LFS-14653; No of Pages 9

Life Sciences xxx (2016) xxx–xxx

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Life Sciences

journal homepage: www.elsevier.com/locate/lifescie

Neurobiology of opioid withdrawal: Role of the endothelin system

a r t i c l e i n f o a b s t r a c t

Article history: Morphine and oxycodone are potent opioid analgesics most commonly used for the management of moderate to
Received 2 November 2015 severe acute and chronic pain. Their clinical utility is limited by undesired side effects like analgesic tolerance, de-
Received in revised form 6 January 2016 pendence, and withdrawal. We have previously demonstrated that endothelin-A (ETA) receptor antagonists po-
Accepted 11 January 2016
tentiate opioid analgesia and eliminate analgesic tolerance. Mechanistically, G proteins and regulatory proteins
Available online xxxx
such as β-arrestins have shown to play an important role in mediating opioid tolerance, dependence, and with-
Keywords:
drawal. Recently, the involvement of central ET mechanisms in opioid withdrawal was investigated. ETA receptor
Morphine antagonist was shown to block majority of the signs and symptoms associated with opioid withdrawal. This re-
Oxycodone view focuses on ET as one of the potential novel strategies to manage the challenge of opioid withdrawal. An
Endothelin ETA receptor antagonist overview of additional players in this process (G proteins and β-arrestin2), and the possible therapeutic implica-
BQ123 tions of these findings are presented.
Withdrawal © 2016 Elsevier Inc. All rights reserved.
Nerve growth factor (NGF)
β-Arrestin 2
G protein coupled receptor (GPCR)

1. Introduction 2. The problem of opioid withdrawal

Chronic pain is an important public health problem that often According to the Journal of the American Medical Association
goes undertreated. Close to 100 million adults in the United States (JAMA) [99] and reports from the National Council on Alcoholism
suffered from chronic pain in 2008 with an estimated cost of $560 and Drug Dependence Inc. (NCADD), the number of teenagers,
to $635 billion in medical treatment and lost productivity [1] Opioids young adults and pregnant women who are addicted to opioids, as
such as morphine and oxycodone are among the most potent analge- well as the infants born with opioid withdrawal symptoms, has
sics used to treat severe acute as well as chronic pain. They are the jumped in the past decade. Further, The New York Times reported
favored drug of choice in clinical situations because of their high an- that people who previously might not have used heroin are more
algesic efficacy; however, a number of side effects develop after their likely to use prescription opioids, and this has the potential to be-
prolonged use. The most serious adverse effects are sedation, drug come a national or international issue. As of 2010, over 12 million
dependence, respiratory depression, hyperalgesia, constipation, Americans reported using prescription pain medications for non-
and miosis [93]. The potential danger of prolonged use of most opi- medical purposes without having obtained a prescription [97]. The
oids is physical and psychological dependence [66]. Mechanisms in- World Health Organization (WHO) also estimates that 2 million people
volved in these negative outcomes are complex and involve opioid in the United States alone are addicted to prescription opioids [98].
and non-opioid systems [8,9]. Opioid receptors are part of the G protein Some of the adverse effects of opioid withdrawal include sedation, re-
coupled receptor (GPCR) family and function via the activation of spiratory depression, cramps, changes in body temperature, diarrhea
adenylyl cyclase (AC)-inhibitory (Gi/o) GTP-binding proteins [6,81]. and emesis [37,73,112,117]. A large number of patients undergoing
Non-opioid systems like gamma butyric acid (GABA), dopamine, nitric opioid withdrawal also exhibit debilitating withdrawal symptoms
oxide (NO), N-methyl-D-aspartate (NMDA) receptors and glutamate such as tachycardia, cardiac arrhythmias, hypertension, anxiety, stroke
receptors also play an important role in the development of adverse and seizures [60,62,87]. The mechanisms implicated in opioid tolerance
effects of opioids [61,126,127,130,131]. and withdrawal are complex and involve signaling pathways, adapta-
tions in receptor expression and interactions between opioid and non-
opioid receptors. Some of the non-opioid systems involved in opioid
tolerance and withdrawal include GABA, NO, NMDA receptors, dopa-
⁎ Corresponding author at: Department of Pharmaceutical Sciences, Midwestern
mine, endothelin (ET) and adrenergic systems [43,49,104,105,142].
University, Chicago College of Pharmacy, 555 31st Street, Downers Grove, IL 60515, USA. Opioid tolerance and dependence result largely from the effects of
E-mail address: [email protected] (S. Bhalla). prolonged drug exposure on brain functionality. Subsequent opioid

http://dx.doi.org/10.1016/j.lfs.2016.01.016
0024-3205/© 2016 Elsevier Inc. All rights reserved.

