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Neuromedin N
Chapter 117
Neurotensin/Neuromedin N Paul R. Dobner and Robert E. Carraway
ABSTRACT Neurotensin (NT) and neuromedin N, two bioactive peptides of brain and gut origin, are processed from a common precursor by prohormone convertases (PCs). Differential processing occurs in some tissues most probably resulting from the tissue-specific expression of distinct PCs, yielding large, N-terminally extended forms of the peptides. Two G protein-coupled receptors show preference for NT but bind both the peptides. NTR-1 signaling elevates intracellular [Ca2+] and can also increase cGMP, cAMP, and arachidonic acid metabolites. NTR-1/sortilin (NTR-3) heterodimers seem to modulate NT signaling and trafficking of internalized receptors, but NT may also signal through sortilin. Intimately associated with dopaminergic systems, NT is most probably an endogenous antipsychotic and has also been implicated in the modulation of psychostimulant responses, pain perception, stress-induced analgesia, appetite control, stress responses, and reproductive functions. The development of NT agonists may provide new pharmaceuticals for the treatment of schizophrenia, drug addiction, and stressrelated neuropathic pain syndromes.
DISCOVERY OF NEUROTENSIN In 1967, Susan Leeman discovered that the extracts of bovine hypothalami induce vasodilation and anaphylactic shock in rats. Using this assay, her laboratory isolated, sequenced, and synthesized the active substance, a 13-residue peptide that they named neurotensin (NT).6 NT is broadly expressed in the limbic nervous system and intestinal endocrine cells and is also found in various glands and in peripheral nerves. NT is a member of a family of peptides that are strikingly similar in their C-terminal regions (Table 1). These peptides share C-terminal residues with NT that have been shown to be critical for NT function and display many of the same pharmacological properties for those that have been tested. For instance, the venom of the cone snail contains the NT-like peptide contulakin G, which is a potent analgesic in rats and dogs.2 Several NT-related peptides are also present in neuronal cells of the protocordate Ciona intestinalis (Table 1). NT most probably has multiple neuronal and endocrine signaling functions, including the modulation of dopamine Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00117-2 Copyright © 2013 Elsevier Inc. All rights reserved.
(DA) signaling, stress-induced analgesia, energy balance, stress, and reproductive functions. This review focuses on the biosynthesis, distribution, receptor signaling, and potential functions of NT in the CNS.
STRUCTURE OF THE NEUROTENSIN/ NEUROMEDIN N PRECURSOR mRNA/GENE The amino acid sequence of the NT precursor was first inferred from canine cDNA sequences and is highly conserved in vertebrates.8 The predicted prepro form of the precursor ranges from 165 to170 amino acids in length, with NT and the related hexapeptide neuromedin N (NMN) lying in tandem near the carboxyl terminus of the precursor, bounded and separated by Lys-Arg processing sites (Fig. 1). The analysis of the rat, mouse, and human NT/NMN genes reveal that the coding region is spread over four exons, separated by three introns spanning just over 10 kb.20 The gene is transcribed to yield two different transcripts (1.0 and 1.5 kb) that differ only in the extent of the 3’-untranslated region, most probably arising from the differential use of alternative polyadenylation signals. The two mRNAs are expressed in about equal amounts in the brain, but the 1.0 kb mRNA greatly predominates in the gastrointestinal tract. The sequences immediately upstream of the transcriptional start site contain a series of response elements (AP-1, CRE, and GRE) that are important for NT/ NMN gene induction in response to environmental stimuli in PC12 cells. A variety of stimuli regulate NT/NMN gene expression in a brain region-specific manner.
NT/NMN mRNA EXPRESSION IN BRAIN NT/NMN mRNA and its peptide products are expressed in many forebrain regions, with higher levels in limbic regions, including hippocampus and subicullum, VP, BNST, medial Acb, and Cput (Fig. 2).1 NT/NMN mRNA-positive neurons are located mainly in the dorsomedial and ventral aspects of the Cput rostrally but are more evenly distributed in the caudal regions. A continuum of positive cells was observed 875
CHAPTER | 117 Neurotensin/Neuromedin N
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TABLE 1 Peptides of the Neurotensin Family Peptide
Source
Sequence
NT
Cow, dog, human
NT
Chicken
NT
Green frog
NMN
Cow, dog, pig, human
Lys-Ile-Pro-Tyr-Ile-Leu*
LANT-6
Chicken
Lys-Asn-Pro-Tyr-Ile-Leu*
XP
Xenopus
XP
Dog, turkey
Phe-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu
Contulakin G
Cone snail
Ci-NTLP-1
Ciona intestinalis
Ci-NTLP-3
Ciona intestinalis
Met-Met-Leu-Gly-Pro-Gly-Ile-Leu*
NT, neurotensin; NMN, neuromedin N; LANT-6, [Lys8, Asn9]-NT 8-13; XP, xenopsin; NTLP, neurotensin-like peptide. *Bordered by double basic cleavage sites in putative precursors. †N-terminal double basic cleavage site only. ‡The side chain is O-glycosylated.
stretching from the S through the preoptic area, including the lateral S, DBB, lateral BNST, and lateral Hpt. Steroid hormones and neurotransmitters have been implicated in the regulation of NT/NMN mRNA expression in several regions of the brain, including the medial preoptic area, Arc, PVN, periventricular nucleus, amygdala, Cput, and Acb. In the brainstem, NT/NMN mRNA-positive neurons are most abundant in the PAG and DR and can also be found in adjacent lateral structures (cuneiform nucleus, microcellular tegmental nucleus).
PROCESSING OF THE PRECURSOR The NT precursor is differentially processed by distinct prohormone convertases (PCs) in the brain and gastrointestinal tract (Fig. 1, reviewed in Ref. 22). PCs are described in detail in the Peptide Biosynthesis/Processing section of this book. In the brain, PC2 results in nearly complete processing of both the peptides, although brain NT levels are only decreased by ~15% in PC2 knockout mice. This is most probably because the precursor can also be processed by PC1 and PC5A, which are colocalized with NT and are capable of generating NT from the precursor. After liberation of the NT sequence from the precursor, the N-terminal Gln residue is converted into pGlu most probably through the action of glutaminyl cyclase.
In the gastrointestinal tract, PC1 inefficiently cleaves the LysArg pair amino terminal to NMN, resulting in the accumulation of amino-terminally extended NMN (large NMN in Fig. 1) that stretches from the signal peptide cleavage site to the carboxyl terminus of NMN (amino acids 23–147 in the rat sequence) and NT in gut endocrine cells. Similarly, colon cancer cell lines expressing both NT and PC5A produce predominantly amino-terminally extended forms of both NMN and NT (large NT in Fig. 1), similar to those found in the adrenal medulla, indicating that differential expression of distinct PCs can account for the observed tissue-specific processing of the NT precursor. The secretion of amino-terminally extended forms of NMN and NT may produce longer lasting biological effects because of their enhanced stability in the bloodstream compared with that of the fully processed peptides. A dibasic amino acid pair near the central region of the precursor is inefficiently processed in the brain and not used in the gastrointestinal tract, and complete processing of the NT precursor results in the production of a relatively stable fragment (Fig. 1, amino acids 24–140 of the human NT precursor) that extends from the signal peptide cleavage site through to the amino acid preceding the Lys-Arg amino terminal processing site of NMN.11 This precursor fragment is detectable in serum from healthy individuals, is stable in both human plasma and serum, and is released into the serum after ingestion of an ordinary meal, opening the possibility that it may serve as a surrogate marker for NT secretion.
