Victor Ruiz-Velasco

A genetic determinant of the striatal dopamine response to alcohol in men

Excessive alcohol use, a major cause of morbidity and mortality, is less well understood than other addictive disorders. Dopamine release in ventral striatum is a common element of drug reward, but alcohol has an unusually complex pharmacology, and humans vary greatly in their alcohol responses. This variation is related to genetic susceptibility for alcoholism, which contributes more than half of alcoholism risk. Here, we report that a functional OPRM1 A118G polymorphism is a major determinant of striatal dopamine responses to alcohol. Social drinkers recruited based on OPRM1 genotype were challenged in separate sessions with alcohol and placebo under pharmacokinetically controlled conditions, and examined for striatal dopamine release using positron emission tomography and [11C]-raclopride displacement. A striatal dopamine response to alcohol was restricted to carriers of the minor 118G allele. To directly establish the causal role of OPRM1 A118G variation, we generated two humanized mouse lines, carrying the respective human sequence variant. Brain microdialysis showed a fourfold greater peak dopamine response to an alcohol challenge in h/mOPRM1-118GG than in h/mOPRM1-118AA mice. OPRM1 A118G variation is a genetic determinant of dopamine responses to alcohol, a mechanism by which it likely modulates alcohol reward.

Calcium Phosphate Nanocomposite Particles for In Vitro Imaging and Encapsulated Chemotherapeutic Drug Delivery to Cancer Cells

Defects change essentially not only the electronic properties but also the chemical properties of graphene, being centers of its chemical activity. Their functionalization is a way to modify the electronic and crystal structure of graphene, which may be important for graphene-based nanoelectronics. Using hydrogen as an example, we have simulated a chemistry of imperfect graphene for a broad class of defects (Stone-Wales (SW) defects, bivacancies, nitrogen substitution impurities, and zigzag edges) by density functional calculations. We have studied also an effect of finite width of graphene nanoribbons on their chemical properties. It is shown that magnetism at graphene edges is fragile, with respect to oxidation, and, therefore, chemical protection of the graphene edges may be required for the application of graphene in spintronics. At the same time, hydrogenation of the SW defects may be a prospective way to create magnetic carbon.Paradigm-shifting modalities to more efficiently deliver drugs to cancerous lesions require the following attributes: nanoscale-size, targetability, and stability under physiological conditions. Often, these nanoscale drug delivery vehicles are limited due to agglomeration, poor solubility, or cytotoxicity. Thus, we have designed a methodology to encapsulate hydrophobic antineoplastic chemotherapeutics within a 20-30 nm diameter, pH-responsive, nonagglomerating, nontoxic calcium phosphate nanoparticle matrix. In the present study, we report on calcium phosphate nanocomposite particles (CPNPs) that encapsulate both fluorophores and chemotherapeutics, are colloidally stable in physiological solution for an extended time at 37 degrees C and can efficaciously deliver hydrophobic antineoplastic agents, such as ceramide, in several cell model systems.

A genetic association study of the functional A118G polymorphism of the human mu-opioid receptor gene in patients with acute and chronic pain.

In this prospective, observational study we explored whether A118G single nucleotide polymorphism in the human &mgr;-opioid receptor (MOR) gene could explain the inter-individual differences in opioid analgesic requirements in patients with acute postoperative pain and chronic pain. The frequency of the wild-type A118 MOR (major) and variant G118 MOR (minor) alleles in the subjects with chronic, noncancer pain (n = 121) and opioid-naïve subjects with acute postoperative pain (n = 101), serving as the control group, were examined. The relationships among the A118G MOR genotype, opioid requirements, and the numerical pain score were analyzed in both groups. The frequency of the minor allele was significantly lower in the subjects with chronic pain when compared with the group with acute postoperative pain (0.079 versus 0.158; P = 0.009 by &khgr;2 test). No statistically significant association was observed between the presence of A118G MOR polymorphism and the average postoperative pain score or the doses of morphine used in the immediate postoperative period. In the high-quartile, opioid utilization, chronic pain patients, the homozygotic carriers of the major allele required significantly higher opioid dose than did the carriers of the minor allele. The results indicate that although the presence of the minor allele does not appear to affect opioid analgesic use in acute postoperative pain, the minor allele is less common in chronic pain patients, especially in those requiring higher doses of opioid analgesics.

Pharmacological consequence of the A118G μ opioid receptor polymorphism on morphine- and fentanyl-mediated modulation of Ca²⁺ channels in humanized mouse sensory neurons.