Please cite this article as: S. Bhalla, et al., Neurobiology of opioid withdrawal: Role of the endothelin system, Life Sci (2016), http://dx.doi.org/
10.1016/j.lfs.2016.01.016
2 S. Bhalla et al. / Life Sciences xxx (2016) xxx–xxx

withdrawal affects multiple brain circuits, including those involved in irritation in mice which was inhibited by morphine through a μ1-opioid
reward and motivation, learning and memory, and inhibitory control receptor subtype mediated pathway [108]. ETA receptors have been re-
over behavior. Therefore, there is still a critical need to identify alterna- ported to be located directly on the nociceptors producing either direct
tive and innovative solutions to meet both, treatment needs of patients depolarization or increased excitability and can be blocked by ETA recep-
and desired level of safety expected of modern drug therapies. tor antagonist, BQ123 [64,147]. Rothman and his group demonstrated
the ET-opioid link through a series of studies. In one study, anti-ET-1
3. Management of patients undergoing opioid withdrawal immunoglobulin-G (IgG) was administered to rats via the i.c.v.
route and its effect on the expression of ET receptors and opioid re-
Current clinical practice for the treatment of dependence and ceptors in the brain was measured by Western blot analysis [138].
withdrawal is focused on α2-adrenergic receptor agonists like cloni- It was reported that anti-ET IgG regulates the expression of κ opioid
dine and lofexidine, and opioid receptor agonists like methadone or receptors, dynorphin levels and ETA receptors, as well as regulates
buprenorphine. These are commonly used pharmacologic methods receptor coupling to G proteins. These observations suggest that
of detoxification. The use of methadone and buprenorphine is endogenous ET, in the extracellular fluid, downregulates the κ opioid
based on the principle of cross-tolerance in which one opioid is replaced system, both by decreased expression of the κ opioid receptor and by
with another and then slowly withdrawn. α2-Adrenergic receptor ago- decreased dynorphin levels, downregulates ETA receptors, and in-
nists appear to be effective in suppressing autonomically mediated creases the efficacy of agonists. These results strongly suggest that
signs and symptoms of abstinence [71], but they are less effective for endogenous ET, circulating in the extracellular fluid of the brain, regu-
subjective symptoms. Another treatment option is Probuphine, a subcu- lates both the ET and opioid systems, emphasizing both the functional
taneous implant delivering 6 months of buprenorphine under the skin. A interactions between these two neuropeptide systems, as well as the
sublingual tablet (Zubsolv) containing a combination of buprenorphine role that CSF neuropeptides play as regulatory substances.
and naloxone was approved in 2013 for the management of opioid Studies have shown that central ET mechanisms are involved in
withdrawal symptoms and cravings. Given the limited treatment op- modulating the analgesic actions of opioids, as well as the development
tions for opioid dependence and withdrawal, further study of biological of tolerance in mice and rats [12,13,17,49]. ETA receptor antagonists sig-
functions that are altered by chronic opioid treatment is necessary. nificantly potentiate opioid analgesia, increase the duration of analgesic
Identifying neuroadaptations during traumatic events such as opioid response, and restore anti-nociceptive effect during opioid tolerance
withdrawal will assist in identifying novel targets for developing effec- [12,13,15,17]. It was observed that ETA receptor antagonists did not
tive combination therapy with opioids that lowers adverse effects such bind directly to opioid receptors in the brain [12]. Mechanistic studies
as tolerance and withdrawal yet achieves adequate analgesia. further revealed that the interaction of ETA receptor antagonists and
opioids was mediated through G proteins [15,49]. It was also shown
4. Endothelin (ET) and its receptors: central nervous system (CNS) that interaction of ETA receptor antagonist occurs at all subtypes of opi-
aspects oid receptors, i.e. mu (μ), delta (δ), and kappa (κ) receptors [17]. In light
of these findings, it was hypothesized that ETA receptor antagonists