RECEPTORS AND SIGNALING CASCADES Four NT receptors have been identified, two that are 7-transmembrane G protein-coupled receptors (NTR-1, NTR-2) and two primarily intracellular proteins related to yeast sorting receptors (NTR-3/sortilin, NTR-4/SorLA). NTR-1 displays the highest affinity (Kd, 0.1 nM) and seems to mediate NT effects on dopaminergic transmission, hotplate latency, temperature regulation, and smooth muscle contractility (reviewed in Ref. 21). First described as a lower affinity receptor (Kd, 3 nM) with a differing specificity, NTR-2 has been implicated in the analgesic effects of NT (reviewed in Ref. 29). Although the role of NTR-3 is less clear, one possibility is that through binding and internalizing NT this receptor serves to regulate turnover of the peptide. Evidence supports the involvement of NTR-3 in the growth-promoting effects of NT and in NT-induced migration of brain microglial cells. Signal transduction studies in transfected cell systems and cancer cell lines indicate that NTR-1 is primarily coupled to Gαq and signals via the activation of phospholipase C and the generation of IP3, causing an elevation in intracellular [Ca2+] (reviewed in Ref. 21). Concomitant generation of DAG leads to the activation of PKC in some cells, stimulating MAP kinases and PI3 kinase via complex mechanisms that can involve EGF receptor transactivation. This
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877
PC2 PC2 PC1 PC1 PC5A PC5A
Signal sequence
Lys/Arg Arg
Lys Arg Lys Arg
PC2 PC1 PC5A Lys Arg
NT/NMN precursor Large NT Large NMN NT NMN Stable fragment FIGURE 1 Schematic diagram of the NT/NMN precursor and its major processing products. The full length precursor is 165–170 amino acids long depending on the species and comprises 169 amino acids in the rat. The positions of the signal peptide (stippled fill, aa 1–23) and the NMN (gray fill) and NT (black fill) coding domains are indicated. The NMN and NT coding domains are bounded and separated by the indicated Lys-Arg processing sites. An additional Lys-Arg pair is located near the center of the precursor, but the available evidence suggests that this is not a major processing site. The prohormone convertases that have been shown to cleave the Lys-Arg processing sites are indicated and the extent to which they cleave a particular site is indicated by the size of the type, with the most efficient cleavage indicated by larger letters and the least efficient by smaller letters. PC1 cleaves the Lys-Arg pair located at the amino terminal end of NMN inefficiently but cleaves the sites surrounding NT efficiently resulting in the production of NT and Large NMN (intestinal pattern). PC5A cleaves the site at the carboxy-terminal end of NT efficiently, but the upstream sites less efficiently leading to the production of Large NT and Large NMN (adrenal pattern). PC2 cleaves all three sites efficiently resulting primarily in the production of NT and NMN (brain pattern).
signaling can have a feed forward component, in that NTinduced MAP kinase activation leads to the upregulation of EGF receptor. In neuroblastoma cells and rat brain slices, NT can elevate cGMP formation, inhibit cAMP synthesis and stimulate PI turnover, and evidence suggests that the third intracellular loop of NTR-1 is required for interactions with Gq, while the carboxy-terminal tail interacts with Gi/o and Gs (reviewed in Ref. 21). Liposomal ceramide has been shown to decrease the interaction of NTR-1 with Gq within structured membrane microdomains, which inhibits the growth stimulatory effects of NT in MDA-MB-231 breast cancer cells. NTR-1 is inducibly palmitoylated on Cys381 and Cys383 (rat sequence), and inhibition of this process by site-directed mutagenesis and pharmacological strategies diminishes NT-induced ERK phosphorylation and reduces the ability of NT to protect against apoptosis in breast cancer cells.17 NT activation of DA neurons appears to involve cAMP signaling, and NT potentiates cAMP formation (via NTR-1) in response to Gs-agonists by facilitating the activation of Ca2+-dependent adenylyl cyclases. Perhaps secondary to its effects on [Ca2+], NT also activates PLA2 and DAG lipase, stimulating arachidonic acid release and the formation of bioactive metabolites via the cyclooxygenase and lipoxygenase pathways. This signaling could also be amplified by feed forward mechanisms because the
expression of PLA2 is upregulated by the activation of MAP kinase (reviewed in Ref. 7). NT is the most potent substance known to stimulate leukotriene formation in rats, and the possible relevance of this to its central effects is not well investigated. Signaling mechanisms for NTR-2 and NTR-3 are not yet clear. NTR-2 constitutively activates IP production, and this process is stimulated by the NTR-1 antagonist SR48692 but suppressed by NT (reviewed in Ref. 29). NTR-3 is primarily localized to ER-Golgi membranes but ≅10% is on the cell surface. NTR-3 can form heterodimers with NTR1, decreasing the potency of NT to stimulate IP formation and to activate MAP kinase. Thus, NTR-3, which cannot promote either response, modulates these effects of NTR-1. Results obtained by site-directed mutagenesis favor a model for NTR-1 activation in which binding of NT to key residues in EC loop 3 and TM 6 and 7 leads to a shift in IC loop 3 that enhances binding to Gq (reviewed in Ref. 21). The relative instability of NTR-1 in detergents has led investigators to use a systematic mutational approach to identify receptor mutants that display enhanced thermostability, resulting in the identification of an NTR-1 variant that is stabilized in an agonist binding conformation that presumably might be used with a wider range of detergent and buffer conditions for crystallization.35
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Cx
Hi CC
OB
CPut
S
Th PAG C BST
DR
PBN
Hpt PVN LC
Acb DMH
SN NST
Arc
SON
VMH
DBB
Amb ME Amy
NL IL
LRN
AP
FIGURE 2 Schematic diagram of the distribution of NT-IR neurons in the rat brain. ( ) NT-IR neurons detected after colchicine treatment. ( ) NT-IR neurons induced by stressful stimuli that are also detected after colchicine treatment. ( ) NT-IR neurons induced by either psychostimulants (dorsomedial CPu, Acc) or dopamine D2 antagonists, including typical antipsychotic drugs (dorsolateral CPu, Acc). ( ) Estrogen-inducible NT-IR neurons in the female MPO and arcuate nucleus. NT gene expression in the rostal MPO nucleus is sexually dimorphic. Quantitative analysis of NT content by RIA (pmole/g wet weight tissue) provides a more comprehensive picture of the distribution of the peptide in various brain regions (OB, 3; Cx, 4-5; Acc, 29; CPut, 10; S, 73; BST, 172; Hi, 7; Th, 5-23; Hyp, 76-128; ME, 128; SN, 13; PAG, 42; LC, 54).
CONFORMATION 2D solid-state NMR spectroscopy has been used to study the conformational changes of NT8-13 on interaction with an N-terminally truncated form of rat NTR-1.27 On binding to the receptor, the peptide rearranges to adopt a β-strand conformation, characterized by a ψ(Pro10) dihedral angle of 146 ± 15°. The NMR spectrum of NT (8–13) in solution is also consistent with the presence of a type II β-turn, whereas the spectrum of a homolog that displays 40-fold less affinity for human NTR-1 does not.34 Furthermore, a lactam-bridged analog of NT8-13 with ψ(Pro10) angles restricted to an angle (130°) close to the experimentally determined one exhibits ~1000-fold higher affinity for porcine NTR-1 than the stereoisomer (−130°), providing further evidence for the NMR-derived bioactive conformation.
BRAIN FUNCTIONS Close Alliance between NT and Dopamine NT has been broadly implicated in the modulation of DA signaling and can produce both psychostimulant- and antipsychotic drug (APD)-like effects after microinjection into different regions of the brain (reviewed in Ref. 5). Most midbrain DA neurons express high levels of NTR-1, and NT can stimulate these neurons to increase striatal DA release in the Acb. NT can also act presynaptically to potentiate DA
release in both the Acb and Cput, most probably by antagonizing DA D2 receptor autoinhibition. Consistent with these observations, NT microinjection in the VTA stimulates locomotor activity and repeated administration results in sensitization to not only NT but also psychostimulant drugs. Prolonged administration with the NT antagonist SR48692 lowers basal DA release in the Acc, consistent with the hypothesis that endogenous NT acts to augment DA release through NTR-1; however, basal and stress-induced DA release in the mPFC are unaffected. NTR-1, but not NTR-2, knockout mice are reported to be hyperdopaminergic with elevated basal DA release in the Cput, although release in the Acc and mPFC was not examined.26 These differences could reflect regionally distinct roles for NT in the control of DA release, but could also result from either adaptive changes in NTR-1 knockout mice or unanticipated effects of prolonged NT antagonist administration. NT can also produce neuroleptic-like effects when administered in the Acc, seemingly inconsistent with the evidence cited above indicating that NT acts presynaptically to augment DA signaling. The most probable explanation for this is that NT either acts postsynaptically or on other presynaptic afferents to oppose DA signaling and electron microscopic immunohistochemical analysis indicates that NTR-1 is expressed on both dopaminergic and excitatory afferents- and on postsynaptic neurons in the Acc; however, NTR-1 seems to be expressed primarily on striatal afferents
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in the Cput. NTR-2 knockout mice have lower levels of basal striatal glutamate release and both NTR-1 and NTR-2 knockout mice display muted amphetamine-stimulated striatal glutamate release,25 consistent with the hypothesis that endogenous NT modulates striatal glutamate release.
