Immunohistochemical absence of adrenergic neurons in the dorsal part of the solitary tract nucleus in sudden infant death

The immunohistochemical distribution of TH and PNMT containing neuronal elements was investigated utilizing peroxidase anti-peroxidase methods in newborn control and sudden infant death syndrome (SIDS) brainstems. The TH immunoreactive neurons, within the medulla oblongata, displayed a similar distribution in both control and SIDS tissue. However, PNMT immunoreactive neurons seen in the dorsal part of the nucleus of tractus solitarius in control tissue were not observed in SIDS tissue. This alteration of adrenergic neurons in the dorsal part of NTS (region reported to be implicated in the control of blood pressure and respiration) could explain the cardiorespiratory disorders in SIDS.     

Beta-endorphin and neurotensin in brainstem and cerebrospinal fluid in the sudden infant death syndrome.

Beta-endorphin (BE) and neurotensin (NT) are two neuropeptides which induce apneas. In infants who died of Sudden Infant Death Syndrome (SIDS) we measured, in brainstem and CSF, BE and NT by IRMA and RIA respectively. BE and NT levels are compared to same aged infant and adult controls. CSF BE level was significantly higher in SIDS than in the two control groups (86 +/- 14 vs 33 +/- 13 and 16 +/- 5 pmol/l). In 6 SIDS victims NT and BE were assayed in 5 brainstem sections, each of them divided in median, intermediate and lateral parts. We found high levels of BE in every fragment (3-11 pmol/mg protein) while NT elevated values were restricted to the mesencephalic regions (1.4-12 pmol/mg), the medial pons (6 pmol/mg) and the intermediate parts of the medulla (including the olive: 1.3-1.6 pmol/mg). These results support the hypothesis that NT and/or BE could induce or participate to the fetal issue of SIDS.     

Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [125I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores, as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.     

Brain adrenoreceptors in rats with inherited arterial hypertension due to emotional stress

For the study of genetic and physiological mechanisms of inherited stress-sensitive arterial hypertension, specific binding of ligands of alpha 1-, alpha 2- and beta-adrenoceptors was measured in 2 strains of rats: Wistar normotensive and ISSAH rats (rats with inherited stress-sensitive arterial hypertension). The maximal binding sites (Bmax) and apparent dissociation constants (Kd) were studied with the alpha 1-adrenergic antagonist 3H-prazosin, alpha 2-adrenergic agonist 3H-clonidine and 3H-dihydroalprenolol, a beta 1-receptor antagonist. Four brain regions were investigated: frontal cortex, hypothalamus, pons and medulla oblongata. In comparison with normotensive controls, hypertensive rats had significantly greater density of the alpha 1-adrenoceptors in the medulla oblongata. However, the number of hypothalamic alpha 1-adrenoceptors was significantly reduced in these animals. The same significantly lower alpha 2-adrenoreceptor density was found in the hypothalamus and the pons, and lower, beta-adrenoceptors density in the medulla oblongata. It was concluded that brain adrenoceptors are involved in the mechanisms of development of inherited stress-sensitive hypertensive syndrome.     

Over-expression of neurotensin high-affinity receptor 1 (NTS1) in relation with its ligand neurotensin (NT) and nuclear beta-catenin in inflammatory bowel disease-related oncogenesis

We investigated the expression of the neurotensin high-affinity receptor 1 (NTS1) during inflammatory bowel disease (IBD)-related colorectal oncogenesis, in colonic samples from 30 patients with IBD-related adenocarcinomas, dysplasias, and inflammatory mucosa (IM). The percentage of NTS1-positive epithelial cells progressively increased from the inflammatory condition to adenocarcinoma and was significantly higher in adenocarcinomas than in IM (p=0.0169). In parallel, the percentage of neurotensin (NT)-positive epithelial cells increased during the IBD-related oncogenesis. Finally, as NTS1 is a ss-catenin inducible gene, we found that a number of preneoplastic lesions and adenocarcinomas co-expressed NTS1 and beta-catenin without NT expression. Therefore, this study suggests two pathways of NTS1 overexpression during IBD-related oncogenesis: one triggered by NT overexpression, and a second associated with an activation of the APC/beta-catenin pathway, these two pathways being not mutually exclusive.     