ET is a potent vasoconstrictor peptide found in endothelial cells and could minimize opioid withdrawal symptoms.
smooth muscle cells [59,144]. Accumulating evidence indicates that ac-
tivation of brain ET receptors is involved in several pathophysiological 6. Endothelin ETA receptors and opioid withdrawal
responses. The ET family consists of three isopeptides (ET-1, ET-2 and
ET-3). The presence of ET receptors in the CNS suggests that ET has The possible involvement of ETA receptors in opioid withdrawal was
neuromodulatory functions in the CNS [72,96]. The biological responses investigated in mice using ETA receptor selective antagonist, BQ123.
of ET-1 are mediated via the activation of two distinct G protein coupled There was a significant decrease in body weight in mice undergoing
receptors (GPCRs): endothelin-A (ETA) [47] and endothelin-B (ETB) morphine and oxycodone withdrawal. However, BQ123 pretreatment
receptors [129]. While all three ETs have equal affinity for the ETB recep- significantly attenuated the weight loss observed during withdrawal
tor, ET-3 has a lower affinity for the ETA receptor compared with either [16]. Severe hypothermia was seen during both morphine and oxy-
ET-1 or ET-2 [115]. The global distribution of ET and its binding sites in codone withdrawal. Pretreatment with BQ123 prevented the severe
the brain suggests that, in addition to being a vasoactive agent, ET acts as hypothermic response observed in mice during withdrawal. Admin-
an important regulatory neuropeptide in the CNS and affects many istration of BQ123 prior to precipitation of withdrawal resulted in at-
other biological functions [54,85,128]. ET has also been shown to signif- tenuation of characteristic jumping behavior [16]. Symptoms such as
icantly regulate the central autonomic nervous system [40,75]. wet dog shakes and rearing behavior were also blocked by BQ123
pretreatment confirming the involvement of ETA receptors. These re-
5. ETA receptors in opioid analgesia and tolerance sults provide evidence that ETA receptors are not only involved in
acute opioid analgesia and tolerance, but they also play a significant
There are several lines of evidence suggesting the interaction of role in the CNS pathway involved in opioid withdrawal.
central and peripheral ET and opioid systems at a functional level. ET- β-Arrestin2 (β-arr2) is an important GPCR regulatory protein that
1 causes nociception and hyperalgesia by binding to ETA receptors local- can regulate the effects of opioids by functioning as a mediator of
ized on nociceptors [101]. Studies have indicated that an ETA receptor GPCR desensitization. GPCRs are activated and phosphorylated by
antagonist, ABT-627, counteracts ET-1 induced hyperalgesia, attenuates GPCR kinases (GRKs) and bind to β-arrestins, which prevents further
tactile allodynia, and blocks neuropathic pain in a diabetic rat model of coupling of the receptor and G proteins [84,107]. Our research group
neuropathic pain [64]. Responses of morphine may be associated with has previously established the role of G proteins in the interaction of
increased systemic and cerebrovascular levels of ET-1 and upregulation ETA receptor antagonists and opioid agonists [11,15]. Therefore, we
of ET-1 and ETA receptor mRNA in the brainstem of newborn piglets wanted to investigate the role of β-arr2 during opioid withdrawal. The
[91]. ET-1 administered intracerebroventricularly (i.c.v.) caused signifi- levels of β-arr2 were determined in the brain of mice undergoing opioid
cant increases in mean arterial pressure and renal sympathetic nerve withdrawal using Western blot analysis. No change was observed in the
activity (RSNA) and these effects were potentiated by naloxone pre- expression of β-arr2 during morphine or oxycodone withdrawal com-
treatment [88]. Reports have also shown that intraperitoneal (i.p.) pared with the control group. BQ123-treated mice did not show altered
administration of ET-1 induced nociception by producing abdominal β-arr2 levels in the brain in control mice. Further, BQ123 treatment dur-
constriction which can be reversed by central administration of mor- ing morphine and oxycodone withdrawal did not affect β-arr2 protein
phine [109,110]. Intraperitoneal ET-1 administration induced abdominal expression (data not shown). Therefore, ETA receptor antagonist did

