Action Of Neurotensin Research Articles

Brainstem, thalamus, and cortex: Microinjection of neurotensin in the RVM produces antinociception mediated by spinally-projecting serotonergic neurons

The rostral ventromedial medulla (RVM) exerts a powerful modulatory effect on the spinal transmission of pain. Microinjection of neurotensin (NT) in the RVM produces biphasic effects; low doses (0.03-0.3 pmol) facilitate nociception, while higher doses (30 pmol-3 nmol) are antinociceptive. We examined the localization of the neurotensin receptor 1 (NTR1) in retrogradely labeled spinally-projecting, serotonergic neurons of the RVM. We found that 96±1% of RVM neurons that express the NTR1 are serotonergic. Of these serotonergic NTR1 expressing cells, 41±5% clearly descend directly to the spinal cord, while 59±5% do not. In addition, 53±2% of the RVM serotonergic neurons express NTR1 (n=4 rats). Furthermore, intrathecal (i.th.) injection of the serotonergic antagonist methysergide attenuates the antinociceptive effect of NT microinjection in the RVM. NT (3 nmol), increased tail and paw withdrawal latencies to a noxious thermal stimulus by 229±74% and 252±136%, respectively (n = 5). Subsequent i.th. injection of methysergide (60 μg/0.05 μl) reduced this antinociceptive effect to 42±23% and 125±69% (n=6 and n=2), but produced no effect in the absence of NT (n=5). Together these findings suggest that high doses of NT act to produce antinociception that is mediated in part by activation of spinally-projecting RVM serotonergic neurons. NTR1-immunoreactive serotonergic neurons that do not project to the spinal cord may also contribute to the antinociceptive actions of NT. Supported by NIH DA 02879-27, T32 DK07663-08, and DA 03980-22.

Electron microscopic dual labeling of high-affinity neurotensin and dopamine D2 receptors in the rat nucleus accumbens shell.

The dopamine D2 receptor (D2R) in the nucleus accumbens (NAc) shell is implicated in schizophrenia and in psychostimulant-induced drug-seeking behavior, both of which are affected by activation of the functionally opposed high-affinity neurotensin receptor (NTS1). To determine the functionally relevant sites, we examined the dual electron microscopic immunocytochemical localization of D2R and NTS1 in the NAc shell of rat brain. Immunolabeling for each receptor was seen in association with cytoplasmic organelles, or more rarely, on the plasma membrane of both axonal and somatodendritic profiles. Some of the axonal and many of the dendritic processes colocalized the two receptors. The dually labeled axon terminals often formed symmetric synapses or appositional contacts with unlabeled dendritic profiles. The morphology of these terminals suggests that they contain either inhibitory amino acids or dopamine. Other axonal profiles expressing exclusively NTS1 or D2R were without synaptic specializations or formed asymmetric, excitatory-type synapses mainly on unlabeled dendritic spines. In addition, however, several D2R-immunoreactive terminals were observed presynaptic to dendrites containing NTS1. The somatodendritic profiles immunolabeled for NTS1 and/or D2R had morphological features typical of inhibitory spiny projection neurons in the NAc. These results suggest that activation of NTS1 and D2R can dually modulate transmitter release from the same or separate phenotypically distinct axon terminals in the NAc shell. These presynaptic receptors as well as the postsynaptic NTS1 distribution in neurons that also contain or receive input from terminals containing D2R may mediate the opposing actions of neurotensin and dopamine in the NAc.

Characteristics of the actions of neurotensin on motor reactions in rats in response to positive and negative conditioned signals.

This study addressed the effects of microinjections of neurotensin into the caudate nucleus and substantia nigra on the performance of motor reactions in response to positive and negative conditioned signals, as well as the post-effects of microinjections in subsequent experiments. Neurotensin had positive effects on the extinction of non-reinforced motor reactions. Neurotensin had no effect on the number of motor responses to the non-reinforced signal, though the number decreased in subsequent experiments. There was an increase in the latent period of responses as compared with controls. The effect of neurotensin at the level of the caudate nucleus was more marked than that at the level of the substantia nigra. Neurotensin microinjections had no marked effect on performance of conditioned responses to positive signals. The behavioral effects of neurotensin are associated with normalization of the interactions of the brain's monoaminergic systems. It is suggested that the positive actions of neurotensin on extinction of motor responses to negative signals result from the formation of a contextual conditioned emotional state in the animals, this facilitating optimization of conditioned reflex activity.

