmacological properties of high- and low-affinity alpha4beta2 receptors using two-electrode voltage clamp procedures on Xenopus oocytes transfected with high (1:10) or low (10:1) ratios of alpha4/beta2 cDNAs, which yield high (1:10)- or low (10:1)- affinity receptors with monophasic ACh concentration- response curves. Furthermore, to determine the stoichiometry of high- and low-affinity receptors expressed heterologously by Xenopus oocytes, we have determined the stoichiometry of high- and low-affinity alpha4beta2 receptors by mutating a highly conserved hydrophobic residue in the middle (position 9') of the pore-lining domain, which increases agonist potency in a manner that allows predictions on subunit composition (Cooper et al., 1991; Revah et al., 1991; Labarca et al., 1995; Boorman et al., 2000).

Acetylcholine↗

PubMed2006

Phosphorylation and function of alpha4beta2 receptor.

The neuronal nicotinic acetylcholine receptor (nAChR) alpha4 and beta2 subunits expressed in heterologous expression systems assemble into high- and low-affinity receptors (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003), which reflects the assembly of two distinct subunit stoichiometries of alpha4beta2 receptor (Nelson et al., 2003). The high-affinity receptor ([alpha4]2[beta2]3) is about 100-fold more sensitive to ACh than the low-affinity receptor ([alpha4]3[beta2]2) (Zwart and Vijverberg, 1998; Buisson and Bertrand, 2001; Houlihan et al., 2001; Nelson et al., 2003). Recent evidence implicated 14-3-3 proteins as modulators of the relative abundance of nAChR subunits in the endoplasmic reticulum (ER), where ligand-gated ion channels assemble. The 14-3-3 proteins influence ER-to-plasma membrane trafficking of multimeric cell-surface proteins (O'Kelly et al., 2002). 14-3-3 proteins bind components of these multimeric proteins, and this interaction overrides dibasic COP1 retention signal to permit forward transport of the protein (O'Kelly et al., 2002). In the case of alpha4beta2 nAChRs, 14-3-3 binds the alpha4 subunit, and this association is dependent on phosphorylation of a serine residue within a protein kinase A(PKA) consensus sequence in the large cytoplasmic domain of the alpha4 subunit, which is also a binding motif recognized by 14-3-3 (Jeancloss et al., 2001; O'Kelly et al., 2002). The interplay among PKA, alpha4 subunits, and 14-3-3 proteins increases cell-surface expression of alpha4beta2 nAChRs by increasing steady-state levels of the alpha4 subunit available for assembly with beta2 subunits (Jeancloss et al., 2001). Because it is not known how 14-3-3-dependent changes in the steady-state levels of the alpha4 subunit might affect the functional type of alpha4beta2 receptors, we have investigated the effects of mutations of the 14-3-3 binding motif in the alpha4 subunit on alpha4beta2 nAChR function.

Animals↗