Delta opioid receptor analgesia: recent contributions from pharmacology and molecular approaches

Abstract

Delta opioid receptors represent a promising target for the development of novel analgesics. A number of tools have been developed recently that have significantly improved our knowledge of delta receptor function in pain control. These include several novel delta agonists with potent analgesic properties, as well as genetic mouse models with targeted mutations in the delta opioid receptor gene. Also, recent findings have further documented the regulation of delta receptor function at cellular level, which impacts on the pain-reducing activity of the receptor. These regulatory mechanisms occur at transcriptional and post-translational levels, along agonist-induced receptor activation, signaling and trafficking, or in interaction with other receptors and neuromodulatory systems. All these tools for in vivo research, as well as proposed mechanisms at molecular level, have tremendously increased our understanding of delta receptor physiology, and contribute to designing innovative strategies for the treatment of chronic pain and other diseases such as mood disorders.

Introduction

Morphine and other related opiates are potent analgesics widely used for pain treatment. These compounds act on opioid receptors to inhibit pain transmission and perception. The three opioid receptors, mu, delta and kappa receptors, are transmembrane G-proteins coupled receptors encoded by Oprm1, Oprd1 and Oprk1 genes, respectively (Kieffer & Gaveriaux-Ruff, 2002; Stevens, 2009). Opioid receptors are activated endogenously by the opioid peptides enkephalins, beta-endorphin and dynorphins processed from large precursor proteins encoded by Penk, Pomc and Pdyn genes (Akil et al., 1998). The opioid system plays a central role in pain control, as well as in reward (Mendez & Morales-Mulia, 2008; Rodriguez-Arias et al., 2010; Shippenberg et al., 2008) and neuroptoection (Chao & Xia, 2010; Johnson & Turner, 2010). In addition this system regulates a number of physiological functions that include respiration and gastrointestinal transit, as well as endocrine and immune systems (Kieffer & Gaveriaux-Ruff, 2002; Sauriyal et al., 2010).

Since the initial molecular cloning (Evans et al., 1992; Kieffer et al., 1992), a number of tools have been developed enabling better understanding of delta receptor function in nociception and pain. Research on delta opioid analgesia has benefited from a series of novel agonists that extend the panel of pharmacological tools available to study delta receptors in pain control. Mouse genetic models have been created, that harbor targeted mutations of the receptor. These mice represent unique tools for assessing both the endogenous pain-reducing delta tone and the implication of delta receptors in the actions of opioid as well as non-opioid analgesics. These tools will be presented here, together with novel mechanisms for the development of therapeutics strategies targeting the delta opioid receptor.

In addition, there was tremendous progress in understanding how delta receptor expression and activity are regulated, and these mechanisms heavily contribute to delta analgesia. Receptor regulation takes place at different levels, including genomic and transcriptional controls in the nucleus, post-translational events in the cytoplasm and endoplasmic reticulum, interaction of delta receptors with other receptors at the cell membrane, and processes that follow delta receptor activation within the cell, and these aspects will be discussed.

Altogether, delta opioid receptor research represents a very active field of investigation, with about 1000 publications within the last five years. This review therefore cannot be exhaustive and only recent reviews or publications are cited.

Delta opioid analgesia: from early pharmacology to novel agonists

In early opioid pharmacology, two peptidic agonists DPDPE ([2-D-penicillamin, 5-D-penicillamin]-enkephalin) (Mosberg et al., 1983) and deltorphin (Kreil et al., 1989) were most frequently used to study delta receptor function in pain control (Chang et al., 2004). However, their peptidic nature prevented their use by systemic administration in vivo. Later, the non-peptidic agonists BW373U86 and SNC80 (Bilsky et al., 1995; Chang et al., 2004), and the antagonist naltrindole (Portoghese et al., 1988) (Chang et al., 2004) proved to be more stable ligands in vivo to investigate delta receptor-mediated analgesia.