Neurotensin depolarizes globus pallidus neurons in rats via neurotensin type-1 receptor

The globus pallidus is a major component in the indirect pathway of the basal ganglia. There is evidence that neurotensin receptors exist in this nucleus. To determine the electrophysiological effects of neurotensin on pallidal neurons, whole-cell patch-clamp recordings were performed in the acutely prepared brain slices. Under current-clamp recordings, neurotensin at 1 μM depolarized pallidal neurons. Voltage-clamp recordings also showed an inward current induced by neurotensin. The depolarizing effect of neurotensin could be mimicked by the C-terminal fragment, neurotensin (8–13), but not by the N-terminal fragment, neurotensin (1–8). Both SR 142948A, a non-selective neurotensin receptor type-1 and type-2 antagonist, and SR 48692, a selective type-1 receptor antagonist, blocked the depolarizing effect of neurotensin, and which themselves had no effect on membrane potential. Thus, neurotensin type-1 receptors appear to mediate the effect of neurotensin. The depolarization evoked by neurotensin persisted in the presence of tetrodotoxin, ionotropic and metabotropic glutamate and GABA receptor antagonists, indicating that neurotensin excited the pallidal neurons by activating the receptor expressed on the neurons recorded. Current-voltage relationship revealed that both the suppression of a potassium conductance and the activation of a cationic conductance are involved in the neurotensin-induced depolarization. Based on the action of neurotensin in the globus pallidus we hypothesize that alterations of the striatopallidal neurotensin system contribute to symptoms of basal ganglia motor disorders.

Involvement of the Neurotensin Receptor-3 in the Neurotensin-Induced Migration of Human Microglia

Microglia motility plays a crucial role in response to lesion or exocytotoxic damage of the cerebral tissue. We used two in vitro assays, a wound-healing model and a chemotaxis assay, to show that the neuropeptide neurotensin elicited the migration of the human microglial cell line C13NJ by a mechanism dependent on both phosphatidylinositol 3-kinase (PI 3-kinase) and mitogen-activated protein (MAP) kinase pathways. The effect of neurotensin on cell migration was blocked by the neurotensin receptor-3 propeptide, a selective ligand of this receptor. We demonstrate, by using RT-PCR, photoaffinity labeling, and Western blot analysis, that the type I neurotensin receptor-3 was the only known neurotensin receptor expressed in these microglial cells and that its activation led to the phosphorylation of both extracellular signal-regulating kinases 1/2 and Akt. Furthermore, the effect of neurotensin on cell migration was preceded by a profound modification of the F-actin cytoskeleton, particularly by the rapid formation of numerous cell filopodia. Both the motility and the filopodia appearance induced by neurotensin were totally blocked by selective inhibitors of MAP kinases or PI 3-kinase pathways. This demonstrates that the neurotensin receptor-3 is functional and mediates the migratory actions of neurotensin.

Characterization of beta-lactotensin, a bioactive peptide derived from bovine beta-lactoglobulin, as a neurotensin agonist.

beta-Lactotensin (beta-LT: His-Ile-Arg-Leu) is an ileum-contracting peptide derived from residues No. 146-149 of bovine beta-lactoglobulin. The ileum-contracting activity of beta-LT was blocked by the NT1 antagonist SR48692. beta-LT was selective for the neurotensin NT2 receptor while neurotensin was selective for the NT1 receptor. beta-LT is the first natural ligand showing selectivity for the NT2 receptor. beta-LT showed hypertensive activity after intravenous administration at a dose of 30 mg/kg in conscious rats, while neurotensin showed hypotensive activity. The hypertensive activity of beta-LT was blocked by levocabastine (1 mg/kg, i.v.), an NT2 antagonist. SR48692, which blocked the hypotensive activity of neurotensin, had no effect on the hypertensive activity of beta-LT. These results suggest that the hypertensive activity of beta-LT is mediated by the NT2 receptor. It was concluded that the NT1 and NT2 receptors mediate the opposite effect on blood pressure.

