The effects of systemic NT69L, a neurotensin agonist, on baseline and drug-disrupted prepulse inhibition.

Centrally administered neurotensin (NT) produces behavioral and biochemical effects that are very similar to the effects of antipsychotic drugs. Therefore, there is much interest in the potential use of NT agonists as antipsychotic drugs. We have previously reported that PD149163, a NT(8-13) analogue, produced effects on prepulse inhibition (PPI) of startle after systemic administration that were suggestive of an atypical antipsychotic-like drug profile. To determine if these effects are shared by other peripherally administered NT agonists, we tested the effects of NT69L, a recently developed NT agonist that penetrates the CNS, on drug-induced PPI deficits. In the first experiment, rats received subcutaneous (s.c.) injections of NT69L (vehicle, 0.08, 0.25, and 1.0mg/kg) followed 30min later by subcutaneous saline or D-amphetamine (2.0mg/kg). In the second experiment, NT69L injections were followed by saline or the non-competitive NMDA antagonist dizocilpine (0.1mg/kg). Both D-amphetamine and dizocilpine significantly decreased PPI as expected. In the first experiment, NT69L significantly increased PPI levels at baseline and after D-amphetamine. In the second experiment, NT69L attenuated PPI deficits produced by dizocilpine, without increasing baseline PPI. In addition, NT69L had no effect on startle magnitude. The effects of NT69L in these studies were similar in some ways to the effects of PD149163 and were also consistent with the preclinical effects of atypical antipsychotic drugs. These data provide further support for the notion that NT agonists may have use as novel antipsychotic drugs. Furthermore, the ability of NT69L and PD149163 to attenuate dizocilpine-disrupted PPI, an antipsychotic drug effect not mediated by dopamine, suggests that NT agonists may produce some of their antipsychotic-like effects by modulating neurotransmitter systems other than dopamine, such as serotonin, noradrenaline or glutamate.

Neurotensin: peptide for the next millennium.

Neurotensin is an endogenous tridecapeptide neurotransmitter (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Try-Ile-Leu-OH) that was discovered by Carraway and Leeman in bovine hypothalami in the early 1970s. Since then this peptide has been the subject of a multitude of articles detailing discoveries related to its activity, receptors, localization, synthesis, and interactions with other systems. This review article does not intend to summarize again all the history of this fascinating peptide and its receptors, since this has been done quite well by others. The reader will be directed to these other reviews, where appropriate. Instead, this review attempts to provide a summary of current knowledge about neurotensin, why it is an important peptide to study, and where the field is heading. Special emphasis is placed on the behavioral studies, particularly with reference to agonists, antagonists, and antisense studies, as well as, the interaction of neurotensin with other neurotransmitters.

In vivo studies with low doses of levocabastine and diphenhydramine, but not pyrilamine, antagonize neurotensin-mediated antinociception.

The present study describes in vivo experiments in the rat addressing the role of levocabastine, and two other specific histamine H1 antagonists, diphenhydramine and pyrilamine, at neurotensin (NT)-mediated hypothermia and antinociception (hotplate). Levocabastine given i.p. or microinjected directly into the periaqueductal gray (PAG) did not cause antinociception or hypothermia. This indicates that despite the results with the recently-cloned levocabastine-sensitive NT receptors (NTR) in the rat (NTR-2) and mouse (NTRL), levocabastine by itself does not mediate either hypothermia or antinociception at NT receptors. However, pretreatment with 5 or 50 microg/kg of levocabastine or 5 microg/kg diphenhydramine all caused over a three-fold reduction in NT-mediated antinociception. Higher doses (500 or 5000 microg/kg) of levocabastine did not cause any antagonism of NT-mediated antinociception. All three antihistamines did not affect NT-mediated hypothermia. In addition, histamine H1 pathways are not involved in NT-mediated antinociception, as pretreatment with the much more potent histamine H1 antagonist pyrilamine did not affect antinociception mediated by NT. Therefore, these data may suggest the presence of yet unidentified NTR subtypes responsible for NT-mediated hypothermia and antinociception.

Chimeric rat/human neurotensin receptors localize a region of the receptor sensitive to binding of a novel, species-specific, picomolar affinity peptide.

