Non-peptide Neurotensin Receptor Research Articles

The amide linker in nonpeptide neurotensin receptor ligands plays a key role in calcium signaling at the neurotensin receptor type 2.

Compounds acting via the GPCR neurotensin receptor type 2 (NTS2) display analgesia in relevant preclinical models. The amide bond in nonpeptide NTS1 antagonists plays a central role in receptor recognition and molecular conformation. Using NTS2 FLIPR and binding assays, we found that it is also a key molecular structure for binding and calcium mobilization at NTS2. We found that reversed amides display a shift from agonist to antagonist activity and provided examples of the first competitive nonpeptide antagonists observed in the NTS2 FLIPR assay. These compounds will be valuable tools for determining the role of calcium signaling in vitro to NTS2 mediated analgesia.

The discovery of indole full agonists of the neurotensin receptor 1 (NTSR1)

Neurotensin (NT) is an endogenous tridecapeptide found in the central nervous system (CNS) and in peripheral tissues. Neurotensin exerts a wide range of physiological effects and it has been found to play a critical role in a number of human diseases, such as schizophrenia, Parkinson’s disease and drug addiction. The discovery of small-molecule non-peptide neurotensin receptor (NTSR) modulators would represent an important breakthrough as such compounds could be used as pharmacological tools, to further decipher the cellular functions of neurotensin, and potentially as therapeutic agents to treat human disease. Herein, we report the identification of non-peptide low-micromolar neurotensin receptor 1 (NTSR1) full agonists, discovered through structural optimization of the known NTSR1 partial agonist 1. In vitro cellular screenings, based on an intracellular Ca2+ mobilization assay, revealed our best hit molecule 8 (SR-12062) to have an EC50 of 2μM at NTSR1 with full agonist behaviour (Emax=100%), showing a higher efficacy and ∼90-fold potency improvement compared to parent compound 1 (EC50=178μM; Emax=17%).

Neurotensin causes tyrosine phosphorylation of focal adhesion kinase in lung cancer cells

The effects of neurotensin on focal adhesion kinase were investigated using lung cancer cells. Neurotensin bound with high affinity to large cell carcinoma cell line NCI-H1299 as did neurotensin-(8–13), but not neurotensin-(1–7) or levocabastine. Addition of 100 nM neurotensin to NCI-H1299 cells caused transient tyrosine phosphorylation of focal adhesion kinase which was maximal after 1–2.5 min. Also, neurotensin-(8–13), but not neurotensin-(1–8) or levocabastine, caused tyrosine phosphorylation of focal adhesion kinase after addition to NCI-H1299 cells. Focal adhesion kinase tyrosine phosphorylation caused by neurotensin was inhibited by the nonpeptide neurotensin receptor antagonist (2-(1-(7-chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1 H-pyrazole-3-carbonyl)amino)-adamantane-2-carboxylic acid) (SR48692). SR48692 inhibited the clonal growth of NCI-H1299 cells, whereas neurotensin-stimulated proliferation and levocabastine had no effect. These results indicate that lung cancer cells have functional neurotensin receptors which regulate focal adhesion kinase tyrosine phosphorylation. It remains to be determined if neurotensin receptors and focal adhesion kinase plays a role in lung cancer cellular adhesion and migration.

Neurotensin: an endogenous psychostimulant?

The neuropeptide neurotensin has been shown to be closely related to central dopaminergic regulation. Although emphasis has been put on its possible neuroleptic-like effects, more recent data--in particular with the use of non-peptide neurotensin receptor antagonists--suggest instead a psychostimulant-like action of endogenous neurotensin. Such contradictory potential effects open an important debate on the possible therapeutic use of compounds related to neurotensin transmission.