NT may be an Endogenous Antipsychotic Considerable evidence suggests that NT mediates at least a subset of responses to certain APDs. APDs elevate striatal NT expression and NT administration in the Acb suppresses amphetamine-stimulated locomotor activity, similar to APDs, consistent with the idea that increases in NT signaling might be involved in APD actions (reviewed in Ref. 5). Although animal models of schizophrenia are necessarily limited, latent inhibition (LI) and prepulse inhibition (PPI) of the acoustic startle response measure aspects of sensorimotor gating that can be influenced by APDs. Enhanced DA transmission and isolation rearing disrupt PPI and APDs can restore PPI to control levels. In mice, APDs can also increase PPI above basal levels without prior disruption by DA agonists. There is increasing evidence that either pharmacological or genetic disruption of NT signaling can have a profound impact on APD responses in these animal models. NT has been implicated in both the acquisition of LI and APD enhancement of LI. NT antagonist administration was shown to interfere with the acquisition of LI, and this was observed with both the relatively selective NTR-1 antagonist SR48692 and the NTR-1/NTR-2 antagonist SR142948A, indicating that NT signaling through NTR-1 may be particularly important (reviewed in Ref. 5). The suppressive effects of SR142948A are ameliorated by coadministration of the D2 antagonist sulpiride, suggesting that NT antagonist treatment may increase DA D2 transmission in the Acb or other key brain regions involved in LI. NT also seems to play a role in APD enhancement of LI, because SR142948A administration 1 hour before the last of 7 daily injections of haloperidol prevents augmentation of LI. Thus, NT likely plays a role in both basal and APDenhanced LI, possibly through effects on DA D2 signaling. NT also appears to play a role in both basal and APDenhanced PPI; however, results from NTR-1 and NTR-2 knockout mice suggest a more complicated picture. Preadministration of the NTR-1/NTR-2 antagonist SR142948A blocks the ability of both the typical APD haloperidol and the atypical APD quetiapine to restore PPI deficits in isolation-reared rats, without affecting basal PPI (reviewed in Ref. 5). Furthermore, NT mRNA levels are decreased by ~45% and NT receptor binding is increased by ~21% in the Acb shell in isolation-reared animals, and NT mRNA levels are restored to those observed in socially reared animals 4 hours after quetiapine treatment. Similarly, both haloperidol and quetiapine fail to augment PPI in NT knockout mice, which also have reduced basal PPI, although the response to
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clozapine is unaffected.19 The decrease in basal PPI is modest and is not observed in either NTR-1 or NTR-2 knockout mice where it is instead elevated.13,31 The indirect DA agonist amphetamine disrupts PPI, but this effect is blocked in NT knockout mice,19 suggesting that NT may play a crucial role in this process. Similar results have been reported for NTR-1 knockout mice;31 however, another report indicates that amphetamine disruption of PPI is normal in these mice,13 as it is in NTR-2 knockout mice.31 These different results most likely reflect methodological differences, because both used NTR-1 knockout mice from the same source. These results provide a preliminary indication that NT signaling is required for amphetamine disruption of PPI in mice. NT participation in processes that either enhance or reduce PPI most likely reflects compartmental specificity in NT action within the circuits that modulate PPI, particularly the DA circuits. The analysis of amphetamine and haloperidol induction of striatal Fos expression has provided direct evidence for such compartmentalized NT signaling.9,12
NT and Psychostimulant Sensitization As discussed above, NT is capable of modulating DA transmission at different levels. In addition to antipsychotic drug-like effects, NT produces psychostimulant-like effects after direct administration into the VTA and increases DA release in the Acb (reviewed in Ref. 5). Several different laboratories have reported that NT antagonist pretreatment blocks or attenuates amphetamine and can delay cocaine sensitization while sparing the acute locomotor stimulation produced by these drugs. By contrast, both amphetamine (PRD, unpublished observations) and cocaine16 sensitization are augmented somewhat in NT knockout mice. Several other lines of evidence also suggest that NT acts to limit psychostimulant sensitization, including the observations that peripheral administration of the NT agonist, NT69L, suppresses nicotine sensitization, and that NT antagonist administration potentiates rather than blocks supersensitive locomotor responses to L-DOPA in dopamine-deficient mice. NTR-1 knockout mice show both increased basal and amphetamine-stimulated locomotor activity and striatal DA release, also consistent with the idea that NT acts to limit DA signaling, although psychostimulant sensitization was not examined.26 However, both basal and cocainestimulated locomotor activities are similar to wildtype in NT knockout mice16 and in rats treated with SR48692 (reviewed in Ref. 5). Thus, gene knockout and several other approaches favor a model in which NT acts mainly to limit psychostimulant sensitization, while experiments with NT antagonists indicate the opposite. Further work will be required to determine whether this discrepancy results from compensatory mechanisms in knockout animals or unanticipated antagonist effects, for instance agonist or partial agonist activity at NTR-2.
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NT and Pain Given centrally, NT enhances nociceptive responses at lower doses, but produces a potent analgesic response at higher doses that in most studies does not require µ-opioid receptor signaling (reviewed in Ref. 10). NT and NT receptors are expressed in the central descending circuitry involved in pain modulation and in the spinal cord, consistent with a possible role in pain modulation. The evidence to date indicates that endogenous NT plays a relatively minor role in basal nociception but is critical for at least certain aspects of stress-induced antinociception (SIAN). Remarkably, SIAN is totally absent in NT knockout mice, where water avoidance/cold-water swim stress instead results in a hyperalgesic response to colonic distension.15 Similar results were obtained in rats pretreated with SR48692, indicating that SIAN probably requires NT signaling through NTR-1, and the defects observed in NT knockout mice are not because of developmental or compensatory changes resulting from the lifelong absence of NT signaling.15 NTR-2 signaling has also been implicated in certain aspects of SIAN. NTR-2 knockout mice display a selective defect in SIAN after cold-water swim stress during the latter part of the second phase of the pain response in both the intraplantar formalin test and the paw withdrawal response to thermal stimuli, while nonstressed responses are comparable with wildtype.23 Similar results occur in the paw withdrawal test after repeated administration of the NTR-1/NTR-2 antagonist SR142948A, indicating that the differences observed in NTR-2 knockout mice also do not result from developmental defects or compensatory changes resulting from the absence of NTR-2. Furthermore, intrathecal administration of both nonselective and NTR-2-selective NT agonists potentiate cold-water swim stress-induced SIAN in the paw withdrawal test. These results are consistent with the previous observations that NTR-2-selective agonists suppress pain responding in the intraplantar formalin test selectively during the latter part of the second phase of the pain response, influencing predominantly paw lifting while sparing paw licking, biting, and shaking behaviors. These results suggest that endogenous NT signaling through both NTR-1 and NTR-2 is particularly important for SIAN. In contrast to SIAN, NT seems to play a relatively minor role in basal pain perception, where it seems to facilitate pain responses. Although both NTR-1 and NTR-2 knockout mice respond similarly to wild type in a variety of pain tests, NTR-2 knockout mice display longer jump latencies in the hot-plate test.28 Viseromotor responses to colorectal distension are also diminished in both NT knockout mice and rats pretreated with SR48692.15 The evidence discussed above suggests that NT not only facilitates basal pain responses but it also plays a key role in SIAN and any theory of NT modulation of pain
CHAPTER | 117 Neurotensin/Neuromedin N
responses must take into account these seemingly contradictory results. Perhaps the simplest explanation is that the direction of pain modulation depends on the intensity of NT signaling, with basal NT signaling facilitating nociception and increased NT signaling being analgesic. The observation that cold-water swim stress increases NT mRNA levels in certain Hpt regions that project to the PAG is consistent with the idea that stress-induced increases in NT signaling to the PAG may underlie SIAN. Increased NT signaling could lead to the selective recruitment of neurons involved in analgesic responses, such as PAG “off” cells.
