Effect of cold perfusion and perfluorocarbons on liver graft ischemia in a donation after cardiac death model.

BACKGROUND: Effects of two perfluorocarbon (PFC) formulations (perfluorodecalin emulsion and perfluorodecalin liquid) on the quality of liver graft preservation, in a donation after cardiac death (DCD) rat model, were investigated. The significance of continuous graft perfusion during cold preservation was also explored. MATERIALS AND METHODS: DCD model: 30 min after cardiopulmonary arrest was initiated, livers were excised and flushed with cold University of Wisconsin (UW) solution (± PFC) and preserved in the same solution for 8 h. The study groups were preserved as follows: group 1: no perfusion; group 2: perfusion with UW; group 3: PFC was administered before cardiac arrest and the liver was perfused with UW alone; and groups 4 and 5: perfused with UW + 1 of two PFCs. In a baseline group used only for comparison of gene expression, livers were quick-frozen after cardiac arrest. Microarrays were used to analyze liver messenger RNA transcripts. Histopathologic, immunohistochemical, and ADP/ATP ratio evaluations were performed to assess the quality of graft preservation. RESULTS: Significant decreases in downregulation and increases in upregulation of hepatic genes (relative to baseline) were demonstrated in all perfusion groups. This trend was most pronounced in the PFC groups. Lower fat content and ADP/ATP ratio and a reduction in Caspase 3 activation were found in all perfusion groups. CONCLUSION: Hypothermic perfusion of rat DCD liver grafts with oxygenated UW solution (± PFC) produced superior preservation compared with nonperfusion storage. The observed changes in expression of hepatic genes may represent a protective effect in the DCD model.

Gß2 and Gß4 participate in the opioid and adrenergic receptor-mediated Ca2+ channel modulation in rat sympathetic neurons.

Cardiac function is regulated in part by the sympathetic branch of the autonomic nervous system via the stellate ganglion (SG) neurons. Neurotransmitters, such as noradrenaline (NA), and neuropeptides, including nociceptin (Noc), influence the excit ability of SG neurons by modulating Ca(2+) channel function following activation of the adrenergic and nociceptin/orphanin FQ peptide (NOP) opioid receptors, respectively. The regulation of Ca(2+) channels is mediated by Gßγ, but the specific Gß subunit that modulates the channels is not known. In the present study, small interference RNA (siRNA) was employed to silence the natively expressed Gß proteins in rat SG tissue and to examine the coupling specificity of adrenergic and NOP opioid receptors to Ca(2+) channels employing the whole-cell variant of the patch-clamp technique.Western blotting analysis showed that Gß1, Gß2 and Gß4 are natively expressed. The knockdown of Gß2 or Gß4 led to a significant decrease of the NA- and Noc-mediated Ca(2+)current inhibition, while Gß1 silencing was without effect. However, sustaining low levels of Gß2 resulted in an increased expression of Gß4 and a concomitant compensation of both adrenergic and opioid signalling pathways modulating Ca(2+) channels. Conversely, Gß4-directed siRNA was not accompanied with a compensation of the signalling pathway. Finally, the combined silencing of Gß2 and Gß4 prevented any additional compensatory mechanisms.Overall, our studies suggest that in SG neurons, Gß2 and Gß4 normally maintain the coupling of Ca(2+) channels with the receptors, with the latter subtype responsible for maintaining the integrity of both pathways.

Pharmacological consequence of the A118G µ opioid receptor polymorphism on morphine- and fentanyl-mediated modulation of Ca²âº 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 µ opioid receptors to voltage-gated Ca 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 receptor's 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.

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 Ca(2+) channels after exposure to four high-affinity NOP receptor blockers [[Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (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 Ca(2+) 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 Ca(2+) 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 Ca(2+) current inhibition. In experiments designed to measure the N/OFQ concentration-response relationship under varying Na(+) concentrations, a leftward shift of IC(50) 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 Ca(2+) 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 mu opioid receptors in sympathetic neurons are similarly modulated.

Muscarinic acetylcholine receptor modulation of mu (mu) opioid receptors in adult rat sphenopalatine ganglion neurons.

