Karine Belleville

Involvement of NTS2 receptors in stress-induced analgesia

Stress activates multiple neural systems that suppress pain sensation. This adaptive phenomenon referred as stress-induced analgesia (SIA) is mediated by the activation of endogenous pain inhibitory systems. Both opioid and non-opioid forms of SIA have been elicited in rodents according to stressor parameters and duration. There is accumulating evidence that the endogenous neurotensin (NT) system plays an important role in SIA. Especially, NT-deficient mice were shown to exhibit reduced SIA following water avoidance or restraint stress. Since central NT produces naloxone-insensitive analgesic effects by acting on spinal and supraspinal NTS2 receptors, we hypothesized that NT might mediate non-opioid SIA through NTS2 activation. Here, we evaluated the influence of an opioid-independent severe stress produced by a cold-water swim for 3 min at 15 degrees C on rodent offsprings pain perception. Our results demonstrated that mice lacking NTS2 exhibit significantly reduced SIA following cold-water swim stress. Indeed, NTS2 knockout mice submitted to both acute (plantar test) and tonic (formalin test) pain stimuli show a greater sensitivity to pain in comparison to wild-type littermates. Accordingly, pretreatment with the NT receptor antagonist SR142948A results in a hyperalgesic response to stress induced by cold-water swim. Endogenous NT regulates hypothalamic-pituitary-adrenal axis activity in stress condition by increasing corticosterone plasma levels. Accordingly, the plasma levels of corticosterone measured by radioimmunoassay are significantly reduced in non-stressed and stressed NTS2-deficient mice in comparison with wild-type mice. To further investigate the site of action of NT in mediating SIA, we microinjected NTS2 agonists in lumbar spinal cord and quantified post-stress sensitivity to pain in rats using the plantar test. Exogenously administered NTS2 analogs, JMV-431, beta-lactotensin and NT69L markedly enhance the magnitude and duration of stress antinociception in both 25- and 60-day-old rats. In sum, by using genetic and pharmacological approaches, we demonstrated here that NTS2 receptors mediate non-opioid SIA. Our results also revealed that the release of endogenous NT in response to stress requires the presence of NTS2 to stimulate corticotropin-releasing factor (CRF)-induced elevation of plasma corticosterone, and that NTS2 receptors localized at the lumbar spinal cord participate to the disinhibition of descending pain control pathways. Therefore, these data highlight the significance of NTS2 as a novel target for the treatment of pain and stress-related disorders.

Behavioral, Medical Imaging and Histopathological Features of a New Rat Model of Bone Cancer Pain

Pre-clinical bone cancer pain models mimicking the human condition are required to respond to clinical realities. Breast or prostate cancer patients coping with bone metastases experience intractable pain, which affects their quality of life. Advanced monitoring is thus required to clarify bone cancer pain mechanisms and refine treatments. In our model of rat femoral mammary carcinoma MRMT-1 cell implantation, pain onset and tumor growth were monitored for 21 days. The surgical procedure performed without arthrotomy allowed recording of incidental pain in free-moving rats. Along with the gradual development of mechanical allodynia and hyperalgesia, behavioral signs of ambulatory pain were detected at day 14 by using a dynamic weight-bearing apparatus. Osteopenia was revealed from day 14 concomitantly with disorganization of the trabecular architecture (µCT). Bone metastases were visualized as early as day 8 by MRI (T1-Gd-DTPA) before pain detection. PET (Na18F) co-registration revealed intra-osseous activity, as determined by anatomical superimposition over MRI in accordance with osteoclastic hyperactivity (TRAP staining). Pain and bone destruction were aggravated with time. Bone remodeling was accompanied by c-Fos (spinal) and ATF3 (DRG) neuronal activation, sustained by astrocyte (GFAP) and microglia (Iba1) reactivity in lumbar spinal cord. Our animal model demonstrates the importance of simultaneously recording pain and tumor progression and will allow us to better characterize therapeutic strategies in the future.

Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor-related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics.

Evidence for a role of NTS2 receptors in the modulation of tonic pain sensitivity.