Appetite Control NT has been implicated as a possible satiety factor and mediator of the central suppressive effects of leptin on food consumption (reviewed in Ref. 3). Leptin administration suppresses food consumption and weight gain, possibly through the decreased expression of orexigenic peptides and increased NT expression in the hypothalamus. Similarly, NT expression is decreased in specific Hpt regions in response to a high-fat diet that results in obesity. Combined central administration of NT and leptin potentiates both the short-term inhibitory effects of NT on spontaneous food intake and early, but not late (24 h) leptin effects. Because the leptin effect is blocked by pretreatment with either NTR-1 antagonist or NT antiserum, it has been suggested that NT mediates the central effect of leptin. Collectively, these results indicate that dynamic regulation of NT in specific Hpt nuclei in response to alterations in the levels of circulating leptin and perhaps other factors is involved in appetite suppression and body weight balance. Other results, showing that the anoretic effect of NT is antagonized by the H1 receptor blocker pyrilamine and in H1 knockout mice, suggest that the histamine H1 system, which is known to inhibit food intake, could be a final common mediator for the effects of both leptin and NT.30
NT and Stress Responses Stress increases NT expression in several Hpt nuclei, suggesting that NT may modulate stress responses (reviewed in Ref. 14). Centrally administered NT was found to rapidly increase the levels of circulating ACTH and corticosterone and these increases were attenuated by corticotrophinreleasing hormone (CRH) antagonists and bilateral lesions of the PVN. Furthermore, SR 48692 administration in the PVN suppresses stress-induced increases in the plasma levels of ACTH and corticosterone and decreases CRH expression in the PVN. Both basal and stress-induced blood corticosterone levels are lower in NTR-2 knockout compared with those of control mice, consistent with NT playing a role in the control of corticosterone release.23
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NT and Reproductive Functions NT has been implicated in the preovulatory luteinizing hormone (LH) surge and the Hpt control of prolactin (PRL) release in rat (reviewed in Ref. 33). Estrogen increases NT mRNA and peptide expression in several Hpt regions that have been implicated in reproductive functions, and NT expression in these regions is sexually dimorphic. In addition, NT mRNA levels vary throughout the estrus cycle in female rats with NT mRNA-positive cells being ~3-fold more numerous at proestrus than at diestrus. These results coupled with microinjection data indicating that NT can augment LH release have led to speculation that NT might mediate the feedforward effects of estrogen on gonadotropin-releasing hormone (GnRH) release from rostral medial preoptic nucleus GnRH neurons expressing NTR-1 in the rat. However, this system may not operate in the mouse, because even though estrogen increases NT mRNA expression similarly in ovariectomized mice, icv NT administration does not augment LH release in ovariectomized mice treated with estrogen.24 GnRH neurons are clearly subject to complex peptide control with kisspeptin playing a crucial role, and it remains to be determined how NT interacts with kisspeptin to modulate reproductive function. Kisspeptin neurons apparently express NTR-2 but not NTR-1 or NT.24 NT has also been implicated in both positive and negative regulation of PRL secretion from pituitary lactotropes. The evidence suggests that NT directly activates tuberoinfundibular DA (TIDA) neurons and mediates feedback inhibition of PRL release through the activation of these neurons.18 Estrogen increases NT expression in TIDA neurons and NT is intensely expressed in virtually all TIDA neurons in nursing rats, but decreases markedly after removal of pups. Increased NT release from TIDA neurons in the median eminence would most probably counteract the inhibitory effects of DA on PRL secretion, because NT stimulates pituitary PRL release. NT knockout mice reproduce and successfully rear their young, indicating that NT is not essential for reproductive functions.9
PATHOPHYSIOLOGICAL IMPLICATIONS There is now considerable evidence from animal models that endogenous NT signaling is both required for certain APD actions and that decreased NT signaling may underlie deficits in PPI. Comparison of NT levels in cerebrospinal fluid (CSF) from schizophrenics both before and after APD treatment indicates that low CSF NT levels are associated with more severe psychopathology and that clinical improvement is associated with increased CSF NT (reviewed in Ref. 5). Some NT agonists produce effects similar to those of atypical APDs and the relatively stable NT analog, NT69L, prevents drug-induced disruption of PPI in rats after either acute or chronic administration.4 These results suggest that
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the development of NT agonists could provide an important new class of APDs. In the shorter term, there could be some therapeutic benefit associated with the use of NT-dependent (e.g. quetiapine, haloperidol) and NT-independent (e.g. clozapine) APDs in combination. NT also seems to play an important role in stressinduced analgesia, suggesting that NT agonists could be effective in treating certain chronic pain syndromes associated with abnormalities in stress responses. The observation that cold-water swim/water avoidance stress in NT knockout mice results in hyperalgesia rather than analgesia is particularly interesting in this regard.15 Stable NT agonists may prove to be particularly effective in treating stress-related and neuropathic pain.32
REFERENCES 1. Alexander MJ, Miller MA, Dorsa DM, Bullock BP, Melloni Jr RH, Dobner PR, et al. Distribution of neurotensin/neuromedin N mRNA in rat forebrain: unexpected abundance in hippocampus and subiculum. Proc Natl Acad Sci USA 1989;86:5202–6. 2. Allen JW, Hofer K, McCumber D, Wagstaff JD, Layer RT, McCabe RT, et al. An assessment of the antinociceptive efficacy of intrathecal and epidural contulakin-G in rats and dogs. Anesth Analg 2007;104:1505–13. 3. Arora S, Anubhuti. Role of neuropeptides in appetite regulation and obesity—a review. Neuropeptides 2006;40:375–401. 4. Briody S, Boules M, Oliveros A, Fauq I, Richelson E. Chronic NT69L potently prevents drug-induced disruption of prepulse inhibition without causing tolerance. Behav Brain Res 2010;207:118–24. 5. Caceda R, Kinkead B, Nemeroff CB. Neurotensin: role in psychiatric and neurological diseases. Peptides 2006;27:2385–404. 6. Carraway R, Leeman SE. The synthesis of neurotensin. J Biol Chem 1975;250:1912–8. 7. Carraway RE, Plona AM. Involvement of neurotensin in cancer growth: evidence, mechanisms and development of diagnostic tools. Peptides 2006;27:2445–60. 8. Dobner PR, Barber DL, Villa-Komaroff L, McKiernan C. Cloning and sequence analysis of cDNA for the canine neurotensin/neuromedin N precursor. Proc Natl Acad Sci USA 1987;84:3516–20. 9. Dobner PR, Fadel J, Deitemeyer N, Carraway RE, Deutch AY. Neurotensin-deficient mice show altered responses to antipsychotic drugs. Proc Natl Acad Sci USA 2001;98:8048–53. 10. Dobner PR. Neurotensin and pain modulation. Peptides 2006;27: 2405–14. 11. Ernst A, Hellmich S, Bergmann A. Proneurotensin 1–117, a stable neurotensin precursor fragment identified in human circulation. Peptides 2006;27:1787–93. 12. Fadel J, Dobner PR, Deutch AY. Amphetamine-elicited striatal Fos expression is attenuated in neurotensin null mutant mice. Neurosci Lett 2006;402:97–101. 13. Feifel D, Pang Z, Shilling PD, Melendez G, Schreiber R, Button D. Sensorimotor gating in neurotensin-1 receptor null mice. Neuropharmacol 2010;58:173–8. 14. Geisler S, Berod A, Zahm DS, Rostene W. Brain neurotensin, psychostimulants, and stress-emphasis on neuroanatomical substrates. Peptides 2006;27:2364–84.