The sphenopalatine ganglion (SPG) neurons represent the parasympathetic branch of the autonomic nervous system involved in controlling cerebral blood flow. In the present study, we examined the coupling mechanism between mu (mu) opioid receptors (MOR) and muscarinic acetylcholine receptors (mAChR) with Ca(2+) channels in acutely dissociated adult rat SPG neurons. Successful MOR activation was recorded in approximately 40-45% of SPG neurons employing the whole cell variant of the patch-clamp technique. In addition, immunofluorescence assays indicated that MOR are not expressed in all SPG neurons while M(2) mAChR staining was evident in all neurons. The concentration-response relationships generated with morphine and [d-Ala2-N-Me-Phe4-Glycol5]-enkephalin (DAMGO) showed IC(50) values of 15.2 and 56.1 nM and maximal Ca(2+) current inhibition of 26.0 and 38.7%, respectively. Activation of MOR or M(2) mAChR with morphine or oxotremorine-methiodide (Oxo-M), respectively, resulted in voltage-dependent inhibition of Ca(2+) currents via coupling with Galpha(i/o) protein subunits. The acute prolonged exposure (10 min) of neurons to morphine or Oxo-M led to the homologous desensitization of MOR and M(2) mAChR, respectively. The prolonged stimulation of M(2) mAChR with Oxo-M resulted in heterologous desensitization of morphine-mediated Ca(2+) current inhibition, and was sensitive to the M(2) mAChR blocker methoctramine. On the other hand, when the neurons were exposed to morphine or DAMGO for 10 min, heterologous desensitization of M(2) mAChR was not observed. These results suggest that in rat SPG neurons activation of M(2) mAChR likely modulates opioid transmission in the brain vasculature to adequately maintain cerebral blood flow.

Gγ7 proteins contribute to coupling of nociceptin/orphanin FQ peptide (NOP) opioid receptors and voltage-gated Ca(2+) channels in rat stellate ganglion neurons.

The nociceptin/orphanin FQ peptide (NOP) opioid receptors regulate neurotransmitter release via inhibition of voltage-gated Ca(2+) channels (CaV2.2) in sympathetic and sensory neurons. Stimulation of NOP receptors by its endogenous agonist, nociception (Noc), leads to membrane-delimited, voltage-dependent (VD) block of CaV2.2 channel currents mediated by Gßγ protein subunits. Previously we reported that the pertussis toxin-sensitive Gαi1 and Gß2/ß4 isoforms mediate the functional coupling of NOP opioid receptors with CaV channels in rat stellate ganglion (SG) sympathetic neurons. In the present report we extended our studies by identifying the Gγ subunit that forms the heterotrimer within this signaling pathway. Small interference RNA (or siRNA) was employed to silence the expression of the natively expressed Gγ subunits. Initial PCR assays indicated that SG neurons expressed seven Gγ subunits. Silencing Gγ3 subunits did not alter signaling between NOP receptors and Ca(2+) channels. However, after Gγ7 isoforms were silenced, the Noc-mediated inhibition of CaV channels was significantly decreased when compared to SG neurons transfected with scrambled siRNA. We observed that Gγ10 and Gγ11 mRNA levels increased 2.5- and 2.7-fold, respectively, after Gγ7 subunits were silenced. However, this compensatory increase in mRNA expression did not appear to fully rescue the NOP receptor coupling efficiency. Additionally, both Gγ2 and Gγ5 levels increased 50 and 75%, respectively, while Gγ3 and Gγ4 expression levels remained relatively unchanged. Taken together, our findings suggest that the Gαi1/Gß2(ß4)/Gγ7 heterotrimeric G protein complex determines the NOP receptor-mediated modulation of CaV channels in SG neurons.

Cocaine-induced suppression of saccharin intake and morphine modulation of Ca²âº channel currents in sensory neurons of OPRM1 A118G mice.

Several studies have shown that human carriers of the single nucleotide polymorphism of the µ-opioid receptor, OPRM1 A118G, exhibit greater drug and alcohol use, increased sensitivity to pain, and reduced sensitivity to the antinociceptive effects of opiates. In the present study, we employed a 'humanized' mouse model containing the wild-type (118AA) or variant (118GG) allele to examine behavior in our model of drug-induced suppression of a natural reward cue and to compare the morphine pharmacological profile in acutely isolated sensory neurons. Compared with 118AA mice, our results demonstrate that homozygous 118GG mice exhibit greater avoidance of the cocaine-paired saccharin cue, a behavior linked to an aversive withdrawal-like state. Electrophysiological recordings confirmed the reduced modulation of Ca(2+) channels by morphine in trigeminal ganglion (TG) neurons from 118GG mice compared to the 118AA control cells. However, repeated cocaine exposure in 118GG mice led to a leftward shift of the morphine concentration-response relationship when compared with 118GG control mice, while a rightward shift was observed in 118AA mice. These results suggest that cocaine exposure of mice carrying the 118G allele leads to a heightened sensitivity of the reward system and a blunted modulation of Ca(2+) channels by morphine in sensory neurons.