Neurotensin-related peptides inhibit spontaneous longitudinal contractions of porcine distal jejunum.

The tridecapeptide neurotensin (NT) and its C-terminal homologs, including xenopsin (XP) and neuromedin N (NM-N), reduced the amplitude of spontaneous contractions in longitudinal smooth muscle strips from the porcine distal jejunum in vitro. The rank order of potency (IC50 in nM) was XP (0.1) greater than NT (0.9) approximately avian XP (1.0) greater than NM-N (1.6), which could not be explained on the basis of differential peptide degradation. Tachyphylaxis and cross-tachyphylaxis were observed after repeated NT and XP addition to muscle strips. The action of NT was mimicked by norepinephrine (NE), but not by opioid peptides, somatostatin, or vasoactive intestinal peptide. NE was nearly 100-fold less potent than NT and did not produce a state of tachyphylaxis to NT. The effects of NT and NE were unaltered by the neuronal conduction blocker tetrodotoxin (70 nM). However, the actions of NE, unlike those of NT, were reduced by the alpha-adrenoceptor blocker phentolamine (70 nM), the K(+)-channel blocker apamin (7 nM) and the Ca2(+)-channel blocker verapamil (0.7 microM). These results suggest that NT and related peptides, through a nonadrenergic mechanism, interact with smooth muscle receptors to modulate jejunoileal motor function in the pig.     

Ghrelin enhances cue-induced bar pressing for high fat food

Ghrelin is an orexigenic hormone produced by the stomach that acts on growth hormone secretagogue receptors (GHSRs) both peripherally and centrally. The presence of GHSRs in the ventral tegmental area (VTA) suggests that ghrelin signaling at this level may increase the incentive value of palatable foods as well as other natural and artificial rewards. The present investigation sought to determine if ghrelin plays a role in relapse to such foods following a period of abstinence. To achieve this, thirty-six male Long Evans rats were trained to press a lever to obtain a high fat chocolate food reward on a fixed ratio schedule of 1. Following an extinction period during which lever presses were not reinforced, rats were implanted with a cannula connected to a minipump that continuously delivered ghrelin, a GHSR antagonist ([d-Lys-3]-GHRP-6), or saline in the VTA for 14 days. One week later, food reward-associated cues, food reward priming, and an overnight fast were used to induce reinstatement of the lever pressing response. Our results indicate that intra-VTA ghrelin enhances cue-induced reinstatement of responses for palatable food pellets. To the extent that the reinstatement paradigm is considered a valid model of relapse in humans, this suggests that ghrelin signaling facilitates relapse to preferred foods in response to food cues through GHSR signaling in the VTA.     

The electrophysiological actions of neurotensin in the central nervous system

The endogenous neuropeptide, neurotensin (NT) alters the firing frequencies of certain neurons in the central nervous system (CNS). This is one of the findings that support the hypothesis that NT is a neurotransmitter substance. The direct application of NT on CNS neurons causes predominantly excitatory effects. These effects occur in a dose-related fashion via a calcium-dependent postsynaptic mechanism. The C-terminal hexapeptide fragment, NT 8-13 exerts similar electrophysiological effects to NT, while the N-terminal octapeptide fragment, NT 1-8 is devoid of such activity. NT produces a significant increase in the firing rates of individual neurons in the substantia nigra (SN), ventral tegmental area (VTA), medial prefrontal cortex (MPF), hypothalamus, and periaqueductal grey (PAG). This excitation occurs with a rapid onset and is readily reversible after cessation of NT application. In contrast, NT has no effect or weak inhibitory effects on the firing rates of neurons in the locus coeruleus (LC) and cerebellum. These electrophysiological actions of NT appear to be unique and not shared by other neurotransmitter and neuropeptide receptor antagonists and agonists that have been studied via direct co-application. NT attenuates dopamine (DA)-induced inhibition associated with direct application onto neurons in the SN and VTA both in vivo and in vitro. Intracellular recordings suggest that direct application of higher concentrations of NT appears to produce 'depolarization block' on individual neurons in the SN, VTA, MPF, and hypothalamus. The electrophysiological consequences of NT application not only show similarities to clinically efficacious antipsychotic medications, but also demonstrate the ability of NT to modulate the activity of dopamine (DA) neurons at the cellular level via specific NT binding sites. These findings further underscore the possibility that NT may play a pre-eminent role in the pathogenesis of, and psychopharmacological management of neurological and psychiatric disorders purportedly related to perturbation of CNS DA systems including schizophrenia.     