Neurotensin-induced modulation of dopamine D2 receptors and their function in rat striatum: counteraction by a NTR1-like receptor antagonist.

The present study investigated the neurotensin (NT) receptor subtype (NTR) involved in the antagonistic neurotensin modulation of striatal dopamine D2 receptors observed in vitro and in vivo. The NT induced increase of the IC50 values of dopamine (DA) competition for [125I]iodosulpiride binding sites was counteracted by the NTR1-like antagonist SR48692 in rat striatal slices. Intrastriatal perfusion of pergolide induced in the awake rat an inhibition of striatal DA release that was antagonized by NT. This action of NT was counteracted by co-perfusion with the NTR1 like antagonist SR48692. These data indicate that there exists in the striatum at the prejunctional level an intramembrane antagonistic NT receptor/DA D2 receptor-receptor interaction where NTR1 like receptor activation reduces the DA D2 autoreceptor function.

Neurotensin analog selective for hypothermia over antinociception and exhibiting atypical neuroleptic-like properties

Neurotensin (NT) is a tridecapeptide neurotransmitter in the central nervous system. It has been implicated in the therapeutic effects of neuroleptics. Central activity of NT can only be demonstrated by direct injection into the brain, since it is readily degraded by peptidases in the periphery. We have developed many NT(8–13) analogs that are resistant to peptidase degradation and can cross the blood–brain barrier (BBB). In this study, we report on one of these analogs, NT77L. NT77L induced hypothermia (ED 50=6.5 mg/kg, i.p.) but induced analgesia only at the highest dose examined (20 mg/kg, i.p.). Like the atypical neuroleptic clozapine, NT77L blocked the climbing behavior in rats induced by the dopamine agonist apomorphine (600 μg/kg) with an ED 50 of 5.6 mg/kg (i.p.), without affecting the licking and the sniffing behaviors. By itself NT77L did not cause catalepsy, but it moderately reversed haloperidol-induced catalepsy with an ED 50 of 6.0 mg/kg (i.p.). Haloperidol alone did not lower body temperature, but it potentiated the body temperature lowering effect of NT77L. In studies using in vivo microdialysis NT77L showed similar effects on dopamine turnover to those of clozapine, and significantly different from those of haloperidol in the striatum. In the prefrontal cortex, NT77L significantly increased serotonergic transmission as evidenced by increased 5-hydroxyindole acetic acid:5-hydroxytryptamine (5-HIAA:5-HT) ratio. Thus, NT77L selectively caused hypothermia, over antinociception, while exhibiting atypical neuroleptic-like effects.

High-affinity neurotensin receptors in the rat nucleus accumbens: subcellular targeting and relation to endogenous ligand.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor-associated NAc core. We examined the electron microscopic immunolabeling of the high-affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR-labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR-immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR-labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter-specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR.

Effects of neurotensin on discharge rates of rat suprachiasmatic nucleus neurons in vitro.

The neuropeptide neurotensin and two classes of its receptors, the neurotensin receptor-1 and 2, are present in the suprachiasmatic nucleus of the mammalian hypothalamus. The suprachiasmatic nucleus houses the mammalian central circadian pacemaker, but the effects of neurotensin on cellular activity in this circadian pacemaker are unknown. In this study, we examined the effects of neurotensin on the spontaneous discharge rate of rat SCN cells in an in vitro slice preparation. Neurotensin (1-10 microM) increased cell firing rate in approximately 50% of cells tested, while approximately 10% of suprachiasmatic cells tested showed a decrease in firing rate in response to neurotensin. These effects of neurotensin were not altered by the GABA receptor antagonist bicuculline (20 microM) or the glutamate receptor antagonists, D-aminophosphopentanoic acid (50 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). The neurotensin receptor selective antagonists SR48692 and SR142948a (10 microM) failed to antagonise neurotensin responses in the majority of cells examined. Compounds that function as agonists selective for the neurotensin-receptor subtypes 1 and 2, JMV-510 and JMV-431 respectively, elicited neurotensin-like responses in approximately 90% of cells tested. Six out of seven cells tested responded to both JMV-510 and JMV-431. Neuropeptide Y (100nM) treatment of suprachiasmatic nucleus slices was found to elicit profound suppression of neuronal firing rate. Co-application of neurotensin with neuropeptide Y significantly (P<0.05) reduced the duration of the response, as compared to that elicited with neuropeptide Y alone. Together, these results demonstrate for the first time the actions of neurotensin in the suprachiasmatic nucleus and raise the possibility that this neuropeptide may play a role in modulating circadian pacemaker function.