BackgroundCentral neurotensin (NT) administration results in a naloxone-insensitive antinociceptive response in animal models of acute and persistent pain. Both NTS1 and NTS2 receptors were shown to be required for different aspects of NT-induced analgesia. We recently demonstrated that NTS2 receptors were extensively associated with ascending nociceptive pathways, both at the level of the dorsal root ganglia and of the spinal dorsal horn. Then, we found that spinally administered NTS2-selective agonists induced dose-dependent antinociceptive responses in the acute tail-flick test. In the present study, we therefore investigated whether activation of spinal NTS2 receptors suppressed the persistent inflammatory pain symptoms observed after intraplantar injection of formalin.ResultsWe first demonstrated that spinally administered NT and NT69L agonists, which bind to both NTS1 and NTS2 receptors, significantly reduced pain-evoked responses during the inflammatory phase of the formalin test. Accordingly, pretreatment with the NTS2-selective analogs JMV-431 and levocabastine was effective in inhibiting the aversive behaviors induced by formalin. With resolution at the single-cell level, we also found that activation of spinal NTS2 receptors reduced formalin-induced c-fos expression in dorsal horn neurons. However, our results also suggest that NTS2-selective agonists and NTS1/NTS2 mixed compounds differently modulated the early (21–39 min) and late (40–60 min) tonic phase 2 and recruited endogenous pain inhibitory mechanisms integrated at different levels of the central nervous system. Indeed, while non-selective drugs suppressed pain-related behaviors activity in both part of phase 2, intrathecal injection of NTS2-selective agonists was only efficient in reducing pain during the late phase 2. Furthermore, assessment of the stereotypic pain behaviors of lifting, shaking, licking and biting to formalin also revealed that unlike non-discriminative NTS1/NTS2 analogs reversing all nociceptive endpoint behaviors, pure NTS2 agonists specifically inhibited paw lifting, supporting a role of NTS2 in spinal modulation of persistent nociception.ConclusionThe present study provides the first demonstration that activation of NTS2 receptors produces analgesia in the persistent inflammatory pain model of formalin. The dichotomy between these two classes of compounds also indicates that both NTS1 and NTS2 receptors are involved in tonic pain inhibition and implies that these two NT receptors modulate the pain-induced behavioral responses by acting on distinct spinal and/or supraspinal neural circuits. In conclusion, development of NT agonists targeting both NTS1 and NTS2 receptors could be useful for chronic pain management.

Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model

Central administration of the neuropeptide neurotensin (NT) was shown to induce antinociceptive responses both spinally and supraspinally. Although NTS2 receptors play an important role in modulating the activity of spinal neurons, we have recently implicated NTS1 receptors in NTs analgesic effects in acute spinal pain paradigms. The current experiments were thus designed to examine the antinociceptive effects of intrathecal administration of NTS1 agonists in formalin‐induced tonic pain in rats. We first established, using immunoblotting and immunohistochemical approaches, that NTS1 receptors were present in small‐ and medium‐sized dorsal root ganglion cells and localized in the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal injection of NT (1–15 μg/kg) or NTS1 preferring agonists on the nocifensive response to intraplantar formalin. Both NTS1‐agonists, PD149163 (10–120 μg/kg) and NT69L (1–100 μg/kg), dose‐dependently attenuated the formalin‐induced behaviors. Accordingly, NTS1 agonists markedly suppressed pain‐evoked c‐fos expression in the superficial, nucleus proprius and neck regions of the spinal dorsal horn. The concomitant administration of PD149163 with the NTS1 antagonist SR48692 (3 μg/kg) significantly reversed PD149163‐induced antinociception, confirming the implication of NTS1 in tonic pain. In contrast, NT69L’s analgesic effects were partly abolished by co‐administration of SR48692, indicating that NT69L‐induced effects may also be exerted through interaction with NTS2. These results demonstrate that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in persistent pain and suggest that NTS1‐selective agonists may represent a new line of analgesic compounds.

Epithelial BMP signaling is required for proper specification of epithelial cell lineages and gastric endocrine cells

Bone morphogenetic protein (BMP) signaling within the gastrointestinal tract is complex. BMP ligands and their receptors are expressed in both epithelial and mesenchymal compartments, suggesting bidirectional signaling between these two entities. Despite an increasing interest in BMP signaling in gut physiology and pathologies, the distinct contribution of BMP signaling in the epithelium vs. the mesenchyme in gastrointestinal homeostasis remains to be established. We aimed to investigate the role of epithelial BMP signaling in gastric organogenesis, gland morphogenesis, and maintenance of epithelial cell functions. Using the Cre/loxP system, we generated a mouse model with an early deletion during development of BMP receptor 1A (Bmpr1a) exclusively in the foregut endoderm. Bmpr1a(ΔGEC) mice showed no severe abnormalities in gastric organogenesis, gland epithelial proliferation, or morphogenesis, suggesting only a minor role for epithelial BMP signaling in these processes. However, early loss of BMP signaling in foregut endoderm did impact on gastric patterning, leading to an anteriorization of the stomach. In addition, numbers of parietal cells were reduced in Bmpr1a(ΔGEC) mice. Epithelial BMP deletion significantly increased the numbers of chromogranin A-, ghrelin-, somatostatin-, gastrin-, and serotonin-expressing gastric endocrine cells. Cancer never developed in young adult (<100 days) Bmpr1a-inactivated mice although a marker of spasmolytic polypeptide-expressing metaplasia was upregulated. Using this model, we have uncovered that BMP signaling negatively regulates the proliferation and commitment of endocrine precursor cells. Our data also indicate that loss of BMP signaling in epithelial gastric cells alone is not sufficient to induce gastric neoplasia.