The identification of several novel delta agonists has further broadened the repertoire of molecules available to study delta receptors in vivo (Pradhan et al., 2011; Vanderah, 2010). Over the past decade, SB-235863 (Petrillo et al., 2003), DValAla-Enk (Brainin-Mattos et al., 2006), NIH 11082 (Aceto et al., 2007), AR-M100390 (Pradhan et al., 2009), ADL5859 (Le Bourdonnec et al., 2008), ADL5747 (Le Bourdonnec et al., 2009), JNJ-20788560 (Codd et al., 2009), compound 8e (Jones et al., 2009) and KNT-127 (Saitoh et al., 2011) have been developed as analgesics in preclinical models. Table 1 summarizes the analgesic effects of these novel agonists in several pain models, as well as analgesic effects of previously described reference molecules such as DPDPE, DSLET, deltorphin, SNC80 or Tan-67. Altogether, the data cited in Table 1 indicate that delta receptor activation diminishes chronic pain in the three mouse, rat and monkey species. Table 1 also shows that several distinct chronic pain models are sensitive to delta agonists, including inflammatory, neuropathic, cancer and diabetic pain. Moreover, delta receptor activation reduces hypersensitivity in heat, cold and mechanical modalities. Altogether, delta opioid agonists efficiently decrease chronic pain in many preclinical models and clinical trials will validate their translational potential to patients.

Table 1.

Analgesic effects of delta opioid agonists

Agonist	Pain model#	Species	Pain Modality	Reference
DPDPE*/ DSLET	Formalin	Rat		(Obara et al., 2009)
	Capsaicin	Rat		(Saloman et al., 2011)
	Inflammatory	Rat	M*	(Stein et al., 1989)
		Mouse	H*	(Hervera et al., 2009)
		Rat	M	(Zhou et al., 1998)
	Neuropathic	Mouse	H, M, C*	(Hervera et al., 2010)
		Rat	H, M	(Obara et al., 2009)
		Rat	C	(Mika et al., 2001)
	Cancer	Mouse	H	(Baamonde et al., 2005)
Deltorphin	Formalin	Mouse		(Morinville et al., 2003)
		Rat		(Bilsky et al., 1996)
		Rat		(Cahill et al., 2001)
		Rat		(Pradhan et al., 2006)
	Inflammatory	Rat	H	(Fraser et al., 2000)
		Rat	H	(Hurley & Hammond, 2000)
		Rat	H	(Cahill et al., 2003)
		Rat	H	(Gendron et al., 2007a)
		Rat	M	(Otis et al., 2011)
		Rat	H	(Beaudry et al., 2009)
		Mouse	H	(Gendron et al., 2007b)
		Mouse	H	(Dubois & Gendron)
	Neuropathic	Rat	C	(Mika et al., 2001)
		Rat	M, C	(Holdridge & Cahill, 2007)
		Rat	M	(Kabli & Cahill, 2007)
	Cancer	Rat	M	(Otis et al., 2011)
SNC80	Formalin	Mouse		(Barn et al., 2001)
		Rat		(Obara et al., 2009)
	GDNF hyperalgesia	Rat	M	(Joseph & Levine)
	NGF hyperalgesia	Rat	M	(Joseph & Levine)
	PGE2 hyperalgesia	Monkey	H	(Brandt et al., 2001)
		Rat	M	(Pacheco & Duarte, 2005)
	Dynorphin A allodynia	Rat	M	(Kawaraguchi et al., 2004)
	Inflammatory	Monkey	H	(Brandt et al., 2001)
		Rat	H	(Fraser et al., 2000)
		Rat	M	(Cao et al., 2001)
		Rat	H	(Gallantine & Meert, 2005)
		Mouse	H	(Gendron et al., 2007b)
		Mouse	H, M	(Gaveriaux-Ruff et al., 2008)
		Mouse	H, M	(Pradhan et al., 2009;
				Pradhan et al., 2010)
	Neuropathic	Mouse	M	(Scherrer et al., 2009)
		Monkey	H	(Brandt et al., 2001)
		Mouse	M	(Scherrer et al., 2009)
		Mouse	H, M	(Gaveriaux-Ruff et al., 2011)
		Rat	H, M	(Obara et al., 2009)
Tan-67	Formalin	Mouse		(Barn et al., 2001)
	Diabetes	Mouse	H	(Kamei et al., 1997)
SB-235863	Inflammatory	Rat	H	(Petrillo et al., 2003)
		Rat	H	(Beaudry et al., 2009)
	Neuropathic	Rat	H	(Petrillo et al., 2003)
DValAla-Enk	Cancer	Mouse	M	(Brainin-Mattos et al., 2006)
AR-M100390	Inflammatory	Mouse	H, M	(Pradhan et al., 2009)
		Mouse	H, M	(Pradhan et al., 2010)
NIH 11082	Inflammatory	Rat	M	(Aceto et al., 2007)
ADL5859	Inflammatory	Rat	M	(Le Bourdonnec et al., 2008)
ADL5747	Inflammatory	Rat	M	(Le Bourdonnec et al., 2009)
JNJ-20788560	Inflammatory	Rat	H	(Codd et al., 2010)
Compound 8e	Inflammatory	Rat	n.i.	(Jones et al., 2009)
KNT-127	Formalin	Mouse		(Saitoh et al., 2011)