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15. Gui X, Carraway RE, Dobner PR. Endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats. Neuroscience 2004;126:1023–32. 16. Hall FS, Centeno M, Perona MTGAdair J, Dobner PR, Uhl GR. Effects of neurotensin gene knockout in mice on the behavioral effects of cocaine. Psychopharmacology 2012;219:35–45. 17. Heakal Y, Woll MP, Fox TSeaton K, Levenson R, Kester M. Neurotensin receptor-1 inducible palmitoylation is required for efficient receptor-mediated mitogenic-signaling within structured membrane microdomains. Cancer Biol Ther 2011;12:427–35. 18. Hentschel K, Cheung S, Moore KE, Lookingland KJ. Pharmacological evidence that neurotensin mediates prolactin-induced activation of tuberoinfundibular dopamine neurons. Neuroendocrinology 1998;68:71–6. 19. Kinkead B, Dobner PR, Egnatashvili V, Murrary T, Deitemeyer N, Nemeroff CB. Neurotensin-deficient mice have deficits in prepulse inhibition: restoration by clozapine but not haloperidol, olanzapine or quetiapine. J Pharmacol Exp Ther 2005;315:256–64. 20. Kislauskis E, Bullock B, McNeil S, Dobner PR. The rat gene encoding neurotensin and neuromedin N: structure, tissue-specific expression, and evolution of exon sequences. J Biol Chem 1988;263:4963–8. 21. Kitabgi P. Functional domains of the subtype 1 neurotensin receptor (NTS1). Peptides 2006;27:2461–8. 22. Kitabgi P. Prohormone convertases differentially process pro-neurotensin/ neuromedin N in tissues and cell lines. J Mol Med 2006;84:628–34. 23. Lafrance M, Roussy G, Belleville K, Maeno H, Beaudet N, Wada K, et al. Involvement of NTS2 receptors in stress-induced analgesia. Neuroscience 2010;166:639–52. 24. Lemko HMD, Naderi R, Adjan V, Jennes LH, Navarro VM, Clifton DK, et al. Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse. Am J Physiol Endocrinol Metab 2010;298:E80–8. 25. Li Z, Liang Y, Boules M, Gordillo A, Richelson E. Effect of amphetamine on extracellular concentrations of amino acids in striatum in neurotensin subtype 1 and 2 receptor null mice: a possible interaction between neurotensin receptors and amino acid systems for study of schizophrenia. Neuropharmacology 2010;58:1174–8.
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26. Liang Y, Boules M, Li Z, Williams K, Miura T, Oliveros A, et al. Hyperactivity of the dopaminergic system in NTS1 and NTS2 null mice. Neuropharmacol 2010;58:1199–205. 27. Luca S, White JF, Sohal AK, Filippov DV, van Boom JH, Grisshammer R, et al. The conformation of neurotensin bound to its G protein-coupled receptor. Proc Natl Acad Sci USA 2003;100: 10706–11. 28. Maeno H, Yamada K, Santo-Yamada Y, Aoki K, Sun Y-J, Sato E, et al. Comparison of mice deficient in the high- or low-affinity neurotensin receptors, Ntsr1 or Ntsr2, reveals a novel function for Ntsr2 in thermal nociception. Brain Res 2004;998:122–9. 29. Mazella J, Vincent J-P. Functional roles of the NTS2 and NTS3 receptors. Peptides 2006;27:2469–75. 30. Ohinata K, Shimano T, Yamauchi R, Sakurada S, Yanai K, Yoshikawa M. The anorectic effect of neurotensin is mediated via a histamine H1 receptor in mice. Peptides 2004;25:2135–8. 31. Oliveros A, Heckman MG, del Pilar Corena-McLeod M, Williams K, Boules M, Richelson E. Sensorimotor gating in NTS1 and NTS2 null mice: effects of d-amphetamine, dizocilpine, clozapine and NT69L. J Exp Biol 2010;213:4232–9. 32. Rossi GC, Matulonis JE, Richelson E, Barbut D, Pasternak GW. Systemically and topically active antinociceptive neurotensin compounds. J Pharm Exp Ther 2010;334:1075–9. 33. Rostene WH, Alexander MJ. Neurotensin and neuroendocrine regulation. Front Neuroendocrinol 1997;18:115–73. 34. Seebach D, Lukaszuk A, Patora-Komisarska K, Podwysocka D, Gardiner J, Ebert MO, et al. On the terminal homologation of physiologically active peptides as a means of increasing stability in human serum–neurotensin, opiorphin, B27-KK10 epitope, NPY. Chem Biodivers 2011;8:711–39. 35. Shibata Y, White JF, Serrano-Vega MJ, Magnani F, Aloia AL, Grisshammer R, et al. Thermostabilization of the neurotensin receptor NTS1. J Mol Biol 2009;390:262–77.
Neurotensin/Neuromedin N Paul R. Dobner and Robert E. Carraway
ABSTRACT Neurotensin (NT) and neuromedin N, two bioactive peptides of brain and gut origin, are processed from a common precursor by prohormone convertases (PCs). Differential processing occurs in some tissues most probably resulting from the tissue-specific expression of distinct PCs, yielding large, N-terminally extended forms of the peptides. Two G protein-coupled receptors show preference for NT but bind both the peptides. NTR-1 signaling elevates intracellular [Ca2+] and can also increase cGMP, cAMP, and arachidonic acid metabolites. NTR-1/sortilin (NTR-3) heterodimers seem to modulate NT signaling and trafficking of internalized receptors, but NT may also signal through sortilin. Intimately associated with dopaminergic systems, NT is most probably an endogenous antipsychotic and has also been implicated in the modulation of psychostimulant responses, pain perception, stress-induced analgesia, appetite control, stress responses, and reproductive functions. The development of NT agonists may provide new pharmaceuticals for the treatment of schizophrenia, drug addiction, and stressrelated neuropathic pain syndromes.
DISCOVERY OF NEUROTENSIN In 1967, Susan Leeman discovered that the extracts of bovine hypothalami induce vasodilation and anaphylactic shock in rats. Using this assay, her laboratory isolated, sequenced, and synthesized the active substance, a 13-residue peptide that they named neurotensin (NT).6 NT is broadly expressed in the limbic nervous system and intestinal endocrine cells and is also found in various glands and in peripheral nerves. NT is a member of a family of peptides that are strikingly similar in their C-terminal regions (Table 1). These peptides share C-terminal residues with NT that have been shown to be critical for NT function and display many of the same pharmacological properties for those that have been tested. For instance, the venom of the cone snail contains the NT-like peptide contulakin G, which is a potent analgesic in rats and dogs.2 Several NT-related peptides are also present in neuronal cells of the protocordate Ciona intestinalis (Table 1). NT most probably has multiple neuronal and endocrine signaling functions, including the modulation of dopamine Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00117-2 Copyright © 2013 Elsevier Inc. All rights reserved.
(DA) signaling, stress-induced analgesia, energy balance, stress, and reproductive functions. This review focuses on the biosynthesis, distribution, receptor signaling, and potential functions of NT in the CNS.
STRUCTURE OF THE NEUROTENSIN/ NEUROMEDIN N PRECURSOR mRNA/GENE The amino acid sequence of the NT precursor was first inferred from canine cDNA sequences and is highly conserved in vertebrates.8 The predicted prepro form of the precursor ranges from 165 to170 amino acids in length, with NT and the related hexapeptide neuromedin N (NMN) lying in tandem near the carboxyl terminus of the precursor, bounded and separated by Lys-Arg processing sites (Fig. 1). The analysis of the rat, mouse, and human NT/NMN genes reveal that the coding region is spread over four exons, separated by three introns spanning just over 10 kb.20 The gene is transcribed to yield two different transcripts (1.0 and 1.5 kb) that differ only in the extent of the 3’-untranslated region, most probably arising from the differential use of alternative polyadenylation signals. The two mRNAs are expressed in about equal amounts in the brain, but the 1.0 kb mRNA greatly predominates in the gastrointestinal tract. The sequences immediately upstream of the transcriptional start site contain a series of response elements (AP-1, CRE, and GRE) that are important for NT/ NMN gene induction in response to environmental stimuli in PC12 cells. A variety of stimuli regulate NT/NMN gene expression in a brain region-specific manner.
NT/NMN mRNA EXPRESSION IN BRAIN NT/NMN mRNA and its peptide products are expressed in many forebrain regions, with higher levels in limbic regions, including hippocampus and subicullum, VP, BNST, medial Acb, and Cput (Fig. 2).1 NT/NMN mRNA-positive neurons are located mainly in the dorsomedial and ventral aspects of the Cput rostrally but are more evenly distributed in the caudal regions. A continuum of positive cells was observed 875
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876
TABLE 1 Peptides of the Neurotensin Family Peptide
Source
Sequence
NT
Cow, dog, human
NT
Chicken
NT
Green frog
NMN
Cow, dog, pig, human
Lys-Ile-Pro-Tyr-Ile-Leu*
LANT-6
Chicken
Lys-Asn-Pro-Tyr-Ile-Leu*
XP
Xenopus
XP
Dog, turkey
Phe-His-Pro-Lys-Arg-Pro-Trp-Ile-Leu
Contulakin G
Cone snail
Ci-NTLP-1
Ciona intestinalis
Ci-NTLP-3
Ciona intestinalis
Met-Met-Leu-Gly-Pro-Gly-Ile-Leu*
NT, neurotensin; NMN, neuromedin N; LANT-6, [Lys8, Asn9]-NT 8-13; XP, xenopsin; NTLP, neurotensin-like peptide. *Bordered by double basic cleavage sites in putative precursors. †N-terminal double basic cleavage site only. ‡The side chain is O-glycosylated.
stretching from the S through the preoptic area, including the lateral S, DBB, lateral BNST, and lateral Hpt. Steroid hormones and neurotransmitters have been implicated in the regulation of NT/NMN mRNA expression in several regions of the brain, including the medial preoptic area, Arc, PVN, periventricular nucleus, amygdala, Cput, and Acb. In the brainstem, NT/NMN mRNA-positive neurons are most abundant in the PAG and DR and can also be found in adjacent lateral structures (cuneiform nucleus, microcellular tegmental nucleus).