Stability and degradation patterns of chemically modified analogs of apelin-13 in plasma and cerebrospinal fluid

Apelin is the endogenous ligand of APJ, which belongs to the superfamily of G protein‐coupled receptors. In recent years, the apelin/APJ system has been detected in many tissues and emerges as a promising target for the treatment of various pathophysiological conditions. Pyr1‐apelin‐13 is the major isoform of apelin in human plasma; however its stability and proteolytic degradation pattern remain poorly understood. The aim of the present study was first to identify the cleavage sites of Pyr1‐apelin‐13 in mouse, rat and human plasma and rat cerebrospinal fluid, then to determine its stability to proteolytic degradation following intravenous administration in rats. Secondly, key residues were substituted by natural and unnatural amino acids in order to examine the impact on in vitro stability and degradation pattern. The kinetics of degradation revealed that the Leu5‐Ser6 peptide bond of Pyr1‐apelin‐13 is the first cleavage observed in plasma, independently of the species. Replacement of Phe13 by unnatural amino acids showed a 10‐fold increase in plasma stability although the hydrolysis of Pro12‐Phe13 bond, previously described as a site of cleavage by ACE‐2, was not observed. In vivo, this Pro12‐Phe13 bond was cleaved yet appears as a minor product compared to hydrolysis of the Pro10‐Met11 bond. This study pinpoints the most critical amino acids targeted by proteases and will be instrumental for the design of Pyr1‐apelin‐13 analogs possessing increased stability.

Spinal NTS2 receptor activation reverses signs of neuropathic pain

Management of painful peripheral neuropathies remains challenging, since patients with chronic pain respond poorly to the available pharmacopeia. In recent years, the G‐protein‐coupled receptor neurotensin (NT) type 2 (NTS2) emerged as an attractive target for treating transitory pain states. To date, however, there is no evidence for its role in the regulation of chronic peripheral neuropathies. Here, we found that NTS2 receptors were largely localized to primary afferent fibers and superficial dorsal horns. Changes in the time course of the gene expression profile of NT, NTS1, and NTS2 were observed over a 28‐d period following the sciatic nerve constriction [chronic constriction injury (CCI) model]. We next determined the effects of central delivery of selective‐NTS2 agonists to CCI‐treated rats on both mechanical allodynia (evoked withdrawal responses) and weight‐bearing deficits (discomfort and quality‐of‐life proxies). The NTS2 analogs JMV431, levocabastine, and β‐lactotensin were all effective in reducing ongoing tactile allodynia in CCI‐treated rats. Likewise, amitriptyline, pregabalin, and morphine significantly attenuated CCI‐induced mechanical hypersensitivity. NTS2 agonists were also efficient in reversing weight‐bearing and postural deficits caused by nerve damage, unlike reference analgesics currently used in the clinic. Thus, NTS2 agonists may offer new treatment avenues for limiting pain associated with peripheral neuropathies and improve functional rehabilitation and well‐being.— Tétreault, P., Beaudet, N., Perron, A., Belleville, K., René, A., Cavelier, F., Martinez, J., Stroh, T., Jacobi, A. M., Rose, S. D., Behlke, M. A., Sarret, P., Spinal NTS2 receptor activation reverses signs of neuropathic pain. FASEB J. 27, 3741–3752 (2013). www.fasebj.org

Ghrelin Inhibition Restores Glucose Homeostasis in Hepatocyte Nuclear Factor-1α (MODY3)–Deficient Mice

Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3 (MODY3). Mice deficient for Hnf1α are hyperglycemic, with their pancreatic β-cells being defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether changes in these hormones play a role in glucose homeostasis in the absence of Hnf1α. An increase in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP) gene transcripts were observed in the gut of Hnf1α-null mice. These changes correlated with an increase of ghrelin and a decrease of GIP-labeled cells. Ghrelin serological levels were significantly induced in Hnf1α-null mice. Paradoxically, GIP levels were also induced in these mice. Treatment of Hnf1α-null mice with a ghrelin antagonist led to a recovery of the diabetic symptoms. We conclude that upregulation of ghrelin in the absence of Hnf1α impairs insulin secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These observations open up on future strategies to counteract ghrelin action in a program that could become beneficial in controlling non–insulin-dependent diabetes.

Altered Morphine-Induced Analgesia in Neurotensin Type 1 Receptor Null Mice

Both neurotensin (NT) and opioid agonists have been shown to induce antinociception in rodents after central administration. Besides, previous studies have revealed the existence of functional interactions between NT and opioid systems in the regulation of pain processing. We recently demonstrated that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in long-lasting pain. In the present study, we therefore investigated whether NTS1 gene deletion affected the antinociceptive action of mu opioid drugs. To this end, pain behavioral responses to formalin were determined following systemic administration of morphine in both male and female NTS1 knockout mice. Acute injection of morphine (2 or 5 mg/kg) produced strong antinociceptive effects in both male and female wild-type littermates, with no significant sex differences. On the other hand, morphine analgesia was considerably reduced in NTS1-deficient mice of both sexes compared to their respective controls, indicating that the NTS1 receptor actively participates in mu opioid alleviating pain. By examining specifically the flinching, licking and biting nociceptive behaviors, we also showed that the functional crosstalk between NTS1 and mu opioid receptors influences the supraspinally-mediated behaviors. Interestingly, sexual dimorphic action of morphine-induced pain inhibition was found in NTS1 null mice in the formalin test, suggesting that the endogenous NT system interacts differently with the opioid network in male and female mice. Altogether, these results demonstrated that NTS1 receptor activation operates downstream to the opioidergic transmission and that NTS1-selective agonists combined with morphine may act synergistically to reduce persistent pain.