Chronic Blockade of Neurotensin Receptors Strongly Reduces Sensitized, but Not Acute, Behavioral Response to D-amphetamine

This study investigated the effect of a chronic blockade of neurotensin (NT) receptors on the sensitized behavioral response to amphetamine using a nonpeptide NT receptor antagonist, SR 48692. Rats received four injections of D-amphetamine (0.5 or 1 mg/kg, IP) every other day (day 1, 3, 5 and 7) and were then challenged with the same dose of amphetamine after a 6-day withdrawal (day 14) to establish the presence of locomotor sensitization. Daily administration of SR 48692 (1 mg/kg, IP) throughout the amphetamine regimen (day 1 to day 14) almost completely blocked the sensitized locomotor response to amphetamine without affecting stereotyped behaviors (experiment 1). The decreased amphetamine-induced sensitization in chronically SR 48692-treated rats did not appear to result from an influence on basal locomotor activity, as chronic SR 48692 treatment did not modify the spontaneous locomotor activity developed in response to mild stresses (experiment 2). Moreover, we showed that chronic pretreatment with SR 48692 (1 mg/kg, 14 daily IP injections) had no effect on the locomotor activation induced by a single IP administration of amphetamine (experiment 3). These data suggest that a sustained blockade of NT receptors considerably reduces the sensitized behavioral response to amphetamine without altering the acute effect of this psychostimulant or the locomotor activation induced by a mild stress. This ability of SR 48692 to specifically reduce the behavioral sensitization to amphetamine suggests that NT receptor antagonists could have potential clinical utility in the treatment of some psychiatric disorders.

Nigral neurotensin receptor regulation of nigral glutamate and nigroventral thalamic GABA transmission: a dual-probe microdialysis study in intact conscious rat brain

Dual-probe microdialysis in the awake rat was employed to investigate the effects of intranigral perfusion with the tridecapeptide neurotensin on local dialysate glutamate and GABA levels in the substantia nigra pars reticulata and on dialysate GABA levels in the ventral thalamus. Intranigral neurotensin (10–300 nM, 60 min) dose-dependently increased (+29±3% and +46±3% vs basal for the 100 and 300 nM concentrations, respectively) local dialysate glutamate levels, while the highest 300 nM concentration of the peptide exerted a long-lasting and prolonged reduction in both local and ventral thalamic (−20±4% and −22±2%, respectively) GABA levels. Intranigral perfusion with the inactive neurotensin fragment neurotensin(1–7) (10–300 nM, 60 min) was without effect. Furthermore, the non-peptide neurotensin receptor antagonist SR 48692 (0.2 mg/kg) and tetrodotoxin (1 μM) fully counteracted the intranigral neurotensin (300 nM)-induced increase in local glutamate. SR 48692 (0.2 mg/kg) also counteracted the decreases in nigral and ventral thalamic GABA release induced by the peptide. In addition, intranigral perfusion with the dopamine D 2 receptor antagonist raclopride (1 μM) fully antagonized the neurotensin (300 nM)-induced decreases in nigral and ventral thalamic GABA levels. The ability of nigral neurotensin receptor activation to differently influence glutamate and GABA levels, whereby it increases nigral glutamate and decreases both nigral and ventral thalamic GABA levels, suggests the involvement of neurotensin receptor in the regulation of basal ganglia output at the level of the nigra.

SR48692 is a neurotensin receptor antagonist which inhibits the growth of small cell lung cancer cells

Neurotensin (NT) is an autocrine growth factor for some small cell lung cancer (SCLC) cells. In this communication, the effects of a non-peptide NT receptor antagonist, SR48692, were investigated using SCLC cells. 3H-SR48692 bound with high affinity (IC 50 = 20 nM) to NCI-H209 cells. Also, NT and SR48692 inhibited specific 125I-NT binding with high affinity (IC 50 values of 2 and 200 nM). In contrast, the NT 2 receptor agonist, levocabastine, had little effect on specific 125I-NT binding, second messenger production and proliferation using NCI-H209 cells. SR48692 (5 μM) antagonized the ability of NT (10 nM) to cause elevated cytosolic Ca 2+ in Fura-2 AM loaded NCI-H209 cells. SR48692 antagonized the ability of NT to cause elevation of c-fos mRNA in these cells. Using a MTT proliferation assay, SR48692 inhibited NCI-H209 and H345 proliferation in a concentration-dependent manner. Using a clonogenic assay, 1 μM SR48692, reduced NCI-H209 colony number. Also, SR48692 (0.4 mg/kg per day) inhibited NCI-H209 xenograft proliferation in nude mice. These results suggest that SR48692 is a NT 1 receptor antagonist which inhibits SCLC growth.

