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B Cusack

Publications and source records attributed to B Cusack.

At least 19 recordsLinked to original sources
PubMed2000

A single amino acid of the human and rat neurotensin receptors (subtype 1) determining the pharmacological profile of a species-selective neurotensin agonist.

The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spanning receptor, forming 3 extracellular and 3 intracellular loops. Previously, we showed that the third outer loop (E3) is the binding site for NT and its analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 [3,1'-naphthyl-l-Ala(11)]NT(8-13) has greater than 60-fold higher affinity for rNTR1 (46 and 60 pM for transiently- and stably-transfected cells, respectively) than for hNTR1 (2.8 and 5.8 nM for transiently- and stably-transfected cells, respectively) isolated from transfected cell membranes. Previously, our molecular modeling studies of rNTR1 and hNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr(339) in the pocket center, as compared with the corresponding residue Phe(344) in the rat binding pocket. Therefore, with site-directed mutagenesis, we derived mutant forms of rNTR1(F344Y) and hNTR1(Y339F). Examination of the mutant receptors from membranal preparations of transfected cells in radioligand binding assays and with intact cells in functional assays (phosphatidyl-4,5-bisphosphate turnover) showed that the human-like rat receptor and the rat-like human receptor bound NT34 with a predicted reverse of binding compared with its binding to the wild-type receptors. These results strongly affirm our molecular modeling studies and demonstrate the importance of the study of even minor structural variations in proteins to determine the basis of significantly different drug responses, an area of focus for pharmacological research in the 21st century.

Amino Acid Substitution↗
PubMed2000

A novel neurotensin peptide analog given extracranially decreases food intake and weight in rodents.

Neurotensin decreases food intake in the rat when injected into the cerebral ventricles. We tested the effect of a novel neurotensin analog (NT69L), injected intra-peritoneally (i.p.), on weight gain and food intake in rats. Sprague-Dawley rats (270 g) were injected i. p. with either saline or NT69L at 0.001 or 0.010 mg/kg. In further experiments, larger rats at a more steady state on the growth curve (400 g) were injected with either saline or 0.010 or 1 mg/kg NT69L. Food intake, water consumption and body weight were recorded daily. Weight gain was significantly reduced in the smaller rats injected with 0.001 or 0.010 mg/kg, showing only a 8.5 and 9.0% increase in original weight, respectively, as compared to a 29% increase for the controls. The larger rats injected with 1 mg/kg, had a significant reduction in body weight with a 3.0% decrease in original body weight as compared to a 2.4% increase for the controls. Food intake was significantly reduced suggesting that the weight loss observed after injection of NT69L was attributable in part to a reduction in food intake. The genetically obese Zucker rats injected with NT69L (1 mg/kg) had a significant reduction in weight gain and food intake. NT69L significantly increased blood glucose and corticosterone levels and decreased TSH and T4 in Sprague-Dawley and Zucker rats, an effect that was only transitory. NT69L also caused a decrease in norepinephrine in both the hypothalamus and nucleus accumbens, and an increase in dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and serotonin. In this study, NT69L exhibited a consistent and dramatic effect on body weight and food intake in Sprague-Dawley and obese Zucker rats, and enabled us to study the role that NT plays in weight control and the functional interactions of NT with brain amines, and metabolic and endocrinological parameters.

Animals↗
PubMed2000

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 microgram/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 microgram/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 microgram/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 microg/kg. There was no significant difference between the ED(50)s 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.

Animals↗
PubMed2000

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.

Animals↗
PubMed2000

Huprine X is a novel high-affinity inhibitor of acetylcholinesterase that is of interest for treatment of Alzheimer's disease.

Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.

Acetylcholinesterase↗
PubMed1999

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.

Animals↗
PubMed1999

In vitro binding and CNS effects of novel neurotensin agonists that cross the blood-brain barrier.

