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B Cusack

Publications and source records attributed to B Cusack.

35 records · Page 2Linked to original sources
PubMed1993

Identification of a polymorphism in the human neurotensin receptor gene.

A complementary DNA (cDNA) clone encoding the neurotensin receptor was isolated from a human substantia nigra cDNA library. The deduced amino acid sequence of this clone was almost identical to that of a cDNA for this receptor cloned from a previously described HT29 human colonic adenocarcinoma cell line. We found three base changes between the previously reported HT29 cDNA clone and the current cDNA clone. We investigated these changes by using polymerase chain reactions to amplify these areas from various human samples. One of the differences, which resulted in an amino acid change at AA194 (a leucine in the HT29 sequence was a phenylalanine in the current sequence), was found in some, but not in all, human samples. This finding represents genetic variability in human neurotensin receptors, the first such report for a peptide receptor. Both of these receptors, however, when expressed separately in transfected cell lines, had similar affinities for neurotensin and some related peptides.

Amino Acid Sequence↗
PubMed1993

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.

Animals↗
PubMed1993

Pharmacological studies on novel neurotensin mimetics: discovery of a pharmacologically unique agent exhibiting concentration-dependent dual effects as antagonist and agonist.

We report the development of two novel neurotensin mimetics (mimics 1 and 2). These compounds were rationally designed and synthesized according to the multiple template approach. We present results of experiments designed to define their pharmacological profiles. In radioligand binding assays with murine neuroblastoma clone N1E-115, we determined the equilibrium dissociation constants for these compounds at the neurotensin receptor. The Kd values for mimic 1 and mimic 2 were 3.3 microM and 1.9 microM, respectively. Functionally, both mimetics antagonized the neurotensin-stimulated production of cGMP, with Kd values in the low micromolar range. Interestingly, mimic 2 displayed a dualistic pharmacological profile, which was concentration dependent. At doses in the 10-100 microM range, mimic 2 became a full agonist, stimulating cGMP production in N1E-115 cells with an EC50 value of 19 microM. Furthermore, mimic 1 did not antagonize the cGMP response elicited by mimic 2. When the neurotensin receptor was desensitized with a neurotensin receptor agonist, mimic 2 failed to stimulate significant cGMP production. We propose that mimic 2 binds to a higher affinity site when acting as an antagonist and binds to a lower affinity and different site when acting as an agonist. Thus, mimic 2 would appear to represent a unique pharmacological tool to characterize the neurotensin receptor and its diverse binding sites in N1E-115 cells.

Amino Acid Sequence↗
PubMed1993

Desensitization and down-regulation of neurotensin receptors in murine neuroblastoma clone N1E-115 by [D-Lys8] neurotensin(8-13).

Clone N1E-115 cells have specific, high-affinity binding sites for neurotensin (NT). These receptors mediate formation of cyclic GMP and other second messengers. We studied the effects of extended exposure of cells to [D-Lys8]NT(8-13) (NT2), a peptidase-resistant NT analog, on NT's ability to stimulate cyclic GMP synthesis and to bind [3H]NT to its sites on these cells. When intact N1E-115 cells were preincubated with NT2 (1 microM) for various times (15 min to 12 hr) at 37 degrees C, maximal desensitization and binding site (Bmax) loss occurred after only 15 min. Receptor affinity for [3H]NT did not change. At 0 degrees C NT2 for 15 min caused no down-regulation. After 15 min of preincubation with NT2 at 37 degrees C, the recovery of receptor binding and function after a 10 to 20 min lag-time was rapid (T1/2 = 15 min and 19 min, for binding and cyclic GMP response, respectively). After 12 hr of NT2 preincubation, it was slow (T1/2 = 212 min). Incubation of cells with cycloheximide (70 microM) for 5 hr after their exposure to NT2 for 15 min did not change the rate or extent of recovery of NT Bmax. However, cycloheximide did decrease the recovery of NT Bmax after exposure of cells to NT2 for 12 hr. Cycloheximide alone did not change NT Bmax. These data suggest that this decrease of NT receptor binding after short (15 min) and long (12 hr) exposure times to agonist involves two consecutive steps: intracellular sequestration of recyclable NT receptors, followed by receptor degradation causing true down-regulation.