The importance of antioxidant status in gastric intestinal metaplasia

BackgroundOxidative stress status in different cancer types was investigated before, but not studied in gastric intestinal metaplasia to the best of our knowledge. Purpose of this study is to examine whether there is a difference between oxidative stress status in patients with intestinal metaplasia (IM) compared to individuals without IM, we compared the serum levels of disulfide (SS), total thiol (TT) and native thiol (NT).MethodsThis was a prospective, non-randomized casecontrol study including 67 patients with histopathologically confirmed IM and 60 individuals demographically matched in terms of age, gender, BMI, smoking status, and chronic diseases as control group.ResultsThe mean NT, TT and NT to TT (NT/TT) ratios were statistically significantly higher in IM group compared to controls ((351.71 ± 81.9 mol/L vs. 271.82 ± 54.13 mol/L, p=0.000), (391.5 ± 92.69 mol/L vs. 308.59 ± 55.53 mol/L, p=0.000) and (0.89 ± 0.6 vs. 0.87 ± 0.29, p=0.022), respectively). The mean SS to TT (SS/TT) ratio was significantly lower in IM group than control group (0.050 ± 0.31 vs. 0.060 ± 0.014, P=0.022). Median SS and mean SS/NT ratio was similar in both groups (16.3 (3.3-78) vs. 18.3 (10-32.7), p=0.271 and 0.055 ± 0.041 vs. 0.070 ± 0.019, p=0.068, respectively). In ROC analysis, cut off value of SS/NT for IM was found 0.062, in regression analysis, SS/NT <0.062 was found as an independently prognostic marker for IM (OR, 2.38; 95%CI: 1.168-4.865, P=0.017).ConclusionsSS/NT ratio lower than 0.062 was found as an independently prognostic marker for IM. This ratio could help to distinguish which patients should be followed closely for gastric cancer.     

Effects of ICV administration of neurotensin and analogs on EEG in rats

The electroencephalographic (EEG) effects of the ICV administration of neurotensin (NT 1-13), NT 1-8 (an inactive neurotensin fragment) and D TYR-11 NT (a long-lasting analog of neurotensin) were studied in rats. In awake rats, NT 1-13 (30 micrograms) and D TYR-11 NT (10 micrograms) induced an increase of the power spectrum in the theta range activity (4-7 Hz). In rats recorded during the sleep-wakefulness cycles, NT 1-13 (10 and 30 micrograms) and D TYR-11 NT (10 micrograms) had an awakening effect and also induced an increase of latency to the first episode of the different sleep stages (intermediate stage and slow wave sleep). NT 1-8 (30 and 90 micrograms in awake rats, 10 and 90 micrograms for sleep-wakefulness cycles) was inactive in all these experiments. Thus, it seems that all these effects can be linked to neurotensin receptors; indeed only fragments which recognize receptors possess an EEG activity.     

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.     

Orexin A in the ventral tegmental area induces conditioned place preference in a dose-dependent manner: Involvement of D1/D2 receptors in the nucleus accumbens