Neurotensin stimulates Cl− secretion in human colonic mucosa in vitro: Role of adenosine

Background & Aims: Previous studies indicated that the peptide neurotensin (NT) stimulates Cl− secretion in animal small intestinal mucosa in vitro. In this study, we investigated whether NT causes Cl− secretion in human colonic mucosa and examined the mechanism of this response. Methods: Human mucosal preparations mounted in Ussing chambers were exposed to NT. Drugs for pharmacologic characterization of NT-induced responses were applied 30 minutes before NT. Results: Serosal, but not luminal, administration of NT (10−8 to 10−6 mol/L) induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive short-circuit current (Isc) increase that was inhibited by the specific nonpeptide NT receptor antagonists SR 48692 and SR 142948A, the neuronal blocker tetrodotoxin, and the prostaglandin synthesis inhibitor indomethacin. The mast cell stabilizer lodoxamide and the histamine 1 and 2 receptor antagonists pyrilamine and ranitidine, respectively, did not significantly alter NT-induced Isc increase. In contrast, the adenosine receptor 1 and 2 antagonists inhibited this secretory response, whereas the adenosine uptake inhibitors S-(4-nitrobenzyl)-6-thioguanosine and S-(4-nitrobenzyl)-6-thioinosine and the adenosine deaminase inhibitor deoxycoformycin potentiated NT-induced Isc increase. Serosal adenosine induced a rapid, monophasic, concentration- and chloride-dependent, bumetanide-sensitive Isc increase. Conclusions: NT stimulates chloride secretion in human colon by a pathway(s) involving mucosal nerves, adenosine, and prostaglandins.GASTROENTEROLOGY 2000;119:348-357

Blockade of neurotensin receptors suppresses the dopamine D1/D2 synergism on immediate early gene expression in the rat brain.

A remarkable feature of dopamine functioning is that the concomitant activation of D1-like and D2-like receptors acts to intensify the expression of various dopamine-dependent effects, in particular the expression of the immediate-early genes, c-fos and zif268. Using non-peptide neurotensin receptor antagonists, including SR48692, we have determined that blockade of neurotensin receptors reduced the cooperative responses of direct acting D2-like (quinpirole) and partial D1-like (SKF38393) dopamine agonists on the expression of Fos-like antigens and zif268 mRNA. Pretreatment with SR48692 (3 and 10 mg/kg) reduced the number of Fos-like immunoreactive cells produced by the combined administration of SKF38393 (20 mg/kg) and quinpirole (1 mg/kg) in the caudate-putamen, nucleus accumbens, globus pallidus and ventral pallidum. High-affinity neurotensin receptors are likely to be involved in these D1-like/D2-like cooperative responses, as compounds structurally related to SR48692, SR48527 (3 mg/kg) and its (-)antipode, SR49711 (3 mg/kg), exerted a stereospecific antagonism in all selected brain regions. Pretreatment with SR48692 (10 mg/kg) also diminished Fos induction by the indirect dopamine agonist, cocaine (25 mg/kg), particularly at the rostral level of the caudate-putamen. In situ hybridization experiments in the caudate-putamen indicated that SR48692 (10 mg/kg) markedly reduced zif268 mRNA labelling produced by SKF38393 plus quinpirole in cells not expressing enkephalin mRNA, but was unable to affect the concomitant decrease of zif268 mRNA labelling in enkephalin-positive cells. Taken together, the results of the present study indicate that neurotensin is a key element for the occurrence of cooperative responses of D2-like and partial D1-like agonists on immediate-early gene expression.