Opening the amino acid toolbox for peptide-based NTS2-selective ligands as promising lead compounds for pain management

Chronic pain is one of the most critical health issues worldwide. Despite considerable efforts to find therapeutic alternatives, opioid drugs remain the gold standard for pain management. The administration of μ-opioid receptor (MOR) agonists is associated with detrimental and limiting adverse effects. Overall, these adverse effects strongly overshadow the effectiveness of opioid therapy. In this context, the development of neurotensin (NT) ligands has shown to be a promising approach for the management of chronic and acute pain. NT exerts its opioid-independent analgesic effects through the binding of two G protein-coupled receptors (GPCRs), NTS1 and NTS2. In the last decades, modified NT analogues have been proven to provide potent analgesia in vivo. However, selective NTS1 and nonselective NTS1/NTS2 ligands cause antinociception associated with hypothermia and hypotension, whereas selective NTS2 ligands induce analgesia without altering the body temperature and blood pressure. In light of this, various structure–activity relationship (SAR) studies provided findings addressing the binding affinity of ligands towards NTS2. Herein, we comprehensively review peptide-based NTS2-selective ligands as a robust alternative for future pain management. Particular emphasis is placed on SAR studies governing the desired selectivity and associated in vivo results.     

Changes in the alpha-adrenoceptors in the medulla oblongata including nucleus tractus solitarii of spontaneously hypertensive rats

The nucleus tractus solitarii (NTS) is a brain stem center mediating depression of blood pressure. In order to elucidate a possible mechanism for the central regulation of blood pressure, we studied noradrenergic indices in the medulla oblongata, a region including the NTS, in spontaneously hypertensive rats (SHR) as compared with normotensive controls of the Wistar Kyoto strain (WKY) at 12 weeks of age. The medulla oblongata was the only brain region showing a significantly low noradrenaline level in the SHR as compared with WKY rats; the level is also significantly decreased at 8 weeks of age. The alpha 1-adrenergic binding sites, as measured with 2-(2, 6-dimethoxy) phenoxyethylamine-methylbenzodioxan [³H]WB4101 showed significant increases inK _D andB _max values in medulla oblongata homogenates from rats of both strains from 4–12 weeks after birth, with no significant interstrain difference. On the other hand, theK _D andB _max of the alpha 2-sites, measured by [³H]yohimbine binding, were reduced in SHR as compared to WKY animals, even at 4 weeks after birth when hypertension was not yet apparent. As expected, the relatively selective alpha 2-antagonist, clonidine, was a potent inhibitor of [³H]yohimbine binding but not of [³H]WB4101 binding in these homogenates. The results suggest that some genetic disorder in the alpha 2-adrenergic transmission system in the NTS region may be involved in the development of hypertension in the SHR rats.Dedicated to Professor Yasuzo Tsukada.     

Distinct regions of C-terminus of the high affinity neurotensin receptor mediate the functional coupling with pertussis toxin sensitive and insensitive G-proteins

The functional coupling of C-terminally truncated mutants of the high affinity rat neurotensin (NT) receptor (NTS1) was characterized in transfected Chinese hamster ovary cells. On cells expressing NTRDelta372 (truncated NTS1 lacking the entire 52 amino acid C-terminus), NT failed to promote [(35)S]guanosine 5'-[gamma-(35)S]triphosphate binding whereas a robust pertussis toxin (PTx) sensitive response was observed in cells expressing a partially truncated receptor (NTRDelta401 lacking the last 23 residues). Similar results were obtained when measuring the ability of NT to induce the production of arachidonic acid. Since neither deletions impaired the NT-induced phosphoinositide hydrolysis, these results indicate that the membrane proximal region of the C-terminus is specifically involved in the functional coupling of the receptor with PTx sensitive G-proteins. This region was also found to be involved in the control of receptor internalization. However, PTx failed to impair internalization, indicating that these two properties are not directly related.     