^#Visceral pain was induced by intraperitoneal acetic acid injection; inflammatory pain by injection of Complete Freund’s Adjuvant (CFA) , zymosan, yeast, or carrageenan ; neuropathic pain was induced by sciatic nerve or spinal nerve ligation; cancer pain by injection of NCTC 2472 osteosarcoma cells,

^*Abbreviations: C, cold: DPDPE, (D-Pen2, D-Pen5)-enkephalin; DSLET, (D-Ser2,Leu5)-enkephalin; GDNF Glial cell line-derived neurotrophic factor; H, heat; M mechanical; NGF, nerve growth factor; PGE2, prostaglandin-E2

Mouse genetic models to study delta receptor function in vivo

Delta opioid receptor knockout mice

A major step for understanding delta receptor function was the generation of mice with a targeted inactivation of the Oprd1 gene, or delta receptor knockout mice (Filliol et al., 2000)(Zhu et al., 1999). A prime finding with these mutant mice was the discovery that delta receptors plays a critical role in the control of emotional responses (Filliol et al., 2000), revealing anxiolytic and antidepressant activities of the receptor that were further confirmed by the pharmacology (Pradhan et al., 2011) (Jutkiewicz, 2006; Noble & Roques, 2007)(Javelot et al., 2010). This mouse line represents a unique genetic tool to assess both the influence of endogenous delta receptor tone on pain physiology, and the in vivo selectivity of known or new delta agonists.

Delta receptor knockout mice showed no change or only subtle alterations in their sensitivity to acute pain (Contet et al., 2006; Filliol et al., 2000; Gaveriaux-Ruff et al., 2008; Martin et al., 2003; Nadal et al., 2006; Pradhan et al., 2011) and stress-induced analgesia developed normally (Contet et al., 2006). Interestingly however, delta receptor knockout mice showed augmented neuropathic and inflammatory pain (Gaveriaux-Ruff et al., 2008; Nadal et al., 2006), suggesting that endogenous delta opioid activity alleviates chronic pain. Altogether, data from the genetic approach are in agreement with the notion that delta agonists barely modulate acute nociception, but are most efficient under conditions of persistent pain (see below). SNC80-induced analgesia was abolished in delta receptor knockout animals in a model of inflammatory hyperalgesia induced by Complete Freund’s Adjuvant (Gaveriaux-Ruff et al., 2008). These data confirmed the in vivo selectivity of the compound previously proposed by the pharmacology (Chang et al., 2004).