Neurotensin and a non-peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice.

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10(-10) to 10(-6) M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (I-II) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54 micromol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 micromol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer.

Electrophysiological evidence for putative subtypes of neurotensin receptors in guinea-pig mesencephalic dopaminergic neurons

Electrophysiologically identified mesencephalic dopaminergic neurons were examined by means of extra- and intracellular microelectrodes in coronal slices of guinea-pig brain. Neurotensin and its C-terminal fragment (8–13) were equipotent in the enhancement of spontaneous neuronal firing rate ( ec 50 values 81.9 and 72.6 nM, respectively). The duration of response was significantly longer and more variable for neurotensin compared to neurotensin fragment (8–13) (mean half-time of recovery 423±44 and 100±14 s, respectively, for peptides applied at 300 nM). The initial fast phase of excitatory responses to neurotensin receptor agonists was associated with membrane depolarization (when assessed in current-clamp mode) or with inward currents (when assessed in voltage-clamp mode), whereas prolonged excitation was associated with a slowly occurring and long-lasting change in the late afterhyperpolarization. Two kinetically distinct components were revealed in responses to neurotensin and neurotensin fragment (8–13) by the use of SR48692 and SR142948, two selective non-peptide neurotensin receptor antagonists. SR142948 (100 nM) potently antagonized responses to both agonists [response was reduced by 66±5% and 74±9% for neurotensin and neurotensin fragment (8–13), respectively] and caused a rightward shift in the concentration–response curve for neurotensin. On the other hand, SR48692 (100 nM) selectively inhibited the slow (late afterhyperpolarization-dependent) component, without altering the response amplitude; the half-time of recovery was reduced by 71±6% and 65±5% of control values for responses induced by neurotensin (300 nM) and neurotensin fragment (8–13) (300 nM), respectively. In addition, neurotensin, but not neurotensin fragment (8–13), provoked SR48692-sensitive and long-lasting attenuation of dopamine-induced inhibitory responses. It is suggested that two subtypes of neurotensin receptors are present in dopaminergic neurons, based on the differences in agonist and antagonist sensitivity, kinetic properties and the membrane mechanisms involved.

Neurotensin and nonpeptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice

Neurotensin and nonpeptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice

Characterization of biphasic modulation of spinal nociceptive transmission by neurotensin in the rat rostral ventromedial medulla.