It has been shown that orexin A in the ventral tegmental area (VTA) is necessary for development of morphine place preference. Additionally, D1 and D2 dopamine receptors in the nucleus accumbens (NAc) have critical roles in motivation and reward. However, little is known about the function of orexin in conditioned place preference (CPP) in rats and involvement of D1/D2 receptors in the NAc. In the present study, we investigated the effect of direct administration of orexin A into the VTA, and examined the role of intra-accumbal dopamine receptors in development (acquisition) of reward-related behaviors in the rats. Adult male Wistar rats were unilaterally implanted by two separate cannulae into the VTA and NAc. The CPP paradigm was used, and, conditioning score and locomotor activity were recorded by Ethovision software. The results showed that unilateral intra-VTA administration of orexin A (27, 53 and 107ng/0.3μl saline) during conditioning phase induced CPP in a dose-dependent manner. The most effective dose of intra-VTA orexin-A in eliciting CPP was 107ng. However, intra-NAc administration of SCH 23390 (0.25, 1 and 4μg/0.5μl saline), a D1 receptor antagonist, and sulpiride (0.25, 1 and 4μg/0.5μl DMSO), a D2 receptor antagonist, inhibited the development of orexin-induced CPP. The inhibitory effect of D2 but not D1 receptor antagonist was exerted in a dose-dependent manner. It is supposed that the activation of VTA dopaminergic neuron by orexin impresses the D2 receptors more than D1 receptors in the NAc.     

Neurotensin potentiates the endogenous dopamine release from striatal synaptosomes of rat

The effect of neurotensin (NT) on the release of endogenous dopamine (DA) of rat striatal synaptosomes was studied. In the basic medium with Ca++ (5mM K+ and 1.2 mM Ca++), spontaneous release of DA was determined to be 12.03 +/- 1.12 pmol/mg protein, while in the Ca++-free basic medium containing EGTA (2.0 mM), the amount of DA released was still up to 11.2 +/- 1.06 pmol/mg protein. NT in 10(-4)-10(-6) M range tested potentiated both the spontaneous and K+-induced release of DA in Ca++-free medium. In addition, NT in 10(-4) M, but not in lower concentrations tested, potentiated the spontaneous, Ca++-dependent release of DA. It is suggested that the effect of NT on DA release is mediated by the specific NT receptors at the DA axonal terminals. The possibility, however, that NT has some influence on the carrier-mediated process of the membrane might not be ruled out.     

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.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

Comparison of neurotensin levels, receptors and actions in LS/Ibg and SS/Ibg mice

Neurotensin (NT), injected centrally, markedly enhances sensitivity to ethanol-induced anesthesia in SS but not in LS mice (4). Since LS and SS mice were bred selectively for differential sensitivity to ethanol, these findings suggest that neurotensinergic neuronal processes mediate some of ethanol's actions and that LS and SS mice might differ genetically in neurotensinergic systems. Indeed, in biochemical studies it was shown that LS and SS mice differ in NT-like immunoreactivity in specific brain regions, i.e., hypothalamus, and in NT receptor densities (Bmax) in frontal cortex and striatum. In other experiments LS and SS mice differed in behavioral responses to centrally administered NT. Intracerebroventricular (ICV) administration of NT produced dose-dependent changes in motor activity, hypothermia, and analgesia in both LS and SS mice. SS mice appeared to be more sensitive than LS to NT-induced analgesia but not hypothermia. Neurotensin increased or decreased locomotor activity in both SS and LS mice following intraventral tegmental area or ICV administration, respectively. The results indicate that LS and SS mice, which were selectively bred for differences in ethanol sensitivity, differ genetically in NT concentrations, receptor densities in specific brain regions, and in some receptor-mediated behavioral responses to NT.     

Neurotensin may function as a regulatory peptide in small cell lung cancer.

Neurotensin (NT) has been postulated to act as a modulatory agent in the central nervous system. Besides its presence in mammalian brain, NT is produced by small cell carcinoma of the lung (SCLC) and cell lines derived from these tumors. Receptors have also been characterized in some SCLC cell lines leading to the suggestion that NT could regulate the growth of SCLC in an autocrine fashion similar to bombesin/GRP. Previously, we had reported that a 10 nM dose of NT and NT(8-13), but not NT(1-8), elevated cytosolic Ca2+, indicating that SCLC NT receptors may use Ca2+ as a second messenger. Using intact SCLC cells we report that time-course incubations with NT lead to the formation of the amino-terminal fragment NT(1-8) and small amounts of the C-terminal fragment NT(9-13). These fragments are formed by metalloendopeptidase 3.4.24.15 cleaving enzyme at the Arg8-Arg9 bond of NT. Significant levels of soluble 3.4.24.15 (10-17 nmoles/mg Pr-/min) are present in SCLC cell lines. Using the in vitro clonogenic assay we tested the effect of 0.5, 5.0 and 10.0 nM doses of NT, NT(1-8) and NT(8-13) on SCLC clonal growth. NT and the C-terminal fragment NT(8-13) stimulated colony formation whereas the N-terminal fragment did not. In summary, NT may function as a regulatory peptide in SCLC through the formation of peptide fragments.     