Animals↗
PubMed1992

The rat neurotensin receptor expressed in Chinese hamster ovary cells mediates the release of inositol phosphates.

To study second messenger synthesis mediated by the cloned rat neurotensin receptor, we derived a cell line stably expressing this receptor. The cDNA clone of this receptor was subcloned into the pcDNA1neo expression vector. This construct was then used to transfect Chinese hamster ovary (CHO)-K1 cells. Colony clones, selected for resistance to antibiotic G-418 sulfate, were isolated and grown separately. Nineteen individual clones were screened for total [3H]neurotensin binding as an indication of neurotensin receptor expression. The clone (CHO-rNTR-10) showing the highest level of specific [3H]neurotensin binding was characterized further. With intact cells, the equilibrium dissociation constant (KD) for specific [3H]neurotensin binding was 18 nM, and the maximal number of binding sites (Bmax) was 900 fmol/mg of protein or 740 fmol/10(6) cells (approximately 4.4 x 10(5) sites on the cellular surface). Whereas the KD was similar to that found in other cellular systems, for example, the murine neuroblastoma clone N1E-115, the Bmax exceeded previously reported values. Incubation of intact CHO-rNTR-10 cells with neurotensin caused the release of inositol phosphates in a dose-dependent manner (EC50 = 3 nM), results indicating that the expressed transfected receptor was functional. Neurotensin did not inhibit cyclic AMP levels stimulated by forskolin. As with other systems, neurotensin (8-13) was more potent than neurotensin Neurotensin-mediated inositol phosphate release is the first report of second messenger synthesis for this receptor expressed in a transfected cell line. These results suggest that the relation between structure and function of the neurotensin receptor can be readily studied in transfected cell lines.

Animals↗
PubMed1992

Antagonism by antimuscarinic and neuroleptic compounds at the five cloned human muscarinic cholinergic receptors expressed in Chinese hamster ovary cells.

We determined the affinity and selectivity of binding for 24 compounds: nine antimuscarinics (including some antiparkinson drugs) and 15 neuroleptics (including the atypical compounds clozapine, fluperlapine, melperone, rilapine, risperidone, tenilapine, tiosperone and zotepine) at the five human muscarinic receptor subtypes expressed in Chinese hamster ovary cells. Equilibrium dissociation constants (Kd) were obtained from competitive radioligand binding studies with [3H]quinuclidinyl benzilate and membranal preparations of these cells. As expected, QNB had the highest affinity of the compounds studied at the five receptor subtypes and was not selective (Kd ranged from 0.027-0.088 nM). Benztropine had the next highest affinity of the antimuscarinic compounds and thioridazine had the highest affinity of the neuroleptics. Among the antiparkinson drugs, biperiden was the only one selective for the m1 subtype; and among the neuroleptics, the atypical drug clozapine was also selective for the m1 subtype. This selectivity may explain clozapine's unusual efficacy in refractory schizophrenic patients and its low incidence of extrapyramidal side effects. However, because most other atypical neuroleptics studied lacked high affinity and selectivity at muscarinic receptor subtypes, it is likely that other mechanisms are involved as well.

Animals↗
PubMed1992

Overcoming problems with polypharmacy and drug misuse in the elderly.

Polypharmacy, the inappropriate use of multiple drug regimens, has a significant impact on the health of elderly individuals. Drug use increases with age, but suitability of therapy is sometimes difficult to define. In ambulatory and hospital care, there is some documentation of poor prescribing practices by physicians and drug misuse by patients. Sound data suggest that polypharmacy and drug misuse are highly prevalent in long-term care facilities. Psychotherapeutic agents are the most commonly misused drugs by physicians in this setting. Polypharmacy is associated with factors such as the number and severity of illnesses, hospitalization, number of physicians seen, number of pharmacies used, and possibly increased patient age. Methods to prevent polypharmacy and drug misuse have not been well studied. There is a need for intensive research to define effective methods to strengthen prescribing practice of physicians for elderly patients, to promote cooperation among health care personnel in ensuring optimal drug use by patients, and to enhance the role of patients as responsible partners in drug therapy. The authors are confident, however, that drug misuse and polypharmacy can be reduced in older persons by the use of current knowledge of risk factors associated with polypharmacy and by improved communication between the physician and patient.