Neurotensin-Induced Bursting of Cholinergic Basal Forebrain Neurons Promotes γ and θ Cortical Activity Together with Waking and Paradoxical Sleep

Cholinergic basal forebrain neurons have long been thought to play an important role in cortical activation and behavioral state, yet the precise way in which they influence these processes has yet to be fully understood. Here, we have examined the effects on the electroencephalogram (EEG) and sleep-wake state of basal forebrain administration of neurotensin (NT), a neuropeptide that has been shown in vitro to potently and selectively modulate the cholinergic cells. Microinjection of (0.1-3.0 mm) NT into the basal forebrain of freely moving, naturally waking-sleeping rats produced a dose-dependent decrease in delta ( approximately 1-4 Hz) and increase in both theta ( approximately 4-9 Hz) and high-frequency gamma activity (30-60 Hz) across cortical, areas with no increase in the electromyogram. These EEG changes were accompanied by concomitant decreases in slow wave sleep (SWS) and transitional SWS (tSWS), increases in wake, and most remarkably, increases in paradoxical sleep (PS) and transitional PS (tPS), despite the virtual absence of SWS. The effects were attributed to direct action on cholinergic neurons as evidenced by selective internalization of a fluorescent ligand, Fluo-NT, in choline acetyltransferase (ChAT)-immunoreactive cells and stimulation by NT of bursting discharge in juxtacellularly recorded, Neurobiotin-labeled, ChAT-immunoreactive neurons. We conclude that NT-induced rhythmic bursting of cholinergic basal forebrain neurons stimulates rhythmic theta oscillations and gamma across the cerebral cortex. With the selective action of NT on the cholinergic cells, their bursting discharge promotes theta and gamma independent of motor activity and thereby also stimulates and enhances PS.

Immunohistochemical localization, biochemical characterization, and biological activity of neurotensin in the frog adrenal gland.

The primary structure of neurotensin has been recently determined for the frog Rana ridibunda (Endocrinology 139: 4140-4146, 1998). In the present study, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an antiserum directed against the conserved C-terminal region of the peptide. Neurotensin-like immunoreactivity was detected in two populations of nerve fibers: numerous varicose fibers coursing between adrenal cells, and a few processes located in the walls of blood vessels irrigating the gland. Reversed-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the same retention time as synthetic frog neurotensin. The possible involvement of neurotensin in the regulation of steroid secretion was studied in vitro using perifused frog adrenal slices. For concentrations ranging from 10(-10) to 10(-5) M, synthetic frog neurotensin increased corticosterone and aldosterone production in a dose-dependent manner (EC50 = 1.2 x 10(-9) M and 5.8 x 10(-10) M, respectively). Repeated administration of neurotensin induced a reproducible stimulation of steroid output without any tachyphylaxis. Prolonged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of corticosteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion from dispersed frog adrenal cells. This study demonstrates that neurotensin is located in nerve processes innervating the adrenal gland of amphibians. The results also show that synthetic frog neurotensin exerts a direct stimulatory effect on corticosteroid output. Taken together, these data support the view that neurotensin, released by nerve fibers, may act as a local regulator of corticosteroid secretion.