Influence of exercise training on resting blood pressures of Dahl rats

To determine whether female Dahl salt-sensitive (SS) hypertensive rats would adapt to chronic treadmill exercise by exhibiting lower resting systolic blood pressures (RSBP), a 12-wk training program was undertaken. Female Dahl salt-resistant (SR) rats were also trained for the same time period a a similar intensity [40-70% maximal O2 consumption (VO2max)] and duration (55 min). Postexperimental treadmill run times and VO2max values [SR: nontrained (NT) 87 +/- 1, trained (T) 97 +/- 2; SS: NT 82 +/- 2, T 92 +/- 3 ml.min-1 X min-1 X kg-1] indicated that the prescribed program had produced a trained state. However, the training program caused no group differences between the SR or the SS and their nontrained controls in measurements associated with sodium chloride intake, fluid consumption, urine production, 24-h sodium excretion, plasma volumes, plasma insulin, or blood volumes. Chronic exercise did significantly lower RSBP in the SR subgroup after 6 wk (NT 123 +/- 4, T 110 +/- 3 mmHg) and 8 wk (NT 120 +/- 4, T 106 +/- 2 mmHg) and remained lower throughout the remaining weeks of the experiment. On the other hand, the RSBP results of the trained SS rats were significantly higher than the nontrained SS rats after 6 wk (NT 155 +/- 8, T 191 +/- 7 mmHg) and were never significantly different than the controls for the remainder of the study.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens

Ghrelin, a powerful orexigenic peptide released from the gut, stimulates feeding when injected centrally and has thus far been implicated in regulation of metabolic, rather than hedonic, feeding behavior. Although ghrelin's effects are partially mediated at the hypothalamic arcuate nucleus, via activation of neurons that co-express neuropeptide Y and agouti-related protein (NPY/Agrp neurons), the ghrelin receptor is expressed also in other brain sites. One of these is the ventral tegmental area (VTA), a primary node of the mesolimbic reward pathway, which sends dopaminergic projections to the nucleus accumbens (Acb), among other sites. We injected saline or three doses of ghrelin (0, 0.003, 0.03, or 0.3 nmol) into the VTA or Acb of rats. We found a robust feeding response with VTA injection of ghrelin, and a more moderate response with Acb injection. Because opioids modulate feeding in the VTA and Acb, we hypothesized that ghrelin's effects in one site were dependent on opioid signaling in the opposite site. The general opioid antagonist, naltrexone (NTX), injected into the Acb did not affect feeding elicited by ghrelin injection into the VTA, and NTX in the VTA did not affect feeding elicited by ghrelin injected into the Acb. These results suggest interaction of a metabolic factor with the reward system in feeding behavior, indicating that hedonic responses can be modulated by homeostatic factors.     

Evidence for nonintercalative complexes formed from the reversible binding of benzo[a]pyrene metabolites to closed-circular, single-stranded M13mp19 DNA