Please cite this article as: S. Bhalla, et al., Neurobiology of opioid withdrawal: Role of the endothelin system, Life Sci (2016), http://dx.doi.org/
10.1016/j.lfs.2016.01.016
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not have any effect on β-arr2 levels in morphine or oxycodone with- our previous studies in SH-SY5Y cells and neuronal membranes, we
drawal although behavioral symptoms were significantly attenuated. have reported that ETA receptor antagonists restore G protein coupling
While β-arr2 levels were unchanged, it remains to be seen whether to opioid receptors. The above findings also support our hypothesis
ETA receptor antagonists influence the recruitment of β-arr2 at the that ETA receptors may share the same pool of inhibitory G proteins
μ-opioid receptor in analgesia, tolerance and withdrawal. This will be that are coupled to μ-ORs, and these Gi proteins are recoupled to the
a topic of future research conducted by this group. μ-ORs in the presence of ETA receptor antagonists (Fig. 1).
Agonist binding to G protein coupled μ-ORs causes a transfer of GDP
7. Mechanism of interaction of ETA receptor antagonists in opioid (guanosine-5′-diphosphate) into GTP (guanosine-3′,5′-cyclic triphos-
withdrawal phate), which leads to a separation of the α from the βγ subunit of
the G protein. This leads to a decrease of AC activity, and decrease in cy-
7.1. G proteins clic adenosine monophosphate (cAMP) levels under acute conditions.
This, in turn, causes reduced Ca2 + influx and increased K+ efflux at
There is a close interaction of ETA and ETB receptors with G proteins ion channels, which results in hyperpolarization of the cell that stops
[83,128]. Actions of ET-1 are mediated by G protein subunits. Effect of the transfer of afferent nociceptive signals and thereby produces analge-
ET-1 on μ and κ opioid receptors mediated through G proteins has also sia (Freye and Latasch, 2003, [57]). Therefore, acute opioid receptor ac-
been demonstrated [138]. The agonist-mediated increase in G protein tivation in cells leads to neuronal inhibition caused by multiple
function is a critical step in receptor activation and function. The extent effectors, including inhibition of AC and voltage-gated Ca2+ channels,
of G protein activation is proportional to the agonist-bound receptors, and stimulation of inwardly rectifying K+ channels. It is well established
and the attenuation of G protein activation is a significant factor in the that opioid tolerance, dependence, and withdrawal are associated with
development of opioid tolerance. upregulation of the cAMP pathway and supersensitization of AC follow-
Studies conducted in neuronal membranes in rats and mice showed ing chronic opioid exposure of opioid agonists [56,81,95,112]. Further, it
that G protein stimulation by morphine and ET-1 was decreased in is known that the altered coupling of opioid receptors to stimulatory G
morphine-tolerant animals. Further, in control animals, ETA receptor proteins (Gs) causes upregulation of the cAMP pathway [18,30,34,137]
antagonist did not affect G protein stimulation (as indicated by by increasing AC activity. It is likely that ETA receptor antagonists also fa-
no change in GTP binding), but enhanced G protein activation in cilitate morphine-induced inhibition of AC and downregulate the cAMP
morphine-tolerant animals [15,49]. Therefore, ETA receptor antagonist pathway during morphine tolerance and withdrawal. Thus, through
recoupled G proteins to μ-opioid receptors (μ-ORs), thereby restoring restoration of the Gi G protein signaling, ETA receptor antagonists aid
the anti-nociceptive effect of morphine [15]. in decreasing cAMP levels and consequently prevent hyperalgesia, re-
In another study, the effects of morphine, ET-1, and ETA receptor an- store analgesic activity of opioids after chronic exposure, and block
tagonists were investigated in SH-SY5Y cells. Acute morphine treatment withdrawal symptoms via Gi protein coupling to μ-ORs.
produced a dose-dependent increase in [35S]GTPγS binding in these
cells. Sensitization of G protein activation by morphine was observed, 7.2. Beta (β) arrestin
implied by the leftward shift of the curve. Chronic morphine treatment
resulted in significantly reduced [35S]GTPγS binding [11]. These findings Physiological actions of morphine and other clinically used opioid