Modulation of spinal nociceptive transmission by neurotensin microinjected in the rostral ventromedial medulla (RVM) was examined in anesthetized, paralyzed rats. Forty-three spinal dorsal horn neurons in the L3-L5 spinal segments responding to mechanical and noxious thermal stimulation (50 degrees C) of the plantar surface of the ipsilateral hind foot were studied. Spinal units were classified as either wide dynamic range or nociceptive specific and were located in spinal laminae I-V. Microinjection of neurotensin (0.03 pmol/0.2 microl) into the RVM produced a significant facilitation (135% of control) of spinal unit responses to noxious thermal stimulation (50 degrees C) that lasted approximately 12 min. In contrast, injection of greater doses of neurotensin (300 or 3,000 pmol) produced an inhibition of spinal unit responses to noxious heat (51.7 and 10.6% of control, respectively) that had a longer duration (60-120 min). The effects of neurotensin on wide-dynamic-range and nociceptive-specific neuron responses to noxious heat were qualitatively and quantitatively similar. Spinal unit responses to graded heating of the skin (42-50 degrees C) were completely inhibited after microinjection of 3,000 pmol of neurotensin into the RVM. Injection of a lesser dose of neurotensin (300 pmol), however, resulted in a partial inhibition of spinal unit responses and significantly reduced the slope of the stimulus-response function to graded heating of the skin. Transection of either the ipsilateral or contralateral dorsolateral funiculus (DLF) significantly reduced the inhibition of spinal nociceptive transmission produced by neurotensin (3,000 pmol) in the RVM, whereas bilateral transection of the DLFs completely blocked the effect. In contrast, bilateral transection of the DLFs had no effect on facilitation of spinal nociception by neurotensin (0.03 pmol) in the RVM. The inhibition of spinal nociceptive transmission by neurotensin (3,000 pmol) in the RVM was completely blocked by injection of the nonpeptide neurotensin receptor antagonist SR48692 (30 fmol) into the RVM 10 min before neurotensin. To confirm a specific block of neurotensin-receptor-mediated effects by the antagonist, a subsequent injection of L-glutamate into the RVM was performed. L-Glutamate (100 nmol) was found to inhibit the nociceptive responses of those spinal units whose responses were no longer inhibited by neurotensin. In contrast, injection of SR48692 (30 fmol) into the RVM failed to block the facilitation of spinal unit responses to noxious heat produced by a subsequent injection of neurotensin (0.03 pmol) into the same site. The present series of experiments demonstrate a specific role for neurotensin in the RVM in the modulation of spinal nociceptive transmission, because the peptide was found to both facilitate and inhibit spinal neuron responses to noxious thermal stimulation. Additionally, the facilitatory and inhibitory effects of neurotensin appear to occur via interaction with multiple neurotensin receptors in the RVM that activate independent systems that descend in the ventrolateral funiculi and DLFs, respectively. The results from these experiments are consistent with prior studies demonstrating that the RVM both facilitates and inhibits spinal nociceptive transmission, and they complement previous work showing that neurotensin in the RVM modulates spinal nociceptive behavioral responses.

Agonist and antagonist modulation of [35S]-GTP gamma S binding in transfected CHO cells expressing the neurotensin receptor.

1. The functional interaction of the cloned rat neurotensin receptor with intracellular G-proteins was investigated by studying the binding of the radiolabelled guanylyl nucleotide analogue [35S]-GTP gamma S induced by neurotensin to membranes prepared from transfected Chinese hamster ovary (CHO) cells. 2. The agonist-induced binding of [35S]-GTP gamma S was only detected in the presence of NaCl in the incubation buffer. However, it was also demonstrated that the binding of [3H]-neurotensin to its receptor was inhibited by NaCl. In the presence of 50 mM NaCl, the binding of the labelled nucleotide was about 2 fold increased by stimulation with saturating concentrations of neurotensin (EC50 value of 2.3 +/- 0.9 nM). 3. The stimulation of [35S]-GTP gamma S binding by neurotensin was mimicked by the stable analogue of neurotensin, JMV-449 (EC50 value of 1.7 +/- 0.4 nM) and the neurotensin related peptide neuromedin N (EC50 value of 21 +/- 6 nM). 4. The NT-induced [35S]-GTP gamma S binding was competitively inhibited by SR48692 (pA2 value of 9.55 +/- 0.28), a non-peptide neurotensin receptor antagonist. SR48692 alone had no effect on the specific binding of [35S]-GTP gamma S. 5. The response to neurotensin was found to be inhibited by the aminosteroid U-73122, a putative inhibitor of phospholipase C-dependent processes, indicating that this drug may act at the G-protein level. 6. Taken together, these results constitute the first characterization of the exchange of guanylyl nucleotides at the G-protein level that is induced by the neuropeptide neurotensin after binding to its receptor.

Inhibition of neurotensin-induced pancreatic carcinoma growth by a nonpeptide neurotensin receptor antagonist, SR48692.

