Kelly M. Standifer

Extracellular Signal-Regulated Kinase 1/2-Mediated Transcriptional Regulation of G-Protein-Coupled Receptor Kinase 3 Expression in Neuronal Cells

Relatively small changes in G-protein-coupled receptor kinase (GRK) 3 expression (∼2-fold) profoundly affect α2-adrenergic receptor (AR) function and preferentially regulate neuronal α2A- and α2B-AR signaling. In the present study, we provide evidence that epinephrine (EPI)-induced up-regulation of GRK3 protein expression in two neuronal cell lines, BE(2)-C cells (endogenously express α2A- and β2-AR) and BN17 cells [endogenously express α2B (NG108) and transfected to express β2-AR] is due in part to increased GRK3 gene expression. In both cell lines, the increase in GRK3 transcription occurred via an extracellular signal-regulated kinase (ERK) 1/2-dependent mechanism because the increase in GRK3 mRNA is eliminated in the presence of the mitogen-activated protein kinase/ERK kinase 1/2 inhibitor, U0126 [1,4-diamino-2,3-dicyano-1,4-bis (2-amino phenylthiobutadiene)]. EPI-induced GRK3 mRNA up-regulation also is prevented in the presence of propranolol or phentolamine. Moreover, GRK3 mRNA did not increase in response to EPI treatment in NG108 cells (endogenously express α2B-AR with no β2-AR). Both these results suggest that simultaneous activation of α2- and β2-AR by EPI is required for the ERK1/2-dependent increase in GRK3 mRNA. The EPI-induced increase in GRK3 mRNA was unaffected in the presence of the protein kinase C inhibitor, chelerythrine chloride. Finally, EPI treatment resulted in increased nuclear translocation and accumulation of the transcription factors, Sp-1 and Ap-2, in BE(2)-C cells. Taken together, our results demonstrate the involvement of the ERK1/2 pathway in selective up-regulation of GRK3 mRNA expression, possibly via activation of Sp-1 and Ap-2 transcription factors in neuronal cells.

Involvement of G Protein-Coupled Receptor Kinase (GRK) 3 and GRK2 in Down-Regulation of the α2B-Adrenoceptor

Increasing the cellular levels of G protein-coupled receptor kinase (GRK) 2 or GRK3 renders the α2B-adrenoceptor (AR) more sensitive to agonist-induced down-regulation (J Pharmacol Exp Ther 312:767–773, 2005). However, an absolute requirement of GRK3 and GRK2 for α2B-AR down-regulation is controversial. In this study, using NG108 cells (endogenous α2B-AR), we provide strong evidence for a critical role of both GRK3 and GRK2 in down-regulation of the α2B-AR. Pretreatment of NG108 cells with 20 μM epinephrine (EPI) begins down-regulating the α2B-AR by 2 h. The translocation of GRK3 and GRK2 to the membrane peaks at 30 min, decreasing by 1 h. Although these results may implicate GRK3 and GRK2 in α2B-AR down-regulation, significant receptor down-regulation is not observed until 2 h, after GRK3 and GRK2 translocation has peaked and is declining. To more directly establish a role for GRK3 and GRK2 in α2B-AR down-regulation, NG108 cells were transfected to express GRK3ct, which binds to liberated Gβγ subunits, preventing GRK3 and GRK2 translocation to the membrane. Overexpression of GRK3ct prevented not only the translocation of GRK3 and GRK2 but also the down-regulation of the α2B-AR caused by 24-h pretreatment with 20 μM EPI. Taken together, these data provide direct evidence for a role of GRK3 and GRK2 in the down-regulation of the α2B-AR and contribute significantly to the increasing evidence in the literature for a pivotal role of GRKs in modulating the agonist-induced down-regulation of the α2-AR.

Cellular Mechanisms of Nociceptin/Orphanin FQ (N/OFQ) Peptide (NOP) Receptor Regulation and Heterologous Regulation by N/OFQ

The nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor is the fourth and most recently discovered member of the opioid receptor superfamily that also includes μ, δ, and κ opioid receptor subtypes (MOR, DOR, and KOR, respectively). The widespread anatomic distribution of the NOP receptor enables the modulation of several physiologic processes by its endogenous agonist, N/OFQ. Accordingly, the NOP receptor has gained a lot of attention as a potential target for the development of ligands with therapeutic use in several pathophysiological states. NOP receptor activation frequently results in effects opposing classic opioid receptor action; therefore, regulation of the NOP receptor and conditions affecting its modulatory tone are important to understand. Mounting evidence reveals a heterologous interaction of the NOP receptor with other G protein–coupled receptors, including MOR, DOR, and KOR, which may subsequently influence their function. Our focus in this review is to summarize and discuss the findings that delineate the cellular mechanisms of NOP receptor signaling and regulation and the regulation of other receptors by N/OFQ and the NOP receptor.