Interestingly, chronic treatment with tricyclic antidepressants, which produce anti-allodynic effects in a neuropathic pain model, was ineffective in delta receptor knockout mice (Benbouzid et al., 2008). These data revealed the implication of delta receptors in the analgesic effects of tricyclic anti-depressants, likely downstream from aminergic transporter systems. The implication of endogenous opioid mechanisms in this particular activity of anti-depressant drugs was specifically mediated by delta receptors, since the compounds were fully effective in mu receptor knockout animals (Bohren et al., 2010). Along this line, anti-allodynia induced by chronic beta2-agonists was blocked by the delta receptor antagonist naltrindole (Yalcin et al., 2010) suggesting an interaction between beta2-adrenergic and delta receptor systems. In the future, delta receptor knockout mice may help to reveal the interaction of delta receptor with other receptor systems in pain control.

Conditional knockout mice lacking delta opioid receptors in Nav1.8 nociceptive neurons

Under chronic pain, delta receptor activation produces analgesia at different sites within nociceptive circuits. Delta agonists are effective when administered systemically, intrathecally, intracerebroventricularly, or into the rostral ventromedial medulla or Nucleus Raphe Magnus (Figure 1). A potent analgesia was also obtained at the periphery where nociceptive processing is initiated, and opioid receptors of the peripheral nervous system are proposed as therapeutic targets to limit the centrally-mediated adverse effects of opiates. First trials have been promising (Stein et al., 1990) and the field of peripheral opioid receptors has gained importance lately (Hua & Cabot, 2010; Stein et al., 2009).

Figure 1.

Sites for delta opioid analgesia. This scheme summarizes sites where delta agonist administration induces analgesia in animal models of chronic pain (see table1).

*Abbreviations: DPDPE, (D-Pen2, D-Pen5)-enkephalin; DSLET, (D-Ser2,Leu5)-enkephalin; NRM, Nucleus Raphe Magnus ; RVM, Rostroventral medulla

To inactivate genes specifically in nociceptive primary afferents, a conditional knockout strategy has been reported for the cannabinoid CB1 receptor (Agarwal et al., 2007) and Nav1.7 channel (Nassar et al., 2004). The approach was based on the Cre-Lox system and used a mouse line expressing Cre recombinase in Nav1.8⁺ sensory neurons that include unmyelinated C and thinly myelinated Ad nociceptive neurons. The same driver Cre line was used recently to inactivate Oprd1 in these neurons and examine whether peripheral delta receptors contribute to pain control (Gaveriaux-Ruff et al., 2011). In conditional knockout mutants, analgesia induced by intraperitoneal SNC80 treatment was abolished under both inflammatory and neuropathic conditions, revealing the essential role of peripheral delta receptors in systemic delta opioid analgesia. These peripheral receptors are not necessarily sufficient to produce the full analgesic response, since delta receptors expressed in peripheral non-Nav1.8 DRG neurons, at the level of spinal cord or in the brain (Ossipov et al., 2010) may also participate in systemic delta analgesia. The results nevertheless support the notion that developing peripherally-acting delta agonists is a feasible strategy for the design of novel effective analgesics devoid of centrally-mediated side effects.

Knock-in mice expressing a functional green fluorescent delta opioid receptor

A unique genetic mouse model was developed, in order to investigate the distribution of delta receptors throughout the nervous system, and the link between receptor localization at a subcellular level and receptor function in vivo. In these mice, endogenous delta receptors are replaced by delta receptors in fusion with green fluorescent protein (DOR-eGFP) using a knock-in strategy (Scherrer et al., 2006). DOR-eGFP mice express fully functional delta receptors, which are directly visible in vivo. Fluorescent delta receptors are expressed in DRGs, spinal cord and brain (Scherrer et al., 2006, 2009,), with profiles in accordance with results from in situ hybridization (Mansour et al., 1995; Mennicken et al., 2003) and ligand binding autoradiography (Goody et al., 2002). In models of inflammatory pain, DOR-eGFP mice respond to delta agonist-induced analgesia as control wild-type mice (Pradhan et al., 2009., 2010). These animals have been instrumental in deciphering differential internalizing properties of the two delta agonists SNC80 and AR-M100390 throughout the nervous system in vivo, and elucidating short-and long-term consequences of ligand-biased receptor trafficking on analgesic responses (see details below and (Pradhan et al., 2009., 2010).