are supported by previous reports indicating that G protein activation is analgesics are mediated primarily through activation of the μ-OR, a
significantly attenuated in opioid tolerance [74,119,120]. Acute treat- known GPCR [69,89,114,124]. Diverse signaling components and com-
ment of SH-SY5Y cells with ET-1 showed a dose-dependent increase plex neuronal adaptations contribute to the development of analgesic
in [35S]GTPγS binding. The binding response was similar to ET-1- tolerance and physical dependence. There is evidence suggesting that
induced [35S]GTPγS binding in control cells. Further, chronic treatment regulation of μ-OR signaling can affect these adaptive responses [106].
with ET-1 did not sensitize or desensitize ET receptors. Increasing doses Binding of agonists to GPCRs activates G protein-linked intracellular sig-
of ETA receptor antagonist, BMS182874, alone did not stimulate GTP naling cascades with subsequent receptor phosphorylation leading to
binding in these cells after acute or chronic treatment. Cells treated recruitment of the scaffolding protein, β-arrestin (β-arr). β-Arrestin2
chronically with morphine + ETA receptor antagonist showed signifi- (β-arr2) is an important GPCR regulatory protein that differentially reg-
cantly higher GTP binding than cells treated chronically with morphine ulates opioid effects in a manner determined by the agonist. GPCRs,
alone. This increase in G protein activation in response to morphine when activated, are phosphorylated by GPCR kinases (GRKs) and subse-
after combined treatment with BMS182874 provides evidence that quently bind to β-arrestins, which prevents further coupling of the re-
morphine-induced G protein activation was restored even at lower ceptor and G proteins [84,107]. GPCRs couple to multiple G protein
concentrations of morphine [11]. This suggests that tolerance did not classes and may also activate signaling pathways such as MAP kinases
develop to chronic morphine exposure in the presence of ETA receptor in a G protein-independent manner via the receptor forming complexes
antagonist similar to in vivo studies. The enhanced binding and G pro- with β-arr1 or β-arr2 [118].
tein signaling by combined treatment of morphine with ETA receptor As a mediator of GPCR desensitization, β-arr2 regulates the degree of
antagonists indicate the ability of these antagonists to restore G protein coupling between the μ-OR and G proteins, and this has been shown in
coupling to opioid receptors [11]. certain brain regions in β-arr2 knock-out (KO) mice [21,23]. Recruit-
μ-ORs couple to pertussis-toxin sensitive G proteins [132] which in- ment of β-arr2 results in uncoupling of opioid receptors from the asso-
hibit adenylyl cyclase (AC). There is evidence demonstrating that μ-ORs ciated G protein (desensitization) and may target the receptor for
and δ opioid receptors (δ-ORs) couple to similar subtypes of Gi and Go subsequent internalization and trafficking resulting in tolerance [36,
proteins which inhibit AC and are pertussis-toxin sensitive [29,77, 38]. Acute morphine analgesia is potentiated and the duration of anal-
102]. In another study conducted on SH-SY5Y cells, which endogenous- gesic response is prolonged in β-arr2 KO mice [20,23]. Further, tolerance
ly express both μ-ORs and δ-ORs [67], it was determined that both does not develop even after chronic treatment with morphine in β-arr2
these receptors activate the same individual G proteins in cell lines co- KO mice [106,107]. Another interesting finding was the increase in
expressing both receptors. These findings strongly supported the [35S]GTPγS binding observed in β-arr2 KO mice versus the wild type,
hypothesis that μ-ORs and δ-ORs share a common G protein pool and suggesting enhanced G protein coupling in the absence of β-arr2 re-
this provides an intracellular mechanism for interaction between cruitment [22,23]. β-Arr2 knockdown mice showed higher morphine
these two receptor subtypes [5]. This finding that μ-ORs and δ-ORs analgesia [82,145], whereas animals overexpressing β-arr2 demonstrat-
share G proteins in the SH-SY5Y cell membranes indicates that both re- ed lower analgesic response [65]. Moreover, morphine analgesia can be
ceptors can access the complete pool of inhibitory G proteins [5,94]. In potentiated by inhibiting β-arr2 recruitment [31,123,135] and by