Immunohistochemical Localization, Biochemical Characterization, and Biological Activity of Neurotensin in the Frog Adrenal Gland

Immunohistochemical Localization, Biochemical Characterization, and Biological Activity of Neurotensin in the Frog Adrenal Gland

Neurotensin regulates intracellular calcium in ventral tegmental area astrocytes: evidence for the involvement of multiple receptors

Recent evidence suggests that some types of neurotensin receptors may be expressed by astrocytes. In order to explore the function of neurotensin receptors in astrocytes, the effect of a neurotensin receptor agonist, neurotensin(8–13), on intracellular Ca 2+ dynamics in mixed neuronal/glial cultures prepared from rat ventral tegmental area was examined. It was found that neurotensin(8–13) induces a long-lasting rise in intracellular Ca 2+ concentration in a subset of glial fibrilary acidic protein-positive glial cells. This response displays extensive desensitization and appears to implicate both intracellular and extracellular Ca 2+ sources. In the absence of extracellular Ca 2+, neurotensin(8–13) evokes only a short-lasting rise in intracellular Ca 2+. The neurotensin-evoked intracellular Ca 2+ accumulation is blocked by the phospholipase C inhibitor U73122 and by thapsigargin, suggesting that it is initiated by release of Ca 2+ from an inositol triphosphate-dependent store. The Ca 2+-mobilizing action of neurotensin(8–13) in astrocytes is dependent on at least two receptors, because the response is blocked in part only by SR48692, a type 1 neurotensin receptor antagonist, and is blocked completely by SR142948A, a novel neurotensin receptor antagonist. The finding that the type 2 neurotensin receptor agonist levocabastine fails to mimic or alter the effects of neurotensin(8–13) on intracellular Ca 2+ makes it unlikely that the type 2 neurotensin receptor is involved. In summary, these results show that functional neurotensin receptors are present in cultured ventral tegmental area astrocytes and that their activation induces a highly desensitizing rise in intracellular Ca 2+. The pharmacological profile of this response suggests that a type 1 neurotensin receptor is involved but that another, possibly novel, non-type 2 neurotensin receptor is also implicated. If present in vivo, such signalling could be involved in some of the physiological actions of neurotensin.

Complex actions of neurotensin in human colon: A binding and functional study

Neurotensin (NT) is a brain-gut peptide with endocrine and neuronal actions. It has complex actions on gastrointestinal muscle, depending on the location and the species studied. In rodents, the actions have been attributed to a direct action on NT receptors on smooth muscle as well as indirect mechanisms involving release of acetylcholine, tachykinins, histamine, nitric oxide and/or prostaglandins. In this study, we have used [lz5I][Tyr]NT to characterize and localize NT binding sites and have investigated the actions of NT in human ascending and sigmoid colon. Normal colon was obtained from patients (age range 60-76 years) undergoing resection for colon carcinoma. Radioligand binding studies were carried out in membranes prepared from sigmoid circular muscle (CM) (which also contained longitudinal muscle and myenteric ganglia), in 50 mM Tris-HCI (pH 7.4) containing I roM MgClz and I mM O-phenanthroline. Nonspecific binding was defined using I pM NT. One site analysis of saturation data gave K, 2.2 :t 0.4 nM and Bma• 3.5 :t 0.4 fmol/mg wet weight tissue (n=13), but 2 distinct sites were found in some specimens. Binding was inhibited by NT(8-13) > NT 2= SR142948A 2= neuromedin N 2= SR48692; levocabastine £artly inhibited binding. In autoradiographic studies, specific binding of [' 5I][Tyr]NT was seen over CM and myenteric ganglia, but not on blood vessels or the mucosa. Strips of CM and taenia coli (TC) were suspended under isometric tension in Krebs-Henseleit solution at 37°C. The magnitude of contractile responses was variable (19-29% in CM; 38-41% in TC, of maximum response to acetylcholine) and on several occasions, biphasic responses were observed. The potency in CM (pDz 6.8-7.1) and TC (pDz 6.4-6.5) was no different in ascending or sigmoid colon. In sigmoid colon CM, contractile responses to NT were potentiated by 1 pM tetrodotoxin (P < 0.05) and inhibited (P < 0.05) by 1 pM MEN10627, the tachykinin NK2 receptor antagonist. In ascending colon CM, a potentiation (P < 0.01) of the contractile response was observed in the presence of indomethacin (l /-LM) as well as IT (l /-LM). Atropine (1 /LM) had no effect in any region of the colon. These inhibitors had little effect on NT responses in TC. Thus, NT appears to have both direct actions on smooth muscle and neuronal actions (supported by the presence of binding on myenteric ganglia). NT may also cause the release of relaxant prostanoids in ascending colon. Many effects of NT appear to be regionally specific. Supported by the National Health and Medical Research Council of Australia

