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.

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.

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 Gα_i/osubunits. However, the identification of the specific Gα 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 Gα subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Gα 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 Gα protein was examined by RT-PCR experiments. Fluorescence imaging showed that Gα_i1, Gα_i3, and Gα_owere expressed in SG neurons. The transfection of Gα_i1-specific siRNA resulted in a significant decrease in Noc-mediated Ca²⁺current inhibition, while silencing of either Gα_i3or Gα_owas without effect. Taken together, these results suggest that in SG neurons Gα_i1subunits selectively couple NOP receptors to N-type Ca²⁺channels.

Postganglionic sympathetic nerve terminals innervate cardiac muscle and express opioid receptor-like 1 (ORL1) receptors, the most recently described member of the opioid receptor subclass. ORL1 receptors are stimulated by the endogenous heptadecapeptide nociceptin (Noc). To better understand how the signaling events by Noc regulate sympathetic neuron excitability, the goal of the present study was to determine whether sympathetic stellate ganglion (SG) neurons, innervating the heart, natively express ORL1 opioid receptors and couple to Ca(2+) channels. SG neurons in adult male rats were retrograde-labeled with a fluorescent tracer via injection of the ventricular muscle employing ultrasound imaging. Thereafter, N-type Ca(2+) channel modulation was investigated using the whole-cell variant of the patch-clamp technique. Exposure of labeled SG neurons to Noc resulted in a concentration-dependent inhibition of Ca(2+) currents (with an estimated EC(50) of 193 +/- 14 nM). Pre-exposure of SG neurons to the ORL1 receptor blocker, [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101), significantly decreased the Noc-mediated Ca(2+) current inhibition. The Ca(2+) current inhibition was also blocked by pertussis toxin pretreatment, indicating that signaling occurs via Galpha(i/o) G proteins. Finally, the full-length ORL1 receptor cDNA in SG neurons was cloned and sequenced. Of the two known alternatively spliced variants in rats, sequencing analysis showed that the ORL1 receptor expressed in SG neurons is the short form. Overall, these results suggest that stimulation of postsynaptic ORL1 receptors by Noc in SG neurons regulate cardiac sympathetic activity.

• Sympathetic stellate ganglion (SG) neurons that innervate cardiac muscle play an important role in regulating heart rate and contractility. • In this study, we examined which Gβ protein subunit couples Ca²⁺ channels to opioid and adrenergic receptors expressed in SG neurons. • We show that Gβ2 and Gβ4 are important signalling elements that maintain the transduction pathway of both receptor subtypes and Ca²⁺ channels, and how their expression levels are differentially compensated. • Our results also indicate that Gβ1 subunits do not appear to play a role in either signalling pathway. • The elucidation of specific G proteins that couple Ca²⁺ channels with opioid and adrenergic receptors will help us better understand how these receptors regulate synaptic transmission of SG neurons that innervate cardiac muscle. Abstract 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 excitability of SG neurons by modulating Ca²⁺ channel function following activation of the adrenergic and nociceptin/orphanin FQ peptide (NOP) opioid receptors, respectively. The regulation of Ca²⁺ channels is mediated by Gβγ, but the specific Gβ subunit that modulates the channels is not known. In the present study, small interference RNA (siRNA) was employed to silence the natively expressed Gβ proteins in rat SG tissue and to examine the coupling specificity of adrenergic and NOP opioid receptors to Ca²⁺ channels employing the whole-cell variant of the patch-clamp technique. Western blotting analysis showed that Gβ1, Gβ2 and Gβ4 are natively expressed. The knockdown of Gβ2 or Gβ4 led to a significant decrease of the NA- and Noc-mediated Ca²⁺ current inhibition, while Gβ1 silencing was without effect. However, sustaining low levels of Gβ2 resulted in an increased expression of Gβ4 and a concomitant compensation of both adrenergic and opioid signalling pathways modulating Ca²⁺ channels. Conversely, Gβ4-directed siRNA was not accompanied with a compensation of the signalling pathway. Finally, the combined silencing of Gβ2 and Gβ4 prevented any additional compensatory mechanisms. Overall, our studies suggest that in SG neurons, Gβ2 and Gβ4 normally maintain the coupling of Ca²⁺ channels with the receptors, with the latter subtype responsible for maintaining the integrity of both pathways.