Recently, a nonpeptide neurotensin (NT) receptor antagonist, SR48692, was developed that selectively antagonizes the high affinity, biologically active NT binding site. The effect of SR48692 on NT-mediated growth of a human pancreatic carcinoma, MIA PaCa-2, was determined both in vitro and in vivo. (125)I-NT binding and Northern blot analyses were performed for evaluation of the NT receptor in MIA PaCa-2 cells. Intracellular calcium ([Ca2+]i) mobilization and inositol phosphate (IP3) levels were measured. Cell growth studies were performed by counting cell numbers. Athymic nude mice were inoculated with MIA PaCa-2 cells and randomized into four groups to receive either vehicle (NT or SR48692) or NT + SR48692. MIA PaCa-2 cells possess both a high affinity, SR48692-sensitive and a levocabastine-insensitive NT binding site; Northern blot analysis demonstrated expression of the NT receptor. SR48692 inhibited [Ca2+]i mobilization, IP3 turnover, and MIA PaCa-2 cell growth induced by NT in a dose-dependent fashion. In in vivo experiments, NT significantly increased the size, weight, and DNA and protein content of xenografted MIA PaCa-2 tumors; SR48692 inhibited the effect of NT. The novel NT receptor antagonist SR48692 will be a valuable agent to delineate further the cellular mechanisms responsible for peptide-mediated growth of normal and neoplastic gut tissues.

Neurotensin induces the release of prostacyclin from human umbilical vein endothelial cells in vitro and increases plasma prostacyclin levels in the rat

Human umbilical vein endothelial cells express high affinity neurotensin receptors which are coupled to phosphoinositide turnover and 45Ca 2+ efflux ( Schaeffer et al., 1995. J. Biol. Chem. 270, 3409-3413). In order to assess the physiological significance of neurotensin receptor activation in endothelial cells, we have compared the in vitro effect of neurotensin on prostacyclin release and cytosolic free calcium increase ([Ca 2+] i) as determined by fura-2 fluorescence experiments to the in vivo effect of neurotensin on blood pressure and haematocrit. Neurotensin increased [Ca 2+] i levels at low concentrations (EC 50=4.2±0.2 nM, n=3). At similar concentrations, neurotensin was also able to induce prostacyclin release from human umbilical vein endothelial cells (EC 50=14±1 nM, n=3) as determined by a 6-keto-prostaglandin F 1α enzyme immunoassay. The neurotensin (100 nM)-induced [Ca 2+] i increase and prostacyclin release were inhibited by the specific non-peptide neurotensin receptor antagonist SR 48692 at similar concentrations (IC 50=41±16 nM and 86±17 nM, respectively, n=3), confirming that these responses were mediated by high affinity neurotensin receptors. Intravenous injection of neurotensin (1–4 nmol/kg i.v.) in the rat resulted in a drop of blood pressure and increased haematocrit, and nearly doubled the plasma levels of 6-keto-prostaglandin F 1α, the stable metabolite of prostacyclin. Whereas indomethacin (10 mg/kg i.v.) pretreatment significantly reduced the effect of neurotensin on blood pressure, it did not alter its effect on haematocrit. These results suggest that prostacyclin release plays a role in the hypotensive effects of neurotensin, but is not involved in its effects on haematocrit. © Elsevier Science B.V. All rights reserved.

In vivo regulation of neurotensin receptors following long-term pharmacological blockade with a specific receptor antagonist

Adaptive changes in brain neurotensin (NT) receptors were investigated in rats after repeated administration of SR 48692, a potent and selective non-peptide NT receptor antagonist. Administration of SR 48692 (1 mg/kg i.p.) for 15 days did not alter NT content in the brain but highly enhanced the expression of NT receptor mRNA as shown by quantitative in situ hybridization. The increase of the signal was observed in numerous areas of the brain, such as the anterior cingulate, perirhinal and retrosplenial cortices, the suprachiasmatic nucleus, the ventral tegmental area, the substantia nigra and the posterior cortical nucleus of the amygdaloid complex. Moreover, the SR 48692 treatment induced the expression of NT receptor mRNA in several nuclei of the diencephalon where it could not be detected in basal conditions. Immunoblot analysis with a specific antibody directed against the rat cloned NT receptor revealed an important increase in NT receptor protein in the brain of SR 48692-treated rats, correlating well with the increase in NT receptor mRNA levels. Surprisingly, the number and the affinity constant of NT binding sites determined on brain membrane homogenates remained unchanged after SR 48692 treatment, even after membrane permeabilization with low concentrations of digitonin. These results suggest that chronic treatment with a specific NT antagonist induces an up-regulation of NT receptors at the level of mRNA and protein. Moreover, they indicate that after a chronic treatment with SR 48692, the number of NT binding sites remains stable in contrast to what is observed after 5-day treatment or with central monoaminergic receptor following their long-term blockade.