Background: The most common functional single nucleotide polymorphism of the human OPRM1 gene, A118G, has been shown to be associated with interindividual differences in opioid analgesic requirements, particularly with morphine, in patients with acute postoperative pain. The purpose of this study was to examine whether this polymorphism would modulate the morphine and fentanyl pharmacological profile of sensory neurons isolated from a humanized mouse model homozygous for either the 118A or 118G allele. Methods: The coupling of wild-type and mutant &mgr; opioid receptors to voltage-gated Ca2+ channels after exposure to either ligand was examined by employing the whole cell variant of the patch-clamp technique in acutely dissociated trigeminal ganglion neurons. Morphine-mediated antinociception was measured in mice carrying either the 118AA or 118GG allele. Results: The biophysical parameters (cell size, current density, and peak current amplitude potential) measured from both groups of sensory neurons were not significantly different. In 118GG neurons, morphine was approximately fivefold less potent and 26% less efficacious than that observed in 118AA neurons. On the other hand, the potency and efficacy of fentanyl were similar for both groups of neurons. Morphine-mediated analgesia in 118GG mice was significantly reduced compared with the 118AA mice. Conclusions: This study provides evidence to suggest that the diminished clinical effect observed with morphine in 118G carriers results from an alteration of the receptors pharmacology in sensory neurons. In addition, the impaired analgesic response with morphine may explain why carriers of this receptor variant have an increased susceptibility to become addicted to opioids.

Functional expression and FRET analysis of green fluorescent proteins fused to G-protein subunits in rat sympathetic neurons

1 cDNA constructs coding for a yellow‐emitting green fluorescent protein (GFP) mutant fused to the N‐terminus of the G‐protein subunit β1 (YFP‐β1) and a cyan‐emitting GFP mutant fused to the N‐terminus of the G‐protein subunit γ2 (CFP‐γ2) were heterologously expressed in rat superior cervical ganglion (SCG) neurons following intranuclear injection of the tagged subunits. The ability of the tagged subunits to modulate effectors, form a heterotrimer and couple to receptors was characterized using the whole‐cell patch‐clamp technique. Fluorescent resonance energy transfer (FRET) was also measured to determine the protein‐protein interaction between the two fusion proteins. 2 Similar to co‐expression of untagged β1/γ2, co‐expression of YFP‐β1/γ2, β1/CFP‐γ2, or YFP‐β1/CFP‐γ2 resulted in a significant increase in basal N‐type Ca2+ channel facilitation when compared to uninjected neurons. Furthermore, the noradrenaline (NA)‐mediated inhibition of Ca2+ channels was significantly attenuated. 3 Co‐expression of YFP‐β1/CFP‐γ2 with G‐protein‐gated inwardly rectifying K+ channels (GIRK1 and GIRK4) resulted in tonic GIRK currents that were blocked by Ba2+. 4 The ability of the tagged subunits to form heterotrimers was tested by co‐injecting either tagged or untagged Gβ1 and Gγ2 with excess GαoA cDNA. Under these conditions, the NA‐mediated Ca2+ current inhibition was significantly decreased when compared to uninjected neurons. 5 Coupling to the α2‐adrenergic receptor was reconstituted in neurons expressing pertussis toxin (PTX)‐insensitive GαoA and either tagged or untagged Gβ1γ2 subunits. Application of NA to PTX‐treated cells resulted in a voltage‐dependent inhibition of N‐type Ca2+ currents. 6 FRET measurements in the SCG revealed an in vivo interaction between YFP‐β1 and CFP‐γ2. Co‐expression of untagged β1 significantly decreased the interaction between the two fusion proteins. 7 In summary, the attachment of GFP mutants to the N‐terminus of Gβ1 or Gγ2 does not qualitatively impair their ability to form a heterotrimer, modulate effectors (N‐type Ca2+ and GIRK channels), or couple to receptors.

Heterologous expression and coupling of G protein-gated inwardly rectifying K+ channels in adult rat sympathetic neurons.

1 G protein‐gated inwardly rectifying K+ (GIRK) channels were heterologously expressed in rat superior cervical ganglion (SCG) neurons by intranuclear microinjection. The properties of GIRK channels and their coupling to native receptors were characterized using the whole‐cell patch‐clamp technique. 2 Following coinjection of either GIRK1–2 or GIRK1–4 cDNA, application of noradrenaline (NA) produced large inwardly rectifying K+ currents. Injection of cDNA encoding individual GIRK subunits produced only small and inconsistent NA‐activated inward currents. Current arising from the native expression of GIRK channels in SCG neurons was not observed. 3 NA‐mediated activation of GIRK channels was abolished by pertussis toxin (PTX) pretreatment, indicating coupling via G proteins of the Gi/Go subfamily. Conversely, vasoactive intestinal peptide (VIP) activated GIRK channel currents via a cholera toxin‐sensitive pathway suggesting coupling through Gαs. Pretreatment of neurons with PTX caused a significant increase in amplitude of the VIP‐mediated GIRK channel currents when compared with untreated cells. 4 Application of adenosine, prostaglandin E2 and somatostatin resulted in activation of GIRK channel currents. Activation of m1 muscarinic acetylcholine receptors (i.e. application of oxotremorine m to PTX‐treated neurons) failed to elicit overt GIRK channel currents. 5 GIRK channel overexpression decreased basal Ca2+ channel facilitation significantly when compared with uninjected neurons. Furthermore, the NA‐mediated inhibition of Ca2+ channels was significantly attenuated. 6 In summary, the ability to heterologously express GIRK channels in adult sympathetic neurons allows the experimental alteration of receptor‐G protein‐effector stoichiometry. Such studies may increase our understanding of the mechanisms underlying ion channel modulation by G proteins in a neuronal environment.