PROCESSING OF THE PRECURSOR The NT precursor is differentially processed by distinct prohormone convertases (PCs) in the brain and gastrointestinal tract (Fig. 1, reviewed in Ref. 22). PCs are described in detail in the Peptide Biosynthesis/Processing section of this book. In the brain, PC2 results in nearly complete processing of both the peptides, although brain NT levels are only decreased by ~15% in PC2 knockout mice. This is most probably because the precursor can also be processed by PC1 and PC5A, which are colocalized with NT and are capable of generating NT from the precursor. After liberation of the NT sequence from the precursor, the N-terminal Gln residue is converted into pGlu most probably through the action of glutaminyl cyclase.
In the gastrointestinal tract, PC1 inefficiently cleaves the LysArg pair amino terminal to NMN, resulting in the accumulation of amino-terminally extended NMN (large NMN in Fig. 1) that stretches from the signal peptide cleavage site to the carboxyl terminus of NMN (amino acids 23–147 in the rat sequence) and NT in gut endocrine cells. Similarly, colon cancer cell lines expressing both NT and PC5A produce predominantly amino-terminally extended forms of both NMN and NT (large NT in Fig. 1), similar to those found in the adrenal medulla, indicating that differential expression of distinct PCs can account for the observed tissue-specific processing of the NT precursor. The secretion of amino-terminally extended forms of NMN and NT may produce longer lasting biological effects because of their enhanced stability in the bloodstream compared with that of the fully processed peptides. A dibasic amino acid pair near the central region of the precursor is inefficiently processed in the brain and not used in the gastrointestinal tract, and complete processing of the NT precursor results in the production of a relatively stable fragment (Fig. 1, amino acids 24–140 of the human NT precursor) that extends from the signal peptide cleavage site through to the amino acid preceding the Lys-Arg amino terminal processing site of NMN.11 This precursor fragment is detectable in serum from healthy individuals, is stable in both human plasma and serum, and is released into the serum after ingestion of an ordinary meal, opening the possibility that it may serve as a surrogate marker for NT secretion.
RECEPTORS AND SIGNALING CASCADES Four NT receptors have been identified, two that are 7-transmembrane G protein-coupled receptors (NTR-1, NTR-2) and two primarily intracellular proteins related to yeast sorting receptors (NTR-3/sortilin, NTR-4/SorLA). NTR-1 displays the highest affinity (Kd, 0.1 nM) and seems to mediate NT effects on dopaminergic transmission, hotplate latency, temperature regulation, and smooth muscle contractility (reviewed in Ref. 21). First described as a lower affinity receptor (Kd, 3 nM) with a differing specificity, NTR-2 has been implicated in the analgesic effects of NT (reviewed in Ref. 29). Although the role of NTR-3 is less clear, one possibility is that through binding and internalizing NT this receptor serves to regulate turnover of the peptide. Evidence supports the involvement of NTR-3 in the growth-promoting effects of NT and in NT-induced migration of brain microglial cells. Signal transduction studies in transfected cell systems and cancer cell lines indicate that NTR-1 is primarily coupled to Gαq and signals via the activation of phospholipase C and the generation of IP3, causing an elevation in intracellular [Ca2+] (reviewed in Ref. 21). Concomitant generation of DAG leads to the activation of PKC in some cells, stimulating MAP kinases and PI3 kinase via complex mechanisms that can involve EGF receptor transactivation. This
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877
PC2 PC2 PC1 PC1 PC5A PC5A
Signal sequence
Lys/Arg Arg
Lys Arg Lys Arg
PC2 PC1 PC5A Lys Arg
NT/NMN precursor Large NT Large NMN NT NMN Stable fragment FIGURE 1 Schematic diagram of the NT/NMN precursor and its major processing products. The full length precursor is 165–170 amino acids long depending on the species and comprises 169 amino acids in the rat. The positions of the signal peptide (stippled fill, aa 1–23) and the NMN (gray fill) and NT (black fill) coding domains are indicated. The NMN and NT coding domains are bounded and separated by the indicated Lys-Arg processing sites. An additional Lys-Arg pair is located near the center of the precursor, but the available evidence suggests that this is not a major processing site. The prohormone convertases that have been shown to cleave the Lys-Arg processing sites are indicated and the extent to which they cleave a particular site is indicated by the size of the type, with the most efficient cleavage indicated by larger letters and the least efficient by smaller letters. PC1 cleaves the Lys-Arg pair located at the amino terminal end of NMN inefficiently but cleaves the sites surrounding NT efficiently resulting in the production of NT and Large NMN (intestinal pattern). PC5A cleaves the site at the carboxy-terminal end of NT efficiently, but the upstream sites less efficiently leading to the production of Large NT and Large NMN (adrenal pattern). PC2 cleaves all three sites efficiently resulting primarily in the production of NT and NMN (brain pattern).
signaling can have a feed forward component, in that NTinduced MAP kinase activation leads to the upregulation of EGF receptor. In neuroblastoma cells and rat brain slices, NT can elevate cGMP formation, inhibit cAMP synthesis and stimulate PI turnover, and evidence suggests that the third intracellular loop of NTR-1 is required for interactions with Gq, while the carboxy-terminal tail interacts with Gi/o and Gs (reviewed in Ref. 21). Liposomal ceramide has been shown to decrease the interaction of NTR-1 with Gq within structured membrane microdomains, which inhibits the growth stimulatory effects of NT in MDA-MB-231 breast cancer cells. NTR-1 is inducibly palmitoylated on Cys381 and Cys383 (rat sequence), and inhibition of this process by site-directed mutagenesis and pharmacological strategies diminishes NT-induced ERK phosphorylation and reduces the ability of NT to protect against apoptosis in breast cancer cells.17 NT activation of DA neurons appears to involve cAMP signaling, and NT potentiates cAMP formation (via NTR-1) in response to Gs-agonists by facilitating the activation of Ca2+-dependent adenylyl cyclases. Perhaps secondary to its effects on [Ca2+], NT also activates PLA2 and DAG lipase, stimulating arachidonic acid release and the formation of bioactive metabolites via the cyclooxygenase and lipoxygenase pathways. This signaling could also be amplified by feed forward mechanisms because the
expression of PLA2 is upregulated by the activation of MAP kinase (reviewed in Ref. 7). NT is the most potent substance known to stimulate leukotriene formation in rats, and the possible relevance of this to its central effects is not well investigated. Signaling mechanisms for NTR-2 and NTR-3 are not yet clear. NTR-2 constitutively activates IP production, and this process is stimulated by the NTR-1 antagonist SR48692 but suppressed by NT (reviewed in Ref. 29). NTR-3 is primarily localized to ER-Golgi membranes but ≅10% is on the cell surface. NTR-3 can form heterodimers with NTR1, decreasing the potency of NT to stimulate IP formation and to activate MAP kinase. Thus, NTR-3, which cannot promote either response, modulates these effects of NTR-1. Results obtained by site-directed mutagenesis favor a model for NTR-1 activation in which binding of NT to key residues in EC loop 3 and TM 6 and 7 leads to a shift in IC loop 3 that enhances binding to Gq (reviewed in Ref. 21). The relative instability of NTR-1 in detergents has led investigators to use a systematic mutational approach to identify receptor mutants that display enhanced thermostability, resulting in the identification of an NTR-1 variant that is stabilized in an agonist binding conformation that presumably might be used with a wider range of detergent and buffer conditions for crystallization.35
CHAPTER | 117 Neurotensin/Neuromedin N
878
Cx
Hi CC
OB
CPut
S
Th PAG C BST
DR
PBN
Hpt PVN LC
Acb DMH
SN NST
Arc
SON
VMH
DBB
Amb ME Amy
NL IL
LRN
AP
FIGURE 2 Schematic diagram of the distribution of NT-IR neurons in the rat brain. ( ) NT-IR neurons detected after colchicine treatment. ( ) NT-IR neurons induced by stressful stimuli that are also detected after colchicine treatment. ( ) NT-IR neurons induced by either psychostimulants (dorsomedial CPu, Acc) or dopamine D2 antagonists, including typical antipsychotic drugs (dorsolateral CPu, Acc). ( ) Estrogen-inducible NT-IR neurons in the female MPO and arcuate nucleus. NT gene expression in the rostal MPO nucleus is sexually dimorphic. Quantitative analysis of NT content by RIA (pmole/g wet weight tissue) provides a more comprehensive picture of the distribution of the peptide in various brain regions (OB, 3; Cx, 4-5; Acc, 29; CPut, 10; S, 73; BST, 172; Hi, 7; Th, 5-23; Hyp, 76-128; ME, 128; SN, 13; PAG, 42; LC, 54).