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Fig. 1. Proposed mechanism of functional interaction between ETA receptors and Gi G proteins coupled to μ-opioid receptors (μ-ORs) in tolerance. The Gi G protein uncouples from the μ-OR
upon chronic exposure to morphine contributing to analgesic tolerance. ETA receptor antagonist restores G protein coupling to the μ-OR and thereby enhances G protein signaling. The
result is restoration of the anti-nociceptive effect during tolerance.

increased G protein coupling and downstream signaling [139]. It is also the highest dose of morphine infusion was administered, the wild
known that β-arr2 binding to GPCRs, both, uncouples receptors from type (WT) and KO mice showed equivalent withdrawal symptoms.
heterotrimeric G proteins and targets them to clathrin-coated pits for However, at two lower doses, the severity of naloxone-precipitated
endocytosis [84]. These reports collectively demonstrate that β-arr2 withdrawal symptoms was significantly attenuated in β-arr2 KO mice
plays a critical role in morphine tolerance by the processes of homolo- compared with their WT counterparts [106]. This was particularly evi-
gous desensitization and GPCR sequestration, leading to the inhibition dent in the jumping behavior post-withdrawal, which is considered a
of G protein activation (Fig. 2). key behavior associated with the severity of dependence and withdraw-
The cellular actions of ET peptides are mediated via interaction with al [68]. The decreased severity of morphine withdrawal in β-arr2 KO
two GPCRs, ETA and ETB [7,116]. Mechanistically, ETA receptor antago- mice suggests that β-arr2 may facilitate μ-OR signaling in neurons in-
nists restore G protein coupling to the μ-OR and this restored coupling volved in the development of dependence and withdrawal behaviors
is the mechanism of potentiation of opioid analgesia [11,15,49]. It is [106].
also known that β-arr2 regulates the G protein coupled ETA and ETB re- Our recent findings show that majority of the withdrawal symptoms
ceptors [25,86]. Therefore it is likely that ETA receptor antagonists re- of opioids are attenuated by ETA receptor antagonists [16]. Because G
store G protein coupling to opioid receptors by modulating the effect protein uncoupling and β-arr2 recruitment is a key mechanism under-
of β-arr2 on μ-ORs. We speculate that blockade of ETA receptors by lying tolerance, we propose that this mechanism may also be involved
ETA receptor antagonists results in a bias of G protein activation and in opioid withdrawal. Based on these collective reports, we believe
μ-OR downstream signaling pathway over β-arr2 recruitment and that ETA receptor antagonists result in biased agonism at the μ-ORs
may contribute to the restoration of opioid analgesia in the tolerant and restore G protein signaling, while simultaneously inhibiting
state (Fig. 3). β-arr2 recruitment, and this functional interaction in vivo may be re-
In addition to acute morphine analgesia and tolerance, the complex sponsible for potentiation of analgesia, reversal of tolerance, and block-
role of β-arr2 has been demonstrated in dependence and withdrawal. ade of opioid withdrawal symptoms. We found no change in β-arr2
Another report indicated that β-arr2 KO mice displayed less severe protein levels in the presence or absence of ETA receptor antagonist
withdrawal symptoms at two infusion doses of morphine [106]. When BQ123 during morphine or oxycodone withdrawal. However, it is

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Fig. 2. A. Morphine binds to the μ-opioid receptor; B. Morphine binding activates G protein signaling. Binding of morphine to the receptor activates coupled G proteins and initiates
signaling that results in anti-nociception; C. G protein receptor kinases (GRK) mediated phosphorylation and β-arrestin (β-arr) mediated desensitization. GRK phosphorylate key
serine/threonine residues primarily at the C-terminus of the agonist-occupied receptor. Phosphorylated receptor recruits β-arr which hinders sterically, the ability of the G proteins to
couple with the receptor and causes desensitization.

possible that protein levels or expression of β-arr2 may not change but mRNA is found in laminae IV–VI in the spinal cord [45], and in a variety
recruitment of β-arr2 may be altered by ETA receptor antagonist. There- of brain regions associated with pain signaling, including the hippocam-
fore, the next step is to study β-arr2 recruitment in the brain and in neu- pus, striatum, amygdala, hypothalamus, raphe nucleus, locus coeruleus,
roblastoma SH-SY5Y cells in the presence of ETA receptor antagonists to cerebral cortex, pontine tegmentum, and lateral reticular formation
determine whether these antagonists block β-arr2 recruitment and [44,78]. ET A receptors are also found in the hypothalamus, locus
allow biased signaling to proceed at Gi G proteins. coeruleus, pontine tegmentum, reticular formation, and substantia
ET is produced by endothelial cells in the CNS and non-endothelial nigra [76,143], and the periaqueductal gray (PAG) area [35]. Distribution
cells such as neurons, astrocytes, and glial cells [53,54,85]. The wide- of μ-OR in the PAG neurons [113], locus coeruleus neurons [63], DRG [10,
spread distribution of binding sites for ET in the CNS is indicative of 111,136], spinal cord [46], and other brain regions [55,70,100] suggest
the role of ET as a regulatory neuropeptide [52,54,85,128]. ETA receptors that μ-ORs and ETA receptors may be co-localized and may even be co-
are expressed on the peripheral endings of nociceptors, on nerve axons, expressed in the same group(s) of neurons. The mechanistic interaction
on nociceptor cell bodies located in the dorsal root ganglion (DRG) of these receptors can be further understood by co-localization/
[101], and on small, medium, and large-sized nociceptors [32]. ET-1 co-immunoprecipitation studies, bioluminescence resonance energy

Fig. 3. Proposed mechanism of functional interactions between ETA and μ-opioid receptors (μ-ORs) in analgesia: biased signaling. ETA receptor antagonism induces biased signaling in the
morphine occupied μ-OR causing diminished β-arr recruitment and signaling and enhanced G protein signaling. The result is an improved coupling with the G protein, an increased anti-
nociceptive effect and decreased desensitization/tolerance.