Impact of polyphenols on receptor–ligand interactions by NMR: the case of neurotensin (NT)–neurotensin receptor fragment (NTS1) complex

Abstract

Ligand–receptor interactions can be implicated in many pathological events such as chronic neurodegenerative diseases. Thus, the discovery of molecules disrupting this type of interactions could be an interesting therapeutic approach. Polyphenols are well known for their affinity for proteins and several studies have characterized these direct interactions. But studying the direct influence of multi-therapeutic drugs on a ligand–receptor complex relevant to a neurodegenerative disorder is a challenging issue. Solution NMR, molecular modeling and iterative calculations were used to obtain information about the interaction between a phenolic compound, α-glucogallin (α-2) and a ligand/fragment receptor complex neurotensin (NT) and its receptor NTS1. The α-2 was shown to bind to NT and a peptidic fragment of its NTS1 receptor, independently. Although the formation of the corresponding ligand–receptor complex did not seem to be affected, this experimental modeling protocol will enable the evaluation of other anti-amyloidogenic compounds such as blockers of NT–NTS1 binding. These types of studies help in understanding the specificity and influence in binding and can provide information to develop new molecules with a putative pharmacological interest.

Communicated by Ramaswamy H. Sarma     

Interactions between neurotensin receptors and G proteins

Three neurotensin (NT) receptors have been cloned to date, two of which, NTS1 and NTS2, belong to the family of seven transmembrane domain receptors coupled to G proteins (GPCRs). NTS1 and NTS2 may activate multiple signal transduction pathways, involving several G proteins. However, whereas NT acts as an agonist towards all NTS1-mediated pathways, this peptide may exert either agonist or antagonist activities, depending on the NTS2-mediated pathway in question. Studies on these receptors reinforce the concept of independence between multiple signals potentially mediated through a single GPCR, generating a wide diversity of functional responses depending on the host cell and the ligand.     

NTS2-selective neurotensin mimetics with tetrahydrofuran amino acids

Stimulation of the NTS2 neurotensin receptor causes antipsychotic effects and leads to a promotion of the μ-opioid-independent antinociception, which is important in the modulation of tonic pain sensitivity. We report the synthesis and properties of a small library of peptidic agonists based on the active neurotensin fragment NT(8–13). Two tetrahydrofuran amino acid derivatives were synthesized to replace Tyr¹¹ in NT(8–13). Additionally, Arg⁸, Arg⁹, and Ile¹² of the lead peptide were exchanged by Lys, Lys, and Gly, respectively. The new compounds showed substantial NTS2 binding affinity and up to 1000-fold selectivity over NTS1. The highest selectivity (K_i(NTS2): 29 nM, K_i(NTS1): 35,000 nM) was observed for the peptide analog 17R^trans.     

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 NT's 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.     

Binding of [3H]Neurotensin in Human Brain: Properties and Distribution

The binding of [3H]neurotensin to membranes from human brain at 0 degrees C was specific, saturable, and reversible. In the frontal cortex, the equilibrium dissociation constant (KD) for [3H]neurotensin determined from the ratio of rate constants (k-1/k1), saturation isotherms, and inhibition binding experiments was 0.80, 2.0, and 2.0 nM, respectively, and the maximum number of binding sites (Bmax) from the saturation isotherms and the competitive binding experiments was 2.4 and 2.2 pmol/g of tissue, respectively. Hill coefficients for binding were equal to 1, indicating the presence of single, noncooperative binding sites. Inhibition of specific binding of [3H]-neurotensin by several analogs of neurotensin showed that [Gln4]neurotensin and neurotensin(8-13) had the highest affinities for these binding sites in human frontal cortex, with each analog being approximately 13-fold more potent than neurotensin. In addition, these data showed that the carboxy-terminal portion of neurotensin played an important part in the binding of this neuropeptide in human brain, a result described for other species. Regional distribution of binding sites was different from that reported for animal brains. Of the 33 different regions investigated, the uncus and substantia nigra showed the highest specific binding of [3H]neurotensin, whereas such areas as the pineal body, medulla, and corpus callosum had few binding sites.     