Recently, we reported the development of a species-specific neurotensin analog that displays selective binding affinity at the rat and human neurotensin (NT) receptor, L-[3,2'-Nal11]NT(8-13) (where Nal is naphthylalanine) (NT19). We have developed another neurotensin analog, L-[3,1'-Nal11]NT(8-13), (NT34), that exhibits a 126-fold difference in binding affinities between the rat and human receptors. This compound differs from our previous reported species-specific ligand in the steric positioning of the naphthyl ring on the L-alanine side chain. For NT34, the observed Kd values at the rat and human neurotensin receptors were 0.046 and 5.8 nM, respectively. In stimulating phosphatidylinositol turnover, the observed EC50 values were 2.8 nM and 130 nM in rat and human, respectively. We constructed a series of chimeric rat/human neurotensin receptor genes and expressed them by transient transfection into human embryonic kidney (HEK-293) cells. Radioligand binding assays were then performed using neurotensin and NT34. Our results led us to propose a region of the neurotensin receptor that may be involved in determining species specificity, i. e. the transmembrane VI, the third extracellular loop, and transmembrane VII regions of the neurotensin receptor.

A method for radioligand binding assays using a robotic workstation.

Radioligand binding assays provide a powerful tool for screening drug candidates at many receptors. We present a method utilizing a robotic workstation, the Biomek 1000, which automates the tedious and repetitive tasks of these assays. First, the robot handles the serial dilution of up to 8 drugs with 11 concentrations per drug. The sequential addition of diluting buffer, non-radioactively labeled ligand, radioactive ligand, and finally the tissue homogenate or membrane preparation, is fully automated. A novel rack design allows the use of tubes with a maximum capacity of 2 ml, providing a total assay volume of 1 ml. Final filtration on a Brandel cell harvester outfitted with a uniquely designed head allows for processing of 48 samples simultaneously from the rack holder. We have employed this method for the determination of equilibrium dissociation constants (Kds) for drugs at the 5 human muscarinic acetylcholine receptor subtypes expressed in cultured cells, as well as histamine H1, dopamine D2, serotonin 5HT1A, alpha 1- and alpha 2-adrenergic receptors in human brain tissue homogenates. Our results compare favorably with manual methods reported for these receptors, and exhibit a very high degree of reproducibility and throughput, with a minimum of operator input.

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Selective loss with subculturing of the low-affinity agonist site mediating cyclic GMP formation.

Cells of the murine neuroblastoma clone N1E-115 possess muscarinic receptors that influence the intracellular level of cyclic nucleotides. The stimulation of [3H]cyclic GMP levels occurs only with intact cells and has an EC50 near the "low-affinity" agonist equilibrium dissociation constant (KL) determined by radioligand binding assays. The inhibition of prostaglandin E1-stimulated [3H]cyclic AMP formation has an EC50 close to the value for the "high-affinity" agonist equilibrium dissociation constant (KH). During sequential subculturing in medium supplemented with newborn bovine serum, the inhibition of [3H]cyclic AMP was maintained, but the [3H]cyclic GMP response declined dramatically, and after 7 subculturings it was essentially absent. The time course for [3H]cyclic GMP formation in a late subculture with an 88% loss of the response was identical with the time course in early subcultures. A normal [3H]cyclic GMP response to bradykinin and histamine was demonstrated to be present in cells that had lost the [3H]cyclic GMP response to carbachol. The EC50 and KD values for the two muscarinic responses and binding sites increased 3- to 4-fold after several subculturings. A 90% loss of low-affinity binding sites was closely correlated with a similar loss of the [3H]cyclic GMP response. High-affinity binding sites did not decline significantly in concentration until the 11th subculture, where the total number of muscarinic sites was only 6% of the earliest subculture. In all subcultures, however, the ability of the muscarinic receptor to decrease [3H]cyclic AMP levels was maintained. These data, which show that the subculturing of N1E-115 cells in medium supplemented with newborn calf serum results in a selective loss of one muscarinic function, strongly support the hypothesis that these cells contain two separate muscarinic receptor-effector systems. One receptor subtype or conformation has a low affinity for the agonist and mediates cyclic GMP formation. The other receptor subtype or conformation has a higher affinity for the agonist and mediates an inhibition of prostaglandin E1-stimulated cyclic AMP formation.

Lack of function of histamine H1 and muscarinic acetylcholine receptors of mouse neuroblastoma cells grown in serum-free medium.

Mouse neuroblastoma cells (Clone NIR-115) were grown in serum-free (defined) medium, defined medium supplemented with serum, and control medium to determine whether serum-free medium could substitute for serum-containing medium in our studies of the histamine H1 and muscarinic acetylcholine receptors of these cells. The function of these receptors as determined by measurement of receptor-mediated cyclic [3H]GMP formation was absent in cells grown in serum-free medium and increased as the percentage of serum was increased in the defined medium, but never attained the levels found with control cells. Muscarinic receptor number for cells grown in defined medium was 60% above that found for control cells with no change in the affinity of the receptor for the radioligand (--)[3H]quinuclidinyl benzilate. Guanylate cyclase and acetylcholinesterase activities for cells grown in defined medium were 23 and 66% of those found in control cells, respectively. This marked reduction of guanylate cyclase activity in large part explains the lack of function of these receptors.