Delta receptor expression and function are regulated at different levels within the cell

A major factor contributing to delta opioid analgesia is the expression level of the receptor protein at the cell surface of neurons, and at the different sites of the pain-processing pathways. Variations in receptor density may result from sequence variations within the Oprd1 gene, transcriptional regulation, post-translational events or receptor trafficking to and from the plasma membrane.

Genetic variability

Sequence variants within the Oprd1 gene may influence delta receptor expression across individuals, and may contribute to inter-individual differences in responses to delta drugs (Lotsch & Geisslinger, 2011). In the human gene, several single nucleotide polymorphisms (SNP) have been identified (Kim et al., 2006). The T80G variant in exon 1, occurring at an allele frequency of 0.08, leads to a Phe27Cys substitution in the extracellular N-terminal domain, and has been associated with pain sensitivity (Kim et al., 2006). In heterologous expression systems, the Cys27 variant showed lower maturation efficiency, increased accumulation of receptor precursors in pre-Golgi compartment and faster constitutive internalization (Leskela et al., 2009). Other human SNP variants showed no association with pain sensitivity (Huang et al., 2008; Kim et al., 2006; Zhang et al., 2010). In the mouse Oprd1 gene, polymorphism was identified in mice selectively bred for high and low stress-induced analgesia, and later shown to differ also in basal nociception and opioid analgesia. In this study, three polymorphic sites were detected in the Oprd1 coding region (Sacharczuk et al., 2010). Among these, the C320T transition resulted in an A107V substitution in the first extracellular loop of the delta receptor protein. The C320T decreased SNC80-induced analgesia in thermal pain tests. Altogether, the identification of natural genetic variants affecting extracellular domains of the delta receptor and altering pain perception, opens the possibility of inter-individual variability in responses to delta agonists.

Transcriptional regulation

Pathological situations like chronic pain, inflammation and nerve damage were shown to induce transcriptional regulation of the delta receptor that may impact on receptor activity. Transcription factors binding to Oprd1 promoter sequences and regulating gene expression, as well as epigenetic aspects, have recently been reviewed extensively (Wei & Loh, 2011). Regulation at the transcriptional level of delta receptor expression was investigated in several models of chronic pain and appears highly variable depending on a series of factors including (i) the pain model, (ii) the receptor localization (brain, spinal cord, DRG, sciatic nerve or skin), (iii) time points considered after pain initiation, (iv) the animal strain (Herradon et al., 2008) and (v) the technique used for detecting receptor expression. Data are summarized in Table 2. In the different models of inflammatory pain, no change, up-regulation or down-regulation of delta receptor mRNA levels were reported. In models of neuropathic pain also, several regulation patterns were observed and together the data indicate that regulation at transcriptional level may be detectable in specific pain situations, and likely impact on delta receptor protein levels (Table 2). Most expression studies were performed at the level of DRG and spinal cord. Delta receptor activity, as measured by agonist-induced G protein activation, was also altered in the cingulate cortex and amygdala under conditions of inflammatory and neuropathic pain (Narita et al., 2006a,b). This particular regulation in the central nervous system was suggested to contribute to chronic pain-induced emotional dysfunction (Narita et al., 2006a,b). Finally, delta receptor expression was highly increased under two clinical pain conditions, in the skin of fibromyalgia patients (Salemi et al., 2007) as well as in hypertrophic scars (Cheng et al., 2008). Collectively the data show that delta receptor expression is regulated under experimental chronic pain, and more investigations in pain patients are needed to correlate findings in animal models with clinical situations.

Table 2.