Effects of a novel neurotensin peptide analog given extracranially on CNS behaviors mediated by apomorphine and haloperidol

Neurotensin (NT) is a neuropeptide neurotransmitter in the central nervous system. It has been implicated in the therapeutic and in the adverse effects of neuroleptics. Activity of NT in brain can only be shown by direct injection of the peptide into that organ. However, we have developed a novel analog of NT(8–13), NT69L, which is active upon intraperitoneal (i.p.) injection. Like atypical neuroleptics, NT69L blocked the climbing behavior in rats, but not the licking and sniffing behaviors of a high dose (600 μg/kg) of the non-selective dopamine agonist apomorphine. Its blockade of climbing was very potent with an ED 50 (effective dose at 50% of maximum) of 16 μg/kg. Both apomorphine and NT69L caused a long-lasting hypothermia, which was greater with the peptide but not synergistic in combination with apomorphine. The ED 50 of NT69L for hypothermia was 390 μg/kg. NT69L (up to 5 mg/kg i.p.) did not produce catalepsy. However, when given before haloperidol, NT69L, but not clozapine, completely prevented catalepsy. When given after haloperidol, NT69L, but not clozapine, reversed haloperidol's cataleptic effects with an ED 50 of 260 μg/kg. There was no significant difference between the ED 50s for hypothermia and anticataleptic effects of NT69L. However, the ED 50 for blocking the effects of apomorphine was significantly lower than the other two. These data suggest that NT69L may have neuroleptic properties in humans and may be useful in the treatment of extrapyramidal side effects caused by typical neuroleptics such as haloperidol.

Colocalization of neurotensin messenger ribonucleic acid (mRNA) and progesterone receptor mRNA in rat arcuate neurons under estrogen-stimulated conditions.

In the female rat, estrogen and progesterone directly or indirectly regulate the activity of neurotensin (NT)-synthesizing neurosecretory cells located in the hypothalamic arcuate nucleus (ARC). To determine whether these NT neurons are subject to direct regulation by ovarian steroids, estrogen-inducible messenger RNA (mRNA) encoding nuclear progesterone receptor (PR) was used as a cellular marker for nuclear estrogen receptor (ER) as well as PR, and double label in situ hybridization was employed to determine the extent to which NT/neuromedin N mRNA and PR mRNA are colocalized in ARC neurons under estrogen-stimulated conditions. In estradiol-treated ovariectomized rats, approximately 80% of NT/neuromedin N mRNA-expressing cells in sections through the dorsomedial division of the ARC and approximately 60% of such cells in sections through the ventrolateral division of the ARC were found to contain PR mRNA. Depending on the ARC division and rostrocaudal level, double labeled cells accounted for approximately 20-50% of PR mRNA-containing cells. These results indicate that under estrogen-stimulated conditions the majority of NT neurons in the ARC express both PR and ER, as previous studies of this region indicate that estrogen-inducible PR occurs only in cells that also express ER. In the rat, NT neurons appear to be a major ARC cell type subject to direct regulation by estrogen and progesterone.

Synthesis and human neurotensin receptor binding activities of neurotensin(8-13) analogues containing position 8 alpha-azido-N-alkylated derivatives of ornithine, lysine, and homolysine.