CONFORMATION 2D solid-state NMR spectroscopy has been used to study the conformational changes of NT8-13 on interaction with an N-terminally truncated form of rat NTR-1.27 On binding to the receptor, the peptide rearranges to adopt a β-strand conformation, characterized by a ψ(Pro10) dihedral angle of 146 ± 15°. The NMR spectrum of NT (8–13) in solution is also consistent with the presence of a type II β-turn, whereas the spectrum of a homolog that displays 40-fold less affinity for human NTR-1 does not.34 Furthermore, a lactam-bridged analog of NT8-13 with ψ(Pro10) angles restricted to an angle (130°) close to the experimentally determined one exhibits ~1000-fold higher affinity for porcine NTR-1 than the stereoisomer (−130°), providing further evidence for the NMR-derived bioactive conformation.
BRAIN FUNCTIONS Close Alliance between NT and Dopamine NT has been broadly implicated in the modulation of DA signaling and can produce both psychostimulant- and antipsychotic drug (APD)-like effects after microinjection into different regions of the brain (reviewed in Ref. 5). Most midbrain DA neurons express high levels of NTR-1, and NT can stimulate these neurons to increase striatal DA release in the Acb. NT can also act presynaptically to potentiate DA
release in both the Acb and Cput, most probably by antagonizing DA D2 receptor autoinhibition. Consistent with these observations, NT microinjection in the VTA stimulates locomotor activity and repeated administration results in sensitization to not only NT but also psychostimulant drugs. Prolonged administration with the NT antagonist SR48692 lowers basal DA release in the Acc, consistent with the hypothesis that endogenous NT acts to augment DA release through NTR-1; however, basal and stress-induced DA release in the mPFC are unaffected. NTR-1, but not NTR-2, knockout mice are reported to be hyperdopaminergic with elevated basal DA release in the Cput, although release in the Acc and mPFC was not examined.26 These differences could reflect regionally distinct roles for NT in the control of DA release, but could also result from either adaptive changes in NTR-1 knockout mice or unanticipated effects of prolonged NT antagonist administration. NT can also produce neuroleptic-like effects when administered in the Acc, seemingly inconsistent with the evidence cited above indicating that NT acts presynaptically to augment DA signaling. The most probable explanation for this is that NT either acts postsynaptically or on other presynaptic afferents to oppose DA signaling and electron microscopic immunohistochemical analysis indicates that NTR-1 is expressed on both dopaminergic and excitatory afferents- and on postsynaptic neurons in the Acc; however, NTR-1 seems to be expressed primarily on striatal afferents
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in the Cput. NTR-2 knockout mice have lower levels of basal striatal glutamate release and both NTR-1 and NTR-2 knockout mice display muted amphetamine-stimulated striatal glutamate release,25 consistent with the hypothesis that endogenous NT modulates striatal glutamate release.
NT may be an Endogenous Antipsychotic Considerable evidence suggests that NT mediates at least a subset of responses to certain APDs. APDs elevate striatal NT expression and NT administration in the Acb suppresses amphetamine-stimulated locomotor activity, similar to APDs, consistent with the idea that increases in NT signaling might be involved in APD actions (reviewed in Ref. 5). Although animal models of schizophrenia are necessarily limited, latent inhibition (LI) and prepulse inhibition (PPI) of the acoustic startle response measure aspects of sensorimotor gating that can be influenced by APDs. Enhanced DA transmission and isolation rearing disrupt PPI and APDs can restore PPI to control levels. In mice, APDs can also increase PPI above basal levels without prior disruption by DA agonists. There is increasing evidence that either pharmacological or genetic disruption of NT signaling can have a profound impact on APD responses in these animal models. NT has been implicated in both the acquisition of LI and APD enhancement of LI. NT antagonist administration was shown to interfere with the acquisition of LI, and this was observed with both the relatively selective NTR-1 antagonist SR48692 and the NTR-1/NTR-2 antagonist SR142948A, indicating that NT signaling through NTR-1 may be particularly important (reviewed in Ref. 5). The suppressive effects of SR142948A are ameliorated by coadministration of the D2 antagonist sulpiride, suggesting that NT antagonist treatment may increase DA D2 transmission in the Acb or other key brain regions involved in LI. NT also seems to play a role in APD enhancement of LI, because SR142948A administration 1 hour before the last of 7 daily injections of haloperidol prevents augmentation of LI. Thus, NT likely plays a role in both basal and APDenhanced LI, possibly through effects on DA D2 signaling. NT also appears to play a role in both basal and APDenhanced PPI; however, results from NTR-1 and NTR-2 knockout mice suggest a more complicated picture. Preadministration of the NTR-1/NTR-2 antagonist SR142948A blocks the ability of both the typical APD haloperidol and the atypical APD quetiapine to restore PPI deficits in isolation-reared rats, without affecting basal PPI (reviewed in Ref. 5). Furthermore, NT mRNA levels are decreased by ~45% and NT receptor binding is increased by ~21% in the Acb shell in isolation-reared animals, and NT mRNA levels are restored to those observed in socially reared animals 4 hours after quetiapine treatment. Similarly, both haloperidol and quetiapine fail to augment PPI in NT knockout mice, which also have reduced basal PPI, although the response to
879
clozapine is unaffected.19 The decrease in basal PPI is modest and is not observed in either NTR-1 or NTR-2 knockout mice where it is instead elevated.13,31 The indirect DA agonist amphetamine disrupts PPI, but this effect is blocked in NT knockout mice,19 suggesting that NT may play a crucial role in this process. Similar results have been reported for NTR-1 knockout mice;31 however, another report indicates that amphetamine disruption of PPI is normal in these mice,13 as it is in NTR-2 knockout mice.31 These different results most likely reflect methodological differences, because both used NTR-1 knockout mice from the same source. These results provide a preliminary indication that NT signaling is required for amphetamine disruption of PPI in mice. NT participation in processes that either enhance or reduce PPI most likely reflects compartmental specificity in NT action within the circuits that modulate PPI, particularly the DA circuits. The analysis of amphetamine and haloperidol induction of striatal Fos expression has provided direct evidence for such compartmentalized NT signaling.9,12
NT and Psychostimulant Sensitization As discussed above, NT is capable of modulating DA transmission at different levels. In addition to antipsychotic drug-like effects, NT produces psychostimulant-like effects after direct administration into the VTA and increases DA release in the Acb (reviewed in Ref. 5). Several different laboratories have reported that NT antagonist pretreatment blocks or attenuates amphetamine and can delay cocaine sensitization while sparing the acute locomotor stimulation produced by these drugs. By contrast, both amphetamine (PRD, unpublished observations) and cocaine16 sensitization are augmented somewhat in NT knockout mice. Several other lines of evidence also suggest that NT acts to limit psychostimulant sensitization, including the observations that peripheral administration of the NT agonist, NT69L, suppresses nicotine sensitization, and that NT antagonist administration potentiates rather than blocks supersensitive locomotor responses to L-DOPA in dopamine-deficient mice. NTR-1 knockout mice show both increased basal and amphetamine-stimulated locomotor activity and striatal DA release, also consistent with the idea that NT acts to limit DA signaling, although psychostimulant sensitization was not examined.26 However, both basal and cocainestimulated locomotor activities are similar to wildtype in NT knockout mice16 and in rats treated with SR48692 (reviewed in Ref. 5). Thus, gene knockout and several other approaches favor a model in which NT acts mainly to limit psychostimulant sensitization, while experiments with NT antagonists indicate the opposite. Further work will be required to determine whether this discrepancy results from compensatory mechanisms in knockout animals or unanticipated antagonist effects, for instance agonist or partial agonist activity at NTR-2.
