Modulation of Ca²⁺Channels by Opioid Receptor-Like 1 Receptors Natively Expressed in Rat Stellate Ganglion Neurons Innervating Cardiac Muscle

Abstract

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 2ϩ 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 2ϩ 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 2ϩ currents (with an estimated EC 50 of 193 Ϯ 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 2ϩ current inhibition. The Ca 2ϩ current inhibition was also blocked by pertussis toxin pretreatment, indicating that signaling occurs via G␣ 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.The opioid receptor-like 1 (ORL1) receptor and the three "classic" opioid receptor subtypes (, , and ␦) are members of the G protein-coupled receptor superfamily. The heptadecapeptide nociceptin, (Noc, also known as orphanin/FQ) is the endogenous ORL1 receptor ligand. ORL1 receptors are coupled to members of the pertussis toxin (PTX)-sensitive

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…Morphine also acts at m-opioid receptors that are expressed in the CNS (Diaz et al, 1995;Goodchild et al, 2004), where a clear correlation between physiologic effects and modulation of Ca V 2.2 channels is more difficult to establish. Although morphine is considered selective for m-opioid receptors, selective agonists of the other three members of the extended opioid receptor family (i.e., d-and k-opioid receptors, and nociceptin receptors) also functionally inhibit Ca V 2.2 channels (Gross and Macdonald, 1987;Moises et al, 1994;Motin et al, 1995;Morikawa et al, 1998;Toselli et al, 1999;Larsson et al, 2000;Yeon et al, 2004;Ruiz-Velasco et al, 2005;Evans et al, 2010). As in the case of m-opioid receptors, their activation induces analgesia in various animal models of pain (King et al, 1997;Darland et al, 1998;Field et al, 1999;Mika et al, 2001;Courteix et al, 2004;Nozaki et al, 2012).…”

Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems

“…NOP receptor inhibition of N-type calcium conductance is likely mediated by binding of the dissociated Gbg subunit directly to the channel. This binding event is thought to reduce voltage activation of channel pore opening Snutch, 1998, 2002;Beedle et al, 2004;Yeon et al, 2004;Ruiz-Velasco et al, 2005). Furthermore, it has also been recently reported that NOP receptors use Rho-associated coiled-coil-containing protein kinase (ROCK) and LIM domain kinase (LIMK) in the regulation of voltage-dependent Ca 2+ channels (Mittal et al, 2013).…”

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

“…1A (right). This protocol has been used routinely to study the agonist-mediated voltage-dependent inhibition of Ca 2ϩ channel currents, including N/OFQ (Ruiz-Velasco et al, 2005). The protocol consists of a test pulse to ϩ10 mV (prepulse, F) followed by a large depolarizing conditioning test pulse to ϩ80 mV, a brief return to Ϫ80 mV, and followed by a test pulse to ϩ10 mV (postpulse, E).…”

“…The plasmid coding for rat NOP receptor was subcloned in pCI and injected at a final concentration of 100 to 800 ng/l. We isolated and cloned this receptor construct previously from SG neurons and found that adult rats express a single NOP receptor subtype or one splice variant (Ruiz-Velasco et al, 2005). The plasmid coding for the human opioid peptide (MOP) receptor was subcloned in pcDNA3.1 (Invitrogen) and injected at a concentration of 500 ng/l.…”

“…We reported previously that the N/OFQ-mediated activation of natively expressed NOP receptors in rat stellate ganglion (SG) neurons results in voltage-dependent inhibition of Ca 2ϩ channels (Ruiz-Velasco et al, 2005). In the present study, we examined whether NOP receptor overexpression in SG neurons would result in tonic receptor activation.…”