A series of neurotensin(8-13) (NT) analogues were synthesized through intermediates in which the N-terminal Arg(8) was replaced by various omega-bromo-2(S)-azido residues spanning 3-5 methylene units in side-chain length. Subsequent nucleophilic substitution of the omega-bromo groups with ammonia, methylamine, dimethylamine, or trimethylamine provided peptides containing N-terminal 2(S)-azido residues containing primary through quaternary N-alkylated side chains corresponding in length to ornithine, Lys, and homolysine. The synthetic procedure appears applicable for incorporation of a wide variety of amine-containing nonnatural amino acids into peptides. The particular modifications were intended to enhance physiochemical properties of NT(8-13) responsible for human NT 1 receptor (hNTR) binding, overall lipophilicity, and stability that may influence the potency of the peptides in vivo. When the peptides were tested for hNTR binding, the affinities in each series followed the order primary > secondary > tertiary > quaternary amine with the homolysine side-chain length being favored. All analogues possess binding affinities between acetyl-NT(8-13) and NT(8-13) indicating that the sterically less bulky alpha-azido may be inherently preferable to the acetyl group for N-terminal protection. This study extends the strategy of modifying amine-containing drugs through alkylations to peptide modification. The set of alkylated side chains also offers a new method of steric selection between receptor subtypes and could be used to modify the properties and biological effects of any peptide that contains cationic residues.

Peptide nucleic acids targeted to the neurotensin receptor and administered i.p. cross the blood-brain barrier and specifically reduce gene expression.

Intraperitoneal injection of an unmodified antisense peptide nucleic acid (PNA) complementary to mRNA of the rat neurotensin (NT) receptor (NTR1) was demonstrated by a gel shift assay to be present in brain, thus indicating that the PNA had in fact crossed the blood-brain barrier. An i.p. injection of this antisense PNA specifically inhibited the hypothermic and antinociceptive activities of NT microinjected into brain. These results were associated with a reduction in binding sites for NT both in brain and the small intestine. Additionally, the sense-NTR1 PNA, targeted to DNA, microinjected directly into the brain specifically reduced mRNA levels by 50% and caused a loss of response to NT. To demonstrate the specificity of changes in behavioral, binding, and mRNA studies, animals treated with NTR1 PNA were tested for behavioral responses to morphine and their mu receptor levels were determined. Both were found to be unaffected in these NTR1 PNA-treated animals. The effects of both the antisense and sense PNAs were completely reversible. This work provides evidence that any antisense strategy targeted to brain proteins can work through i. p. delivery by crossing the normal blood-brain barrier. Equally important was that an antigene strategy, the sense PNA, was shown in vivo to be a potentially effective therapeutic treatment.

Ionic mechanisms of action of neurotensin in acutely dissociated neurons from the diagonal band of Broca of the rat.

Whole cell recordings were performed on acutely dissociated neurons from the horizontal limb of the diagonal band of Broca (hDBB) from rats to elucidate the ionic mechanisms of action of neurotensin. Neurotensin caused a decrease in whole cell voltage-activated outward currents and failed to elicit a response when Ca2+ influx was blocked by changing the external solution to the one containing 0 mM Ca2+ and 50 microM Cd2+, suggesting the involvement of Ca2+-dependent conductances. Charybdotoxin, a specific blocker of voltage-sensitive calcium-activated K+ channels (IC), caused a decrease in outward currents comparable with that caused by blocking calcium influx and occluded the neurotensin-induced decrease in outward currents. Similarly, 50 microM tetraethylammonium ions also blocked the neurotensin response. Also neurotensin reduced whole cell barium currents (IBa) and calcium currents (ICa). Amiloride and omega-conotoxin GVIA, but not nimodipine, were able to eliminate the neurotensin-induced decrease in IBa. Thus T- and N- but not L-type calcium channels are subject to modulation by neurotensin, and this may account for its effects on IC. The predicted changes in action potential as a result of the blockade of currents through calcium channels culminating into changes in IC were confirmed in the bridge current-clamp recordings. Specifically, neurotensin application led to depolarization of the resting membrane potential, broadening of spike and a decrease in afterhyperpolarization and accommodation. These alterations in action potential characteristics that resulted in increased firing rate and excitability of the hDBB neurons also were produced by application of charybdotoxin. Neurotensin effects on these properties were occluded by 2 - [(1 - 7 - chloro - 4 - quinolinyl) - 5 - (2, 6 - di - methoxyphenyl) pyrazol-3-yl) carbonylamino] tricyclo (3.3.1.1.)decan-2-carboxylic acid, a nonpeptide high-affinity neurotensin receptor antagonist. Neurotensin blockade of IC, possibly through ICa, is a potential physiological mechanism whereby this peptide may evoke alterations in the cortical arousal, sleep-wake cycle, and theta rhythm.