Modulation of Silent and Constitutively Active Nociceptin/Orphanin FQ Receptors by Potent Receptor Antagonists and Na+ Ions in Rat Sympathetic Neurons

The pharmacology of G protein-coupled receptors can be influenced by factors such as constitutive receptor activation and Na+ ions. In this study, we examined the coupling of natively and heterologously expressed nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors with voltage-dependent Ca2+ channels after exposure to four high-affinity NOP receptor blockers [[Nphe1Arg14Lys15]N/OFQ-NH2 (UFP-101), 1-[1-(cyclooctylmethyl)-1,2,3,6-tetrahydro-5-(hydroxymethyl)-4-pyridinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (Trap-101), 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl}pyrrolidine-2-carboxamide (compound 24), and N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride (JTC-801)] in sympathetic neurons. The enhanced tonic inhibition of Ca2+ currents in the absence of agonists, indicative of constitutively active NOP receptors in transfected neurons, was abolished after pretreatment with pertussis toxin. In control neurons, the four antagonists did not exert any effects when applied alone but significantly blocked the N/OFQ-mediated Ca2+ current inhibition. Exposure of transfected neurons to UFP-101 resulted in partial agonist effects. In contrast, Trap-101, compound 24, and JTC-801 exerted inverse agonism, as measured by the loss of tonic Ca2+ current inhibition. In experiments designed to measure the N/OFQ concentration-response relationship under varying Na+ concentrations, a leftward shift of IC50 values was observed after Na+ exposure. Although similar N/OFQ efficacies were measured with all solutions, a significant decrease of Hill coefficient values was obtained with increasing Na+ concentrations. Examination of the allosteric effects of Na+ on heterologously overexpressed NOP receptors showed that the tonic Ca2+ current inhibition was abolished in the presence of the monovalent cation. These results demonstrate that constitutively active NOP receptors exhibit differential blocker pharmacology and allosteric regulation by Na+. Data are also presented demonstrating that heterologously expressed μ opioid receptors in sympathetic neurons are similarly modulated.

Endoperoxide 4 receptors play a role in evoking the exercise pressor reflex in rats with simulated peripheral artery disease

•  In decerebrated rats, the exercise pressor reflex arising from a hindlimb whose femoral artery was occluded for 72 h was significantly higher than that arising from a hindlimb whose femoral artery was freely perfused. •  Blockade of endoperoxide 4 receptors, but not blockade of endoperoxide 3 receptors, prevented the exaggerated exercise pressor reflex in rats with ligated femoral arteries. •  Blockade of endoperoxide 3 or 4 receptors in rats with freely perfused femoral arteries had no effect on the exercise pressor reflex. •  Western immunoblots showed that ligation of the femoral artery for 72 h increased the endoperoxide 4 receptor protein in the L4 and L5 dorsal root ganglia over their freely perfused counterparts by 24% (P < 0.05).

Coupling Specificity of NOP Opioid Receptors to Pertussis-Toxin-Sensitive Gα Proteins in Adult Rat Stellate Ganglion Neurons Using Small Interference RNA

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Galphai/o subunits. However, the identification of the specific Galpha subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Galpha subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Galpha subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Galpha protein was examined by RT-PCR experiments. Fluorescence imaging showed that Galphai1, Galphai3, and Galphao were expressed in SG neurons. The transfection of Galphai1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Galphai3 or Galphao was without effect. Taken together, these results suggest that in SG neurons Galphai1 subunits selectively couple NOP receptors to N-type Ca2+ channels.

Use of a Third-Generation Perfluorocarbon for Preservation of Rat DCD Liver Grafts

BACKGROUND Cold storage in any of the commonly used preservation solutions is not always adequate for donation after cardiac death (DCD) liver grafts due to prolonged warm ischemic time. In this study, we used a third-generation perfluorocarbon (PFC), Oxycyte, for DCD liver graft preservation in a rat model. MATERIALS AND METHODS Twenty-eight rats (14 in each group) were used. Thirty minutes after cardiopulmonary arrest, livers were harvested and flushed with a cold and pre-oxygenated solution of either University of Wisconsin (UW) or UW + 20% PFC. After 8 h of cold preservation in either of the investigated solutions, liver graft specimens were analyzed for evidence of ischemic injury. Hemotoxylin and eosin staining (H and E), as well as immunohistochemical analysis with anti-cleaved caspase 3 antibody, was performed. Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the preservation solution were analyzed at 1 and 8 h during preservation. RESULTS In the PFC group, the degree of cell congestion, vacuolization and necrosis were all significantly less than in the UW group (P = 0.002-0.004). The number of cells with a positive cleaved caspase 3 antibody reaction was reduced by about 50% in comparison with the UW group (P < 0.006). The AST level in the PFC group was significantly less than in the UW group after 8 h of preservation (P < 0.048). CONCLUSION The addition of PFC to UW solution significantly decreases the degree of histologic damage in rat DCD liver grafts. This preservation strategy can be potentially helpful for organ preservation after prolonged warm ischemia.