880
NT and Pain Given centrally, NT enhances nociceptive responses at lower doses, but produces a potent analgesic response at higher doses that in most studies does not require µ-opioid receptor signaling (reviewed in Ref. 10). NT and NT receptors are expressed in the central descending circuitry involved in pain modulation and in the spinal cord, consistent with a possible role in pain modulation. The evidence to date indicates that endogenous NT plays a relatively minor role in basal nociception but is critical for at least certain aspects of stress-induced antinociception (SIAN). Remarkably, SIAN is totally absent in NT knockout mice, where water avoidance/cold-water swim stress instead results in a hyperalgesic response to colonic distension.15 Similar results were obtained in rats pretreated with SR48692, indicating that SIAN probably requires NT signaling through NTR-1, and the defects observed in NT knockout mice are not because of developmental or compensatory changes resulting from the lifelong absence of NT signaling.15 NTR-2 signaling has also been implicated in certain aspects of SIAN. NTR-2 knockout mice display a selective defect in SIAN after cold-water swim stress during the latter part of the second phase of the pain response in both the intraplantar formalin test and the paw withdrawal response to thermal stimuli, while nonstressed responses are comparable with wildtype.23 Similar results occur in the paw withdrawal test after repeated administration of the NTR-1/NTR-2 antagonist SR142948A, indicating that the differences observed in NTR-2 knockout mice also do not result from developmental defects or compensatory changes resulting from the absence of NTR-2. Furthermore, intrathecal administration of both nonselective and NTR-2-selective NT agonists potentiate cold-water swim stress-induced SIAN in the paw withdrawal test. These results are consistent with the previous observations that NTR-2-selective agonists suppress pain responding in the intraplantar formalin test selectively during the latter part of the second phase of the pain response, influencing predominantly paw lifting while sparing paw licking, biting, and shaking behaviors. These results suggest that endogenous NT signaling through both NTR-1 and NTR-2 is particularly important for SIAN. In contrast to SIAN, NT seems to play a relatively minor role in basal pain perception, where it seems to facilitate pain responses. Although both NTR-1 and NTR-2 knockout mice respond similarly to wild type in a variety of pain tests, NTR-2 knockout mice display longer jump latencies in the hot-plate test.28 Viseromotor responses to colorectal distension are also diminished in both NT knockout mice and rats pretreated with SR48692.15 The evidence discussed above suggests that NT not only facilitates basal pain responses but it also plays a key role in SIAN and any theory of NT modulation of pain
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responses must take into account these seemingly contradictory results. Perhaps the simplest explanation is that the direction of pain modulation depends on the intensity of NT signaling, with basal NT signaling facilitating nociception and increased NT signaling being analgesic. The observation that cold-water swim stress increases NT mRNA levels in certain Hpt regions that project to the PAG is consistent with the idea that stress-induced increases in NT signaling to the PAG may underlie SIAN. Increased NT signaling could lead to the selective recruitment of neurons involved in analgesic responses, such as PAG “off” cells.
Appetite Control NT has been implicated as a possible satiety factor and mediator of the central suppressive effects of leptin on food consumption (reviewed in Ref. 3). Leptin administration suppresses food consumption and weight gain, possibly through the decreased expression of orexigenic peptides and increased NT expression in the hypothalamus. Similarly, NT expression is decreased in specific Hpt regions in response to a high-fat diet that results in obesity. Combined central administration of NT and leptin potentiates both the short-term inhibitory effects of NT on spontaneous food intake and early, but not late (24 h) leptin effects. Because the leptin effect is blocked by pretreatment with either NTR-1 antagonist or NT antiserum, it has been suggested that NT mediates the central effect of leptin. Collectively, these results indicate that dynamic regulation of NT in specific Hpt nuclei in response to alterations in the levels of circulating leptin and perhaps other factors is involved in appetite suppression and body weight balance. Other results, showing that the anoretic effect of NT is antagonized by the H1 receptor blocker pyrilamine and in H1 knockout mice, suggest that the histamine H1 system, which is known to inhibit food intake, could be a final common mediator for the effects of both leptin and NT.30
NT and Stress Responses Stress increases NT expression in several Hpt nuclei, suggesting that NT may modulate stress responses (reviewed in Ref. 14). Centrally administered NT was found to rapidly increase the levels of circulating ACTH and corticosterone and these increases were attenuated by corticotrophinreleasing hormone (CRH) antagonists and bilateral lesions of the PVN. Furthermore, SR 48692 administration in the PVN suppresses stress-induced increases in the plasma levels of ACTH and corticosterone and decreases CRH expression in the PVN. Both basal and stress-induced blood corticosterone levels are lower in NTR-2 knockout compared with those of control mice, consistent with NT playing a role in the control of corticosterone release.23
SECTION | X Handbook of Biologically Active Peptides: Brain Peptides
NT and Reproductive Functions NT has been implicated in the preovulatory luteinizing hormone (LH) surge and the Hpt control of prolactin (PRL) release in rat (reviewed in Ref. 33). Estrogen increases NT mRNA and peptide expression in several Hpt regions that have been implicated in reproductive functions, and NT expression in these regions is sexually dimorphic. In addition, NT mRNA levels vary throughout the estrus cycle in female rats with NT mRNA-positive cells being ~3-fold more numerous at proestrus than at diestrus. These results coupled with microinjection data indicating that NT can augment LH release have led to speculation that NT might mediate the feedforward effects of estrogen on gonadotropin-releasing hormone (GnRH) release from rostral medial preoptic nucleus GnRH neurons expressing NTR-1 in the rat. However, this system may not operate in the mouse, because even though estrogen increases NT mRNA expression similarly in ovariectomized mice, icv NT administration does not augment LH release in ovariectomized mice treated with estrogen.24 GnRH neurons are clearly subject to complex peptide control with kisspeptin playing a crucial role, and it remains to be determined how NT interacts with kisspeptin to modulate reproductive function. Kisspeptin neurons apparently express NTR-2 but not NTR-1 or NT.24 NT has also been implicated in both positive and negative regulation of PRL secretion from pituitary lactotropes. The evidence suggests that NT directly activates tuberoinfundibular DA (TIDA) neurons and mediates feedback inhibition of PRL release through the activation of these neurons.18 Estrogen increases NT expression in TIDA neurons and NT is intensely expressed in virtually all TIDA neurons in nursing rats, but decreases markedly after removal of pups. Increased NT release from TIDA neurons in the median eminence would most probably counteract the inhibitory effects of DA on PRL secretion, because NT stimulates pituitary PRL release. NT knockout mice reproduce and successfully rear their young, indicating that NT is not essential for reproductive functions.9
PATHOPHYSIOLOGICAL IMPLICATIONS There is now considerable evidence from animal models that endogenous NT signaling is both required for certain APD actions and that decreased NT signaling may underlie deficits in PPI. Comparison of NT levels in cerebrospinal fluid (CSF) from schizophrenics both before and after APD treatment indicates that low CSF NT levels are associated with more severe psychopathology and that clinical improvement is associated with increased CSF NT (reviewed in Ref. 5). Some NT agonists produce effects similar to those of atypical APDs and the relatively stable NT analog, NT69L, prevents drug-induced disruption of PPI in rats after either acute or chronic administration.4 These results suggest that
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the development of NT agonists could provide an important new class of APDs. In the shorter term, there could be some therapeutic benefit associated with the use of NT-dependent (e.g. quetiapine, haloperidol) and NT-independent (e.g. clozapine) APDs in combination. NT also seems to play an important role in stressinduced analgesia, suggesting that NT agonists could be effective in treating certain chronic pain syndromes associated with abnormalities in stress responses. The observation that cold-water swim/water avoidance stress in NT knockout mice results in hyperalgesia rather than analgesia is particularly interesting in this regard.15 Stable NT agonists may prove to be particularly effective in treating stress-related and neuropathic pain.32
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