Aged↗
PubMed1991

Developmental regulation of neurotensin receptor expression and function in murine neuroblastoma clone N1E-115.

Murine neuroblastoma cells (clone N1E-115) during their growth from log to late stationary phase, expressed no specific neurotensin binding sites until day 7 in culture. From day 7 to day 20, binding sites increased 6-fold in number/cell and more than 4-fold in sites/mg protein. Neurotensin-mediated cyclic [3H]GMP synthesis was not detected until day 17. For these cells these data show that neurotensin receptor binding and function are regulated with respect to growth cycle and that presence of neurotensin binding sites is not sufficient for receptor function.

Animals↗
PubMed1985

Protein binding and disposition of lignocaine in the elderly.

Single dose studies were performed in six young and six elderly nonsmokers using lignocaine as a model drug with high intrinsic clearance. Subjects received lignocaine 250 mg orally and 50 mg intravenously in random order and drug concentrations in blood and plasma were measured for up to 8 h after dose. Protein binding was estimated at 37 degrees C by equilibrium dialysis. Indocyanine green kinetics were also calculated in each individual following 0.15 mg/kg intravenously. Bioavailability of lignocaine was greater in the elderly but there was no apparent difference in the rate of absorption. Intrinsic clearance of lignocaine was lower in the aged. Elimination half-life was longer in the elderly but there was no significant difference in apparent volume of distribution or systemic clearance of lignocaine. Plasma clearance of indocyanine green showed no correlation with systemic lignocaine clearance and was lower in the aged subjects. Blood/plasma lignocaine ratio was less than unity in both groups. Binding of lignocaine to plasma proteins showed concentration-dependence and was higher in the geriatric group. Maximum binding capacity of lignocaine was greater in the elderly but the binding affinity did not significantly change with age. Greater oral bioavailability of drugs like lignocaine may produce higher plasma concentrations in the elderly. Unlike indocyanine green, the systemic clearance of lignocaine was unaltered by age in this group of non-smokers. The protein-binding of lignocaine, like many other basic drugs, is increased in elderly subjects.

Adult↗
PubMed1980

Theophylline kinetics in relation to age: the importance of smoking.

1 Single dose studies of theophylline kinetics were compared in groups of young and elderly smokers and non-smokers to assess the effect of age on theophylline absorption and the effect of smoking on drug metabolising enzyme activity in old age. 2 The rate and extent of absorption was not affected by age. Distribution and elimination kinetics were similar in young and elderly non-smokers. 3 In young subjects the elimination half-life of theophylline was shorter and clearance was significantly greater in smokers than in non-smokers. 4 In the elderly mean elimination half-life was significantly shorter in smokers and their plasma clearance was 40% higher than in non-smokers. The statistical difference for clearance was at the 7% level of significance. 5 These data indicate that ageing per se does not affect theophylline elimination and also that induction of theophylline metabolism due to smoking occurs in old age. Smoking is a variable that should be taken account of when assessing drug metabolism in elderly patients.

Absorption↗
PubMed1980

Effects of caffeine on anterior pituitary and thyroid function in the rat.

We studied the effects of acute, intraperitoneal administration of caffeine on serum thyrotropin (TSH), growth hormone, prolactin, thyroxine and 3,3',5-triiodothyronine in rats. Caffeine lowered serum TSH and GH in a dose-dependent manner with ED50 values of 30 and approximately 50 mg/kg, respectively. TSH levels were depressed 1 to 6 hr after injection and correlated with serum caffeine levels greater than 20 micrograms