Changes in delta opioid receptor expression in chronic pain models

Pain model	Species	Time and region/cell type examined	mRNA or protein - technique	Change	Reference
Inflammatory pain
CFA* into paw	rat	DRG*	mRNA Q-RT-PCR*	No	(Gendron et al., 2006)
CFA into paw	rat	DRG	mRNA Q-RT-PCR	No	(Puehler et al., 2004)
CFA into paw	rat	DRG	Protein immunohistochemistry	No	(Brack et al., 2004)
CFA into paw	rat	DRG	Protein immunohistochemistry	Down	(Zhang et al., 1998)
CFA into paw	rat	Spinal cord	mRNA Q-RT-PCR	No	(Obara et al., 2009)
CFA into paw	rat	Spinal cord	mRNA ISH* Protein Western blot	Up	(Cahill et al., 2003)
CFA into paw	rat	Spinal cord	mRNA ISH	No	(Maekawa et al., 1996)
CFA into paw	rat	Spinal cord	Protein radioligand binding	No	(Millan et al., 1988)
CFA into paw	mouse	DRG	mRNA Q-RT-PCR	Up	(Gaveriaux-Ruff et al., 2011)
Carrageenan into paw	rat	DRG	Protein immunohistochemistry	Down	(Ji et al., 1995)
		Spinal cord		Down
CFA into tail	rat	Spinal cord	mRNA Q-RT-PCR	Up	(Calza et al., 2000)
CFA into joint	rat	Thalamus, reticular nucleus	mRNA ISH	Down	(Neto et al., 2008)
		Brainstem, dorsal reticular nucleus		Down
		Brainstem, parvocellular reticular nucleus		Up
CFA into joint	rat	Spinal cord	Protein radioligand binding	Up	(Besse et al., 1992b)
Incision - post-operative	mouse	DRG	mRNA Q-RT-PCR	Down	(Cabanero et al., 2009)
		Spinal cord		Down
Intestinal, croton oil	mouse	Spinal cord	mRNA Q-RT-PCR	Up	(Pol et al., 2003)
			Protein Western blot
		Whole gut		Up
		Brain		No
Neuropathic pain
CCI*	rat	DRG Spinal cord	mRNA Q-RT-PCR	Down No	(Obara et al., 2009)
CCI	rat	DRG	mRNA Q-RT-PCR	Down	(Herradon et al., 2008)
		Spinal cord		No
CCI	rat	Spinal cord	Protein immunohistochemistry	Down	(Tseng et al., 2008)
CCI	rat	Spinal cord	Protein western blot	No	(Holdridge & Cahill, 2007)
CCI, Spinal NL*	rat	Spinal cord	Protein immunohistochemistry	Down	(Stone et al., 2004)
CCI	rat	Spinal cord	Protein radioligand binding	Down	(Stevens et al., 1991)
CCI	rat	Spinal cord	Protein radioligand binding	Down	(Besse et al., 1992a)
CCI	mouse	DRG	mRNA Q-RT-PCR	Down	(Hervera et al., 2010)
		Spinal cord		No
Cuff	rat	DRG	Protein western blot	Up	(Kabli & Cahill, 2007)
pSNL*	mouse	DRG	mRNA Q-RT-PCR	No	(Gaveriaux-Ruff et al., 2011)
pSNL	mouse	DRG & spinal cord	mRNA Q-RT-PCR	No	(Pol et al., 2006)
Multiple sclerosis
TMEV* infection	mouse	Spinal cord	mRNA Q-RT-PCR	Down	(Lynch et al., 2008)

^*Abbreviations: CCI, chronic constriciton injury; CFA, Complete Freund’s Adjuvant; DRG, dorsal root ganglion; ISH, In Situ hybridization; NL, nerve ligation; pSNL, partial sciatic nerve ligation; Q-RT-PCR, quantitative reverse transcriptase-PCR; TMEV, Theiler’s murine encephalomyelitis virus