Neurotensin induces Fos and Zif268 expression in limbic nuclei of the rat brain

The endogenous tridecapeptide neurotensin exerts a wide range of behavioral, electrophysiological and neurochemical effects when administered directly into the brain. These effects are thought to result from the activation of distinct populations of neurotensin receptors distributed throughout the central nervous system. We have mapped the sites of functional change in the rat brain associated with the central administration of neurotensin using the induction of the nuclear protein products of the immediate early genes c-fos and zif268 as markers of cellular activation. The administration of neurotensin into the lateral ventricle of rats produced an increase in the number of nuclei positive for Fos and Zif268 immunoreactivity in the central and basolateral nuclei of the amygdala and the paraventricular and supraoptic nuclei of the hypothalamus. Neurotensin also produced an increase in serum corticosterone concentration and decrease in body temperature. The intraperitoneal administration of SR48692, a non-peptide neurotensin receptor antagonist, blocked the neurotensin-induced corticosterone secretion and significantly reduced the number of neurotensin-induced Fos-positive and Zif268-positive neurons in the amygdaloid complex. A significant positive correlation wash found between the number of Fos-positive nuclei in the central or basolateral nucleus of the amygdala and the serum corticosterone concentration. A significant positive correlation was also found between the number of Zif-positive cells in the paraventricular nucleus of the hypothalamus and change in body temperature following treatment. Our findings indicate that the central role of neurotensin in increasing serum corticosterone involves the induction of Fos in the central and basolateral nuclei of the amygdala. In contrast, the neurotensin-induced hypothermia, which was unaffected by pretreatment with SR48692, involves Zif induction in the paraventricular nucleus of the hypothalamus. These data support further the existence of central neurotensin receptor subtypes which may regulate distinct immediate early genes.

Indirect inhibitory effect of a neurotensin receptor antagonist on human colon cancer (LoVo) growth

The effect of the nonpeptide neurotensin (NT) receptor antagonist SR48692 on NT-mediated growth of xenografted human colon cancers (LoVo) was determined. Sixty-four athymic nude mice, inoculated with LoVo cells at a single site, were randomized into four groups of 16 mice each to receive either vehicle NT (600 microg/kg), SR48692 (2 mg/kg) or NT+SR48692 administered s.c., t.i.d., for 25 days. Treatment with NT significantly stimulated LoVo tumour growth, weight, DNA and protein content; SR48692 blocked this NT-mediated effect but had no effect when administered as a single agent. In addition, normal jejunum and ileum were removed and assessed. Similar to the effects on LoVo tumours, NT stimulated jejunal and ileal growth; SR48692 blocked this NT-mediated effect. In contrast to our in vivo findings, NT had no effect on LoVo cell growth in vitro. Also, Northern blot analysis demonstrated no expression for the NT receptor in either LoVo tumour cells or xenografted tumours. Our findings suggest that the trophic effect of NT on LoVo may be through an indirect effect; one possibility is that administration of NT may stimulate the release of other trophic factors which then stimulate tumour growth. The nonpeptide NT receptor antagonist SR48692 will be a useful agent to delineate the specific effects of NT on neoplastic as well as normal gastrointestinal tissues.

Effect of nonpeptide neurotensin receptor antagonist (SR48692) on growth of mouse small intestine

Effect of nonpeptide neurotensin receptor antagonist (SR48692) on growth of mouse small intestine