The fluorescence excitation spectrum of complexes formed from the reversible binding of the proximate carcinogen, trans-7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene (BP78D) to closed-circular, single-stranded, viral M13mp19 DNA (SS M13 DNA) exhibits a red-shift of 5 nm compared to the spectrum of BP78D measured without DNA or with native, calf thymus DNA. In SS M13 DNA which is 0.10 mM in PO4-, the fluorescence intensity of BP78D is 2.3 times smaller than the intensity measured without DNA; however, the fluorescence lifetime (42.7 nsec) of BP78D with SS M13 DNA is 1.7-1.8 times larger than the lifetimes of BP78D measured without DNA or with calf thymus DNA. These results are consistent with the conclusion that, in addition to binding sites which cause fluorescence quenching, SS M13 DNA contains sites which permit formation of BP78D inclusion complexes that have weaker interactions with nucleotide bases than those occurring in intercalated complexes. The association constant (1.45 +/- 0.01 x 10(5) M-1) for the binding of BP78D to SS M13 DNA is more than 9.0 times larger than that for binding to calf thymus DNA. It is 7.1 times larger than that for the binding of the less genotoxic metabolite, trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene (BP45D) to SS M13 DNA. UV Photoelectron data and results from ab initio molecular orbital calculations suggest that a difference in polarizability contributes to the greater SS M13 DNA binding of BP78D compared to that of BP45D.     

Clozapine interacts with the glycine site of the NMDA receptor: electrophysiological studies of dopamine neurons in the rat ventral tegmental area

Clozapine has a remarkable efficacy in treatment-resistant schizophrenia and is one of the most effective antipsychotic drugs used today. The clinical effects of clozapine are suggested to be related to a unique interaction with a variety of receptor systems, including the glutamatergic receptors. Kynurenic acid (KYNA) is an endogenous blocker of alpha7 nicotinic receptors and a glutamate-receptor antagonist, preferentially blocking N-methyl-D-aspartate (NMDA) receptors. In the present in vivo electrophysiological study, changes in endogenous concentration of brain KYNA were utilized to analyze an interaction between clozapine and the glycine site of NMDA receptors. In control rats intravenously administered clozapine (0.078-10 mg/kg) increased the firing rate and the burst firing activity of dopamine (DA) neurons in the ventral tegmental area (VTA). Pretreatment with indomethacin (50 mg/kg, i.p., 1-3.5 h), a cyclooxygenase (COX)-inhibitor with a preferential selectivity for COX-1, which produced a significant elevation in brain KYNA levels, reversed the excitatory action of clozapine into an inhibitory response. In contrast, pretreatment with the COX-2 selective inhibitor parecoxib (25 mg/kg, i.v., 1-1.5 h) decreased brain KYNA formation and furthermore, clearly potentiated the excitatory effect of clozapine. Our results show that endogenous levels of brain KYNA are of importance for the response of clozapine on VTA DA neurons. On the basis of the present data we propose that clozapine is able to interact with glutamatergic mechanisms, via actions at the NMDA/glycine receptor.     

Effect of renal medullary H2O2 on salt-induced hypertension and renal injury

Dahl salt-sensitive (SS) and consomic, salt-resistant SS-13(BN) rats possess substantial differences in blood pressure salt-sensitivity even with highly similar genetic backgrounds. The present study examined whether increased oxidative stress, particularly H2O2, in the renal medulla of SS rats contributes to these differences. Blood pressure was measured using femoral arterial catheters in three groups of rats: 1) 12-wk-old SS and consomic SS-13(BN) rats fed a 0.4% NaCl diet, 2) SS rats fed a 4% NaCl diet and chronically infused with saline or catalase (6.9 microg x kg(-1) x min(-1)) directly into the renal medulla, and 3) SS-13(BN) fed high salt (4%) and infused with saline or H2O2 (347 nmol x kg(-1) x min(-1)) into the renal medullary interstitium. After chronic blood pressure measurements, renal medullary interstitial H2O2 concentration ([H2O2]) was collected by microdialysis and analyzed with Amplex red. Blood pressure and [H2O2] were both significantly higher in SS (126 +/- 3 mmHg and 145 +/- 17 nM, respectively) vs. SS-13(BN) rats (116 +/- 2 mmHg and 56 +/- 14 nM) fed a 0.4% diet. Renal interstitial catalase infusion significantly decreased [H2O2] (96 +/- 41 vs. 297 +/- 52 nM) and attenuated the hypertension (146 +/- 2 mmHg catalase vs. 163 +/- 4 mmHg saline) in SS rats after 5 days of high salt (4%). H2O2 infused into the renal medulla of consomic SS-13(BN) fed high salt (4%) for 7 days accentuated the salt sensitivity (145 +/- 2 mmHg H2O2 vs. 134 +/- 1 mmHg saline). [H2O2] was also increased in the treated group (83 +/- 1 nM H2O2 vs. 44 +/- 9 nM saline). These data show that medullary production of H2O2 may contribute to salt-induced hypertension in SS rats and that chromosome 13 of the Brown Norway contains gene(s) that protect against renal medullary oxidant stress.     