Post-translation regulation

A third level of regulation occurs post-translationally in receptor protein maturation and transport. Mechanisms for delta receptor folding and integration into membranes were investigated using heterologous expression systems. Tuusa et al. (2010) recently showed that delta receptor biogenesis is regulated early after translation at the level of endoplasmic reticulum by the molecular chaperone calnexin and the sarcoendoplasmic reticulum calcium ATPase SERCA2b in a calcium and ATP dependent manner. Delta receptor targeting from the endoplasmic reticulum to the cell surface was further proposed to require the golgi chaperone receptor transport protein 4 RTP4. In vitro experiments showed that this protein participates in folding of delta-mu receptor heterodimers, enhancing the trafficking of delta-mu receptor complexes from the Golgi apparatus to the cell surface and decreasing the expression of delta monomers (Decaillot et al., 2008). Future experiments will determine whether these processes occur in vivo.

Receptor activity at the cell surface

Receptor activity is also modulated at the plasma membrane, via interactions with other membrane proteins. In particular, delta receptors may associate with other GPCRs to form heterodimers or larger heteromers at the cell surface. Based on several approaches and criteria used to define potential GPCR heterodimers (see (Massotte, 2010), delta receptors have been proposed to interact with GPCRs implicated in pain control, including mu and kappa opioid receptors (Gupta et al., 2010; Kabli et al., 2010; van Rijn et al., 2010), CB1 cannabinoid receptors (see Bushlin et al., 2010) and alpha2-adrenergic receptors (see van Rijn et al., 2010). Delta receptors may also interact with chemokine receptors (Parenty et al., 2008; Pello et al., 2008) with potential consequences on inflammation, including inflammatory pain (Chen et al., 2007). These studies, mainly performed in heterologous cell recombinant systems, show new pharmacological properties for delta receptor-GPCR heterodimers (reviews (Bushlin et al., 2010; Rozenfeld & Devi, 2010; van Rijn et al., 2010). Opioid receptor heterodimers may also be involved in the activities of bi-functional opioid drugs investigated as novel classes of analgesics (Ansonoff et al., 2010; Schiller, 2010).

Receptor activation and intracellular effectors

Finally, receptor density and activity at the cell surface is tightly regulated by intracellular effectors, which engage the receptor into both signaling and trafficking processes upon activation. The best-known signaling effectors of the delta receptor are inhibitory heterotrimeric Gi/o proteins, which further modulate ion channels and second messengers leading ultimately to reduced neuronal activity (Williams et al., 2001). In addition to signaling, agonist binding to the receptor also induces phosphorylation, internalization, trafficking and redistribution of the receptor, and these events represent key mechanisms for the regulation of receptor activity (Cahill et al., 2007; Ritter & Hall, 2009). Unlike the mu opioid receptor, which rapidly recycles to the cell surface, delta receptors are targeted towards lysosomal degradation (Pradhan et al., 2009) via the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, using ubiquitination-dependent or independent mechanisms (Henry et al., 2010). Other factors upstream of the ESCRT pathway also contribute to delta receptor proteolysis, including G protein-coupled receptor Associated Sorting Proteins (GASPs) shown to interact with the delta receptor (Abu-Helo & Simonin, 2010; Moser et al., 2010).