Animal studies with neurotensin (NT) directly injected into brain suggest that it has pharmacological properties similar to those of antipsychotic drugs. Here, we present radioligand binding data for some novel hexapeptide analogs of NT(8-13) at the molecularly cloned rat and human neurotensin receptors (NTR-1), along with behavioral and physiological effects of several of these peptides after intraperitoneal (i.p.) administration in rats. One unique analog, NT66L, which had high affinity (0.85 nM) for the molecularly cloned rat neurotensin receptor (NTR-1), caused a drop in body temperature and antinociception at doses as low as 0.1 mg/kg after i.p. injection. At 30 min post-injection, the ED50 for NT66L-induced hypothermia (rectal temperature) and antinociception (hot plate test) was 0.5 and 0.07 mg/kg, respectively. At a dose of 1 mg/kg i.p., NT66L caused 100% of the maximum possible effect for antinociception for up to 2 h after administration. At this dose body temperature lowering was greater than -2.5 degrees C from 20 to 120 min after i.p. administration. These results in animals suggest that NT66L has agonist properties at NTR-1 in vivo after extracranial administration and provide support for its further study in behavioral tests predictive of neuroleptic activity.

Animals↗
PubMed1998

Evidence for additional neurotensin receptor subtypes: neurotensin analogs that distinguish between neurotensin-mediated hypothermia and antinociception.

Neurotensin (NT), a tridecapeptide, is a neurotransmitter that elicits potent effects including hypothermia and antinociception in mice and rats. To date, there are two types of the neurotensin receptor (NTR) that have been molecularly cloned from the rat. However, several lines of evidence suggest the presence of additional NTR subtypes. We have identified a NT analog of the NT(8-13) fragment, NT27, that selectively causes only the hypothermic response in vivo, when microinjected into the periaqueductal gray (PAG) of rats. A dose of 18 nmol of NT or NT27 caused a body temperature lowering of 1.8 and 1.2 degrees C, respectively. This same dose of NT or NT27 yielded a hotplate maximum physiological effect of 75% and 25%, respectively. Interestingly, despite its high KD (620 nM) at the cloned NTR-1, NT27-I (the iodinated form of NT27) exerted a potent hypothermic effect even at a very low dose (0.6 nmol). Equally intriguing, was that NT24, a sterioisomer of NT27, with a much higher affinity (KD=0. 5 nM) at NTR-1, did not selectively induce hypothermia in mice, but did selectively induce hypothermia in rats.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo.

Peptide nucleic acids (PNAs) are DNA analogs that can hybridize to complementary sequences with high affinity and stability. Here, we report the first evidence of intracellular delivery of PNAs in vivo. Two CNS receptors, an opioid (mu) and a neurotensin (NTR-1), were targeted independently by repeated microinjection of PNAs into the periaqueductal gray. Behavioral responses to neurotensin (antinociception and hypothermia) and morphine (antinociception) were lost in a specific manner. Binding studies confirmed a large reduction in receptor sites. The loss of behavioral responses was long lasting but did fully recover. The implications of specifically and readily turning off gene expression in vivo are profound.

Animals↗
PubMed1998

Refined radiation hybrid map of mouse chromosome 17.

We have made a radiation hybrid map of mouse Chromosome (Chr) 17 with 75 microsatellite markers, including those from McCarthy et al. (Genome Res 7, 1153-1161, 1997). Seventy-four of the markers are linked at LOD > 9, and all link at LOD > 5. A LOD 3 framework of 18 markers was used to construct a placement map. The order obtained is in good agreement with genetic maps, and distance estimates give an idea of how recombination rates vary across the chromosome. Recombination is remarkably low with respect to RH break frequency in the region from the centromere to the end of H2. This is similar in interspecific and intersubspecific crosses despite the inversion of a substantial part of this region in Mus spretus with respect to Mus musculus.

Animals↗
PubMed1997

Managing TMD.

Explore the source record for details and available documents.

American Dental Association↗
PubMed1996

Proposed ligand binding site of the transmembrane receptor for neurotensin(8-13).