Balance of glutamate and dopamine in the nucleus accumbens modulates self-stimulation behavior after injection of cholecystokinin and neurotensin in the rat brain.

Subpopulations of dopamine (DA) neurons in the ventral mesencephalon have been reported to contain cholecystokinin (CCK) and neurotensin (NT), giving rise to DA, DA/NT, NT/CCK and DA/CCK/NT projections. More precisely, colocalized DA/CCK neurons project mainly to the caudal part of the medial nucleus accumbens, whereas its rostral portion receives CCK and DA nerve terminal networks that are structurally independent. We investigated the respective effects of both CCK and NT on the intracranial self-stimulation behavior (ICSS) from the posterolateral hypothalamus after their direct administration into the lateral ventricle (ICV), into both portions of the nucleus accumbens, into the ventral tegmental area (VTA), and into the subiculum of the hippocampal formation (SUB). The ICV injection of 150 pmol CCK8 induced a decrease in the rate of ICSS. By contrast, the direct administration of 150 pmol CCK8 into the mediocaudal part of the nucleus accumbens induced an enhanced rate of ICSS while a similar injection into its rostral portion gave rise to a slight transient decrease of ICSS. When injected into the SUB, both CCK8 and glutamate produced decreased rates of ICSS at femtomolar doses one thousand-fold under the picomolar concentrations used for ICV injections. Neurotensin induced similar behavioral profiles to that observed after the ICV injection of CCK8 or into both portions of the nucleus accumbens. Neurotensin and CCK8 displayed opposite effects on ICSS when administered into the SUB or into the VTA, suggesting they may regulate ICSS most probably through different synaptic mechanisms and through different anatomical pathways.     

Topography of the neurotensin (NT)(8-9) binding site of human NT receptor-1 probed with NT(8-13) analogs.

A series of neurotensin (NT)(8-13) analogs featuring substitution of the Arg8 and/or Arg9 residues with non-natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor-1 (hNTR-1). The modifications were designed to probe specific steric and electrostatic requirements in the N-terminal cationic region of NT(8-13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non-natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the alpha-amino acid carbon. Substitution of Arg8 with N(G)-alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8-13) to the hNTR-1. Position 8 incorporation of Hlys produced the most favorable primary amine side-chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys8, DAB9]NT(8-13), in which DAB is 2,4-diaminobutyric acid, was also prepared and tested as the shorter side-chain of DAB is known to be favored in position 9 of NT(8-13). This analog maintained 60% of NT(8-13) binding affinity making it the most favored des-guanidinium-containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR-1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8-13) and related analogs.     

Neurotensin increases endogenous glutamate release in rat cortical slices

In the present study, the effects of the tridecapeptide neurotensin [NT(1-13)] and its fragments, NT(1-7) and NT(8-13), on endogenous glutamate release from rat cortical slices, were evaluated. NT(1-13) (100-1000 nM) slightly increased spontaneous glutamate release, while it was ineffective at 1 and 10 nM concentrations. Neither the biologically active NT fragment NT(8-13) nor the inactive one NT(1-7) affected basal glutamate release. NT(1-13) (1-1000 nM) enhanced potassium (35 mM)-evoked glutamate release displaying a bell-shaped concentration response curve. In addition NT(8-13) (10 nM) increased K+-evoked-glutamate release similarly to the parent peptide (10 nM), while the biologically inactive fragment NT(1-7) (10-100 nM) was ineffective. The effects of NT(1-13) and NT(8-13) were fully counteracted by the selective neurotensin receptor antagonist SR48692 (100 nM). These findings suggest that NT plays a role in regulating cortical glutamate transmission.