Orphanin FQ/Nociceptin Activates Nuclear Factor Kappa B

Endogenous neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, nociceptin orphanin FQ peptide receptor (NOPr), play a modulatory role throughout the body including nociceptive sensitivity, motor function, spatial learning, and the immune system. NOPr is an inhibitory G protein coupled receptor (GPCR) that modulates expression and release of inflammatory mediators from immune cells and in the CNS. Inhibitory GPCRs have been shown to activate the immune and central nervous system regulator, nuclear factor kappa B (NFκB), whose family consists of several subunits. When activated, NFκB translocates to the nucleus and can modify transcription. To determine if OFQ/N modulates NFκB activity, SH-SY5Y human neuroblastoma cells were treated with OFQ/N and assessed for changes in nuclear accumulation, DNA binding, and transcriptional activation. For the first time, we show that OFQ/N increases the nuclear accumulation (1.9–2.8-fold) and the DNA binding of NFκB (2.9-fold) by 2xa0h as determined by immunoblotting and electromobility shift assay, respectively. OFQ/N induction of NFκB binding to DNA is protein kinase C-dependent and NOPr-specific. OFQ/N stimulated binding of both NFκB p50 and p65 subunits to their consensus binding site on DNA. OFQ/N also induces transcriptional activation of an NFκB reporter gene 2.2-fold by 2xa0h with an EC50 of 6.3xa0nM. This activation of NFκB by OFQ/N suggests a likely mechanism for its modulation of the central nervous and immune systems.

Methylnaltrexone in the treatment of opioid-induced constipation.

Constipation is a significant problem related to opioid medications used to manage pain. This review attempts to outline the latest findings related to the therapeutic usefulness of a μ opioid receptor antagonist, methylnaltrexone in the treatment of opioid-induced constipation. The review highlights methylnaltrexone bromide (Relistor™; Progenics/Wyeth) a quaternary derivative of naltrexone, which was recently approved in the United States, Europe and Canada. The Food and Drug Administration in the United States approved a subcutaneous injection for the treatment of opioid bowel dysfunction in patients with advanced illness who are receiving palliative care and when laxative therapy has been insufficient. Methylnaltrexone is a peripherally restricted, μ opioid receptor antagonist that accelerates oral–cecal transit in patients with opioid-induced constipation without reversing the analgesic effects of morphine or inducing symptoms of opioid withdrawal. An analysis of the mechanism of action and the potential benefits of using methylnaltrexone is based on data from published basic research and recent clinical studies.

Sex differences in the Nociceptin/Orphanin FQ system in rat spinal cord following chronic morphine treatment

Nociceptin/Orphanin FQ (N/OFQ) appears to contribute to the development of morphine tolerance, as blockade of its actions will block or reverse the process. To better understand the contribution of N/OFQ to the development of morphine tolerance, this study examined the effect of chronic morphine treatment on levels of N/OFQ and levels and activity of the N/OFQ peptide (NOP) receptor in spinal cord (SC) from male and female rats. Both male and female Wistar rats showed less responsiveness to morphine after subcutaneous injection of escalating doses of morphine (10, 20, 40, 60 and 80 mg/kg, respectively) twice daily for five consecutive days. Male rats were more tolerant to the antinociceptive actions of morphine than females. The N/OFQ content of SC extracts was higher in females than in males, regardless of treatment; following chronic morphine treatment the difference in N/OFQ levels between males and females was more pronounced. N/OFQ content in cerebrospinal fluid (CSF) was reduced 40% in male and 16% in female rats with chronic morphine exposure, but increased in periaqueductal grey of both sexes. Chronic morphine treatment increased NOP receptor levels 173% in males and 137% in females, while decreasing affinity in both. Chronic morphine increased the efficacy of N/OFQ-stimulated [³⁵S]GTPγS binding to SC membranes from male rats, consistent with increased receptor levels. Taken together, these findings demonstrate sex differences in N/OFQ-NOP receptor expression and NOP receptor activity following chronic morphine treatment. They also suggest interplay between endogenous N/OFQ and chronic morphine treatment that results in nociceptive modulation.

Nociceptin/Orphanin FQ

Abstract Nociceptin/orphanin FQ (N/OFQ) is an endogenous peptide that is found in brain, immune, vascular endothelial and sertoli cells. It binds to and activates its cognate receptor, opioid receptor-like 1 receptor (ORL1) that is now more commonly referred to as the N/OFQ peptide (NOP) receptor. NOP agonists, such as this peptide, are active on cells in the central and peripheral nervous systems, immune system, vascular endothelium and testes. N/OFQ actions include, but are not limited to, modulation of: nociceptive sensitivity, anxiety, feeding, heart rate, diuresis, locomotion, learning and memory, female reproductive behavior and stress and immune responses.

Nociceptin Orphanin FQ 1-13 -NH 2

Nociceptin/orphanin FQ(1–13) amide is the smallest fragment of nociceptin/orphanin FQ (N/OFQ) that binds to and activates its cognate receptor, ORL1 ( Calo et al ., 1996 xa0; Dooley and Houghten, 1996 ). ORL1 agonists, such as this peptide, are active on cells in the central nervous and immune systems. Their actions include, but are not limited to, modulation of nociceptive sensitivity, anxiolytic actions, orexogenic actions, bradycardia, and diuresis ( Calo et al., 2000 ). Unlike other opioid agonists, N/OFQ-like agonists do not appear to have addictive potential ( Devine et al., 1996 ).

[Phe 1psi(CH2-NH)-Gly2]Nociceptin-(1-13)-NH2

[Phe1-psi-(CH2NH)-Gly2]-Nociceptin-(1-13)-NH2 is a pseudopeptide that was synthesized in an attempt to develop a selective antagonist for the target, ORL1. Though the pseudopeptide appears to be an ORL1 antagonist in many preparations, it exhibits full agonist activity at that receptor in other preparations. The nature of this discrepancy is still unclear.