大鼠延髓至下丘脑腹内侧核的儿茶酚胺能投射神经元在胃伤害性刺激后的Fos表达

本实验用ＨＲＰ注入下丘脑腹内侧核结合逆行追踪与抗ＦＯＳ蛋白和抗酪氨酸羟化酶（ＴＨ）抗血清双重免疫细胞化学相结合的三重标记方法,对大鼠孤束核和延髓腹外侧区至下丘脑腹内侧核的儿茶酚胺能投射神经元在胃伤害性刺激后的ｃ－ｆｏｓ表达进行了观察。本文发现孤束核和延髓腹外侧区有七种不同的标记细胞：ＨＲＰ、Ｆｏｓ、ＴＨ单标细胞Ｆｏｓ／ＨＲＰ、Ｆｏｓ／ＴＨ、ＨＲＰ／ＴＨ双标细胞和Ｆｏｓ／ＨＲＰ／ＴＨ三标细胞。上述七种标记细胞主要分布在延髓中段和尾段孤束核的内侧亚核和延髓腹外侧区以及两者之间的网状结构。ＨＲＰ标记细胞以注射侧为主,对侧有少量分布。本文结果证明,大鼠孤束核、延髓腹外侧区和网状结构内儿茶酚胺能神经元有些至下丘脑腹内侧核的投射,其中一部分儿茶酚胺能神经元参与了胃伤害性刺激的传导和调控。     

Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors.

We have developed a neurotensin analog, L-[3,1'-naphthylalanine11]NT(8-13), NT34, that can distinguish between rat and human neurotensin receptors, and exhibits more than a 100-fold difference in binding affinities and a 60-fold difference in functional coupling to phosphatidylinositol turnover. Using cells transfected with different numbers of the appropriate receptors, we measured the changes in phosphatidylinositol production, and then evaluated the efficiency of receptor-effector coupling based on Furchgott's design. The binding of NT34 at both rat and human neurotensin receptors stably expressed in CHO-K1 cells was to two sites, while the binding of NT was to one site. At the rat receptor the equilibrium dissociation constant (Kd) for NT34 at the high-affinity site was 0.058 nM, while that at the low-affinity site was 3.1 nM. For the human receptor at the high-affinity site, the Kd for NT34 was 18 nM, while that at the low-affinity site was 180 nM. For both species the percentage of receptors representing the high-affinity site was approximately 60-70% with 30-40% at the low-affinity site. We derived agonist dissociation constants (Ka) for NT and NT34, which suggest that for NT34, the low-affinity site is functionally coupled to phosphatidylinositol turnover. Finally, we compared the relative efficacies of both compounds and found that NT34 was about 2-fold and 4-fold more efficacious than NT in stimulating phosphatidylinositol turnover in rat and human NT receptors, respectively.     

Quantitation and characterization of thyrotropin-releasing hormone in vagal nuclei and other regions of the medulla oblongata of the rat