We report here the first proposed ligand binding site of the transmembrane receptor for neurotensin(8-13) in human and rat, the corresponding bound conformation of the peptide ligand, and site-directed mutagenesis studies that support the binding site model. These three-dimensional structures were generated by using a heuristic approach in conjunction with experimental data. The proposed neurotensin(8-13) binding site is primarily composed of eight residues (i.e., Phe326, Ile329, Trp334, Phe337, Tyr339, Phe341, Tyr342, and Tyr344 in the human receptor; Phe331, Ile334, Trp339, Phe342, Phe344, Phe346, Tyr347, and Tyr349 in the rat receptor) located in the third extracellular loop. The seven aromatic residues form an aromatic pocket on the extracellular surface of the neurotensin receptor to accommodate its ligands apparently by cation-pi, pi-pi, and hydrogen bonding interactions. The neurotensin(8-13) ligand adopts a compact conformation at the proposed binding site. In the bound conformation of neurotensin(8-13), the backbone of Arg9-Pro10-Tyr11-Ile12 forms the proline type I turn, and the hydroxy group of Tyr11 interacts with the two guanidinium groups of Arg8 and Arg9. These guanidinium groups are curled toward the hydroxy group so that they interact electrostatically with the hydroxy group, and that the guanidinium group of Arg9 forms an intra-hydrogen bond with the hydroxy group. The proposed three-dimensional structure may not only provide a basis for rationalizing mutations of the neurotensin receptor gene but also offer insights into understanding the binding of many neurotensin analogs, biological functions of the neurotensin receptors, and structural elements for species specificity of the neurotensin receptors, and may expedite developing nonpeptidic neurotensin mimetics for the potential treatment of the neuropsychiatric diseases.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1995

Pharmacological and biochemical profiles of unique neurotensin 8-13 analogs exhibiting species selectivity, stereoselectivity, and superagonism.

Recently, the rat neurotensin receptor and the two human neurotensin receptor clones (differing by one amino acid residue) have been isolated. We present results with 33 newly synthesized neurotensin analogs. We have evaluated their binding potency at the three neurotensin receptor clones by determining equilibrium dissociation constants and coupling to phosphatidylinositol turnover. Our work focused on position 8 and 9 substitutions as well as position 11 of the neurotensin hexamer NT8-13. The results presented include: 1) the development of a compound that is species selective, with a binding potency at the rat receptor that is 20-fold more potent than at the human receptor; 2) the development of a pair of stereoselective compounds with the L-isomer exhibiting 190-700-fold more potency than the D-isomer; and 3) the development of an agonist that has a Kd of 0.3 and 0.2 nM at the human and rat neurotensin receptor, respectively, ranking it as among the most potent tested. Also, we present the first evidence that 1) the effect of pi electrons at position 11 (L-Tyr) are important for binding to the neurotensin receptor, and 2) the length of the side chain on position 9 (L-Arg) changes binding potency.

Amino Acid Sequence↗
PubMed1994

Rational design of novel neurotensin mimetics: discovery of a pharmacologically unprecedented agent exhibiting concentration-dependent dual effects as antagonist and full agonist.

We report the rational design of novel neurotensin mimetics through use of the Multiple Template Approach. This approach is based on our notion that a flexible peptide can be replaced by a partially flexible molecule, identified through testing a comparatively small number of molecules possessing a different intrinsic availability of conformations of the native peptide. The Multiple Template Approach has culminated in the discovery of a pharmacologically unprecedented agent, which behaves as a neurotensin antagonist at low concentration and as a full neurotensin agonist at high concentration.

Amino Acid Sequence↗
PubMed1994

Binding of antidepressants to human brain receptors: focus on newer generation compounds.

Using radioligand binding assays and post-mortem normal human brain tissue, we obtained equilibrium dissociation constants (Kds) for 17 antidepressants and two of their metabolites at histamine H1, muscarinic, alpha 1-adrenergic, alpha 2-adrenergic, dopamine D2, serotonin 5-HT1A, and serotonin 5-HT2 receptors. Several newer antidepressants were compared with older drugs. In addition, we studied some antimuscarinic, antiparkinson, antihistamine, and neuroleptic compounds at some of these receptors. For the antidepressants, classical tricyclic antidepressants were the most potent drugs at five of the seven receptors (all but alpha 2-adrenergic and 5-HT1A receptors). The chlorophenylpiperazine derivative antidepressants (etoperidone, nefazodone, trazodone) were the most potent antidepressants at alpha 2-adrenergic and 5-HT1A receptors. Of ten antihistamines tested, none was more potent than doxepin at histamine H1 receptors. At muscarinic receptors antidepressants and antihistamines had a range of potencies, which were mostly weaker than those for antimuscarinics. From the in vitro data, we expect adinazolam, bupropion, fluoxetine, sertraline, tomoxetine, and venlafaxine not to block any of these five receptors in vivo. An antidepressant's potency for blocking a specific receptor is predictive of certain side effects and drug-drug interactions. These studies can provide guidelines for the clinician in the choice of antidepressant.

Antidepressive Agents↗