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transfer (BRET) and fluorescence resonance energy transfer (FRET) morphine and oxycodone withdrawal [16]. Although receptor expres-
studies in neuronal membranes and cell lines. sion is unaffected, it is possible that blockade of ETA receptors by a selec-
tive antagonist enables endogenous ET-1 to act on ETB receptors, and
8. Neuroadaptation and opioid withdrawal that ETB receptor activation may play a role in attenuation of withdraw-
al symptoms.
In recent studies, there is evidence of neurogenesis during opioid While the above findings indicate that opioid-induced neurogenesis
withdrawal. Serum levels of nerve growth factor (NGF) were increased may not directly involve ETB receptors, however NGF and other mecha-
in patients suffering from heroin withdrawal, while vascular endothelial nisms may be involved. Neuroadaptive processes and changes in the
growth factor (VEGF) levels were not altered [58]. The NGF family of NGF signaling system could likely be causing a decrease in expression
neurotrophins include NGF, brain derived neurotrophic factor (BDNF), of brain NGF during oxycodone withdrawal. It is known that chronic ex-
neurotrophin-3 (NT3) and neurotrophin-4 (NT4) [2]. Accumulating ev- posure to drugs of abuse produces adaptive changes in various neuro-
idence suggests that the NGF family of neurotrophins have important trophic factors in the ventral tegmental neurons and other regions of
modulatory roles in opioid analgesia and addiction [92]. BDNF knock- the brain [3,24]. In particular, expression of neurotrophic factors is
out mice demonstrated reduction in the intensity of morphine with- significantly affected during drug withdrawal [26,121,134], suggesting
drawal symptoms [3]. Intrathecal administration of NGF restored the a direct connection between the aversive state of withdrawal and
effectiveness of morphine in attenuating pain hypersensitivity follow- neuronal plasticity [134]. Therefore, it is likely that during oxycodone
ing peripheral nerve injury [27]. Moreover, marked attenuation of mor- withdrawal, there is a decrease in NGF levels due to the switch from in-
phine tolerance in NT4 deficient transgenic mice was observed [122], hibitory to excitatory neuronal response resulting from chronic drug ex-
indicating that neurotrophin activity is involved in opioid tolerance posure. However, the fact that NGF changes are not seen in morphine
and dependence. withdrawal indicates that NGF may not be the sole mechanism involved
Due to the fact that neuroadaptations and neurorestorative events in opioid withdrawal symptoms, and it is possible that ETB receptors
occur during insults to the brain and attempt to repair and reduce may not be involved in the early stages of morphine withdrawal. More-
damage, it is of value to determine VEGF and NGF expression in opioid over, it is possible that changes in brain NGF and VEGF take place at dif-
withdrawal. Endogenous opioids such as Met-enkephalin decrease an- ferent time intervals. Therefore, in future studies, it would be interesting
giogenesis and both the number of blood vessels and the total length to see if VEGF and NGF expression are altered at later time points after
of vessels are significantly decreased [19] indicating the involvement morphine and oxycodone withdrawal.
of VEGF. Further, NGF plays a functional role in reparative neovascular- There are reports suggesting that chronic administration of mor-
ization through a VEGF-mediated mechanism [28]. Recent studies on phine involves 5-HT1 receptors [50]. It is known that hypothalamic
brain expression of VEGF in mice undergoing morphine and oxycodone 5-HT1A receptors are downregulated in morphine abstinence and
withdrawal showed that VEGF levels did not change with or without the withdrawal [51], and upregulated in the cerebral cortex [50]. Anti-
administration of BQ123 [16]. Further, BQ123 did not produce any depressants, specifically selective serotonin reuptake inhibitors (SSRIs)
change in the brain expression of NGF during morphine withdrawal. have been shown to induce neurogenesis [4]. The role of neurotrophins
However, in mice undergoing oxycodone withdrawal, NGF expression has been discussed in the pathology of depression, with BDNF being a
significantly decreased in the brain. A similar decrease in NGF expres- critical modulator. Both signaling systems, serotonin and BDNF, are in-
sion was also observed with BQ123 pretreatment in mice undergoing volved in regulating neural circuitries and anti-depressant action, and