Abstract: Since evidence is now available to support a nonendocrine autonomic function for thyrotropin-releasing hormone (TRH), quantitative measurements of TRH were made in nuclei of the vagal complex and other areas of the caudal medulla oblongata of the rat. Regions containing the dorsal motor nucleus of the vagus (DMN), nucleus tractus solitarius (NTS), hypoglossal nucleus, dorsal column nuclei, descending nucleus V (DNV), nucleus ambiguus (NA), raphe nuclei (MR) dorsomedial and ventromedial reticular formation, and inferior olivary nuclei were isolated from 300-μm-thick frozen sections of medulla by the micropunch technique. Each region was pooled bilaterally, homogenized in 0.1 M HCl, and vacuum-dried. Extracts were assayed for TRH by specific radioimmunoassay (RIA). TRH levels varied 100-fold among medulla nuclei. Highest content (ng/mg protein ± SEM) was found in DMN (14 ± 1.38) and NTS (4.7 ± 0.68), whereas lowest levels occurred in the DNV and MR (0.13, 0.06). Nearly 65% of the total medullary TRH was localized in nuclei associated with vagal complex (DMN, NTS, NA). Characterization of tissue immunoreactivity (TRHi) in these regions suggests the presence of TRH, since (1) medullary tissue extracts competed with ¹²⁵I-TRH for antibody binding sites with the same affinity as authentic TRH; (2) TRHi in tissue extracts co-migrated with synthetic TRH when subjected to reverse-phase high performance liquid chromatography and Sephadex G-10 chromatography; and (3) rat serum TRH peptidases degraded TRHi and authentic TRH at similar rates. Another group of rats was subjected to unilateral (right side) vagotomy. At 33 weeks post-vagotomy, the vagal preganglionic cell population in the ipsilateral DMN was depleted 50–75%, while the contralateral side was unaffected. Interestingly, the content of TRH in the ipsilateral (right) DMN remained unchanged, whereas TRH in the contralateral DMN increased by 50%. In contrast, TRH was significantly elevated in the NA on the ipsilateral side of the lesion. TRH in both ipsi- and contralateral NTS was unchanged when compared with sham-operated controls. These results indicate that (1) TRH is present in several specific loci of the medulla; (2) very high levels are found in the vagal complex; and (3) vagotomy may alter TRH in the contralateral DMN and ipsilateral NA.     

Increased number of muscarinic binding sites in brain following chronic barbiturate treatment to rat

Rats received as their only drinking fluid a solution of sodium barbital (3.33 mg/ml) for more than 40 weeks. In two groups (A3, A12) the barbital solution was withheld and replaced by water 3 and 12 days before sacrifice. Two other groups consisted of animals drinking barbital until sacrifice (B) and untreated controls (C). Synaptosomes from different parts of the brain were incubated with radioactive quinuclidinyl benzilate (³H-QNB) (0.2 nM) for 60 min. A significantly increased number of ³H-QNB binding sites was found in the striatum and midbrain + medulla oblongata + cerebellum of rats abstinent for 3 days (A3) in comparison with controls (C). Saturation studies indicated that group A3 had significantly more receptors in the midbrain + medulla oblongata + cerebellum than group C, while there was no differences in receptor affinity.     

Evidence for the dual coupling of the rat neurotensin receptor with pertussis toxin-sensitive and insensitive G-proteins

We previously demonstrated the functional coupling of the rat neurotensin receptor NTS1 with G-proteins on transfected CHO cell homogenates by showing modulation of agonist affinity by guanylyl nucleotides and agonist-mediated stimulation of [(35)S]GTP gamma S binding. In the present study, we observed that G(i/o)-type G-protein inactivation by pertussis toxin (PTx) resulted in a dramatic reduction of the NT-induced [(35)S]GTP gamma S binding whereas the effect of guanylyl nucleotide was almost not affected. As expected, NT-mediated phosphoinositide hydrolysis and intracellular calcium mobilization were not altered after PTx treatment. This suggests the existence of multiple signaling cascades activated by NT. Accordingly, using PTx and the PLC inhibitor U-73122, we showed that both signaling pathways contribute to the NT-mediated production of arachidonic acid. These results support evidence for a dual coupling of the NTS1 with PTx-sensitive and insensitive G-proteins.     

Stability of the Neurotensin Receptor NTS1 Free in Detergent Solution and Immobilized to Affinity Resin

Background

Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1.

Methods and Findings

To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [³H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein.

Conclusion

Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.