The link between agonist-induced internalization, downregulation and in vivo function of delta receptors was investigated using the DOR-eGFP mouse model (Pradhan et al., 2009., 2010). Data indicated that a single injection of a high- (SNC80) and a low (AR-M100390)-internalizing compound to DOR-eGFP mice produced equally efficient analgesia in a model of inflammatory pain. SNC80 also concomitantly induced receptor internalization throughout the nervous system, accompanied by G-protein uncoupling and acute behavioral desensitization, such that a second drug injection was inefficient. Receptor desensitization was transient and receptor responsiveness returned to basal levels after one day. In contrast, AR-M100390 induced none of the regulatory responses, since no internalization, G-protein uncoupling or acute desensitization were detectable (Pradhan et al., 2009). Interestingly further chronic treatment with the two agonists both induced the development of tolerance, but the expression of tolerance differed. The high-internalizing agonist (SNC80) induced generalized tolerance, so that all agonist effects were blunted in chronically-treated mice, and the low-internalizing agonist (AR-M100390) produced a partial tolerance that was restricted to analgesic responses, while anxiolytic or locomotor effects of delta agonists remained intact (Pradhan et al., 2010). This set of data is in accordance with previous in vitro studies showing ligand-specific conformational changes of the delta receptor (Audet et al., 2008) and definitely establish the in vivo relevance of delta receptor ligand-biased agonism in drug efficacy. The critical consequences of biased agonism at the delta receptor add further physiological support to the rapidly growing field of functional selectivity at GPCRs (Bosier & Hermans, 2007; Galandrin et al., 2007; Mailman, 2007; Vaidehi & Kenakin 2010; Zheng et al., 2010) and may be considered for the development of effective therapeutic drugs.

Functional interactions between delta and other neuromodulatory systems

An active research field is the elucidation of respective contributions of mu and delta receptors in nociceptive processing, particularly to control heat and mechanical pain modalities (Basbaum et al., 2009; Scherrer et al., 2009; Woolf, 2009), and the identification of molecular and cellular mechanisms underlying functional interactions between the two receptors. Interactions may occur either at cellular or systems levels within nociceptive pathways. This has been extensively discussed in the past (Kieffer, 1999; Smith & Lee, 2003; Zaki et al., 1996) and more recent anatomical analysis has provided evidence for potential interactions at the cellular level in vivo. Data from in situ mRNA hybridization (Mansour et al., 1995; Mennicken et al., 2003; Wang & Wessendorf, 2001), the analysis of DOR-eGFP mice (Scherrer et al., 2009), and local pharmacology in peripheral neurons (Joseph & Levine, 2010) suggest that mu and delta receptors may be co-localized in a limited number of peripheral nociceptor neurons. This co-expression opens the possibility for within-cell functional interactions in primary nociceptive processing, which may occur between receptors or their downstream signaling pathways. Delta and mu receptor co-localization in the dorsal spinal cord remains debated (Overland et al., 2009; Scherrer et al., 2009; Wang et al., 2010).

A large array of evidence indicates that delta receptor function is increased either after chronic morphine or under conditions of chronic pain. A suggested common basis for this phenomenon lies in the observation of increased delta receptor density at the cell surface in both cases, leading to higher number of receptors available for activation (Bie & Pan, 2007; Cahill et al., 2007; Morgan et al., 2009; Schramm & Honda, 2010). In the case of chronic morphine, the enhancement of delta receptor function likely involves a functional link between mu receptor activation and delta receptors, whose mechanism remains open (see above). In situations of persistent pain, other mechanisms involving bradykinin (Patwardhan et al., 2005), protease activated receptor-2 (Patwardhan et al., 2006), arachidonic acid (Rowan et al., 2009) and nerve growth Factor (Bie et al., 2010) have been shown to augment delta receptor activity. Collectively, interactions between delta receptors and several other opioid or non-opioid receptor systems have been reported to influence delta opioid analgesia, and a great diversityof GPCRs involved pain control (Pan et al., 2008; Stone & Molliver, 2009) may potentially interact with, or modulate, delta receptor analgesic activity.

Conclusion

The field of delta opioid receptor analgesia has benefited from recent contributions in pharmacology, molecular and cellular approaches. Novel selective delta agonists with potent in vivo activities are now available to strengthen approaches targeting delta receptors. The field of delta receptors and pain control may also benefit from the development of animal models more closely related to clinical pain (Finley et al., 2008). In particular the evaluation of delta receptor activity in spontaneous pain and in the emotional or affective dimensions of pain (King et al., 2009; Minami, 2009) may provide new elements for the development of delta receptor-based therapeutic strategies.