oxycodone withdrawal [16]. also co-regulate each other [42,90]. A decrease in BDNF signaling is
It is known that neurogenesis continues to take place throughout the linked to neurodegeneration and changes related to chronic stress,
lifespan of human beings, and new neuronal formation occurs in the while re-establishing BDNF levels may be responsible for therapeutic
adult brain as well [41,125]. ETB receptors are a necessary component responses to anti-depressants [39]. Since neurogenesis is an important
of the developing nervous system [80,103]. Neuroadaptations occur part of opioid tolerance and withdrawal, and 5-HT receptors have
during traumatic brain events and there is evidence that ET receptors been shown to play a role in adult neurogenesis and in morphine toler-
may be involved in providing neuroprotection during these processes ance and withdrawal, it could be possible that opioids may act via 5-HT1
[41,125]. In the adult brain, there are endogenous mechanisms which receptors to regulate neurogenesis.
attempt to repair damage resulting from insults to the brain, and this In summary, neurogenesis plays an important role in the develop-
neurorestorative process involves angiogenesis, neurogenesis, and ment of opioid tolerance as well as withdrawal. Further, due to the
oligodendrogenesis [48], possibly mediated by ETB receptors in the critical role of ETB receptors in neurogenesis, ETB receptor agonists
CNS. Since opioid withdrawal is a CNS phenomenon that involves vari- may possibly be used as a strategy to manage tolerance.
ous brain regions, we speculate that neuroadaptations and regeneration
of neurons may occur during withdrawal. Stimulation of central ETB re- 9. Future prospects: challenges and perspectives
ceptors play an important role in providing neuroprotection and lead to
neurovascular remodeling, thereby enhancing the neurorestorative A deeper understanding of the basic mechanisms underlying opioid
processes inherent in the adult brain. Our group has found that this withdrawal and relapse can assist in identifying novel targets for pre-
neurovascular remodeling occurs via ETB receptor agonist, IRL-1620 in- vention and treatment. Especially given the concern about the rising
duced increase in the expression of VEGF and NGF in the brain at differ- misuse of opioid analgesics and subsequent withdrawal, there is a crit-
ent time points during traumatic brain events. These findings reaffirm ical need to develop effective alternatives to the current treatments for
the important role of ETB receptors in the vascular and neuronal devel- opioid dependence and withdrawal. There is increasing evidence that
opment of the CNS [79,80]. Therefore, it will be of immense value to biased agonism or antagonism and ligand-specific pathway modulation
explore the role of ETB receptors in long-term opioid tolerance and may have important therapeutic potential in opioid analgesia, tolerance,
withdrawal. and withdrawal [140,141,146]. Therefore, studies on β-arr2 recruitment
Our research group has established that ETA receptors are involved in the brain and in neuroblastoma SH-SY5Y cells in the presence of ETA
in opioid analgesia, tolerance, and withdrawal. ETA receptor antagonists receptor antagonists and opioids will help determine whether these
restore analgesic activity of opioids after chronic exposure. ETA receptor antagonists block β-arr2 recruitment and allow biased signaling to pro-
antagonist BQ123 attenuated key withdrawal symptoms of morphine ceed at Gi G proteins. Further, the mechanistic interaction of ETA recep-
and oxycodone. Our initial studies using an ETB receptor agonist showed tor antagonists and μ-ORs can be better understood by co-localization/
that ETB receptors may not be useful in acute opioid analgesia [14]. Fur- co-immunoprecipitation and BRET/FRET studies in neuronal mem-
ther, we found that ETB receptor expression is unchanged during branes and cell lines.

Please cite this article as: S. Bhalla, et al., Neurobiology of opioid withdrawal: Role of the endothelin system, Life Sci (2016), http://dx.doi.org/
10.1016/j.lfs.2016.01.016
 S. Bhalla et al. / Life Sciences xxx (2016) xxx–xxx 7

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Please cite this article as: S. Bhalla, et al., Neurobiology of opioid withdrawal: Role of the endothelin system, Life Sci (2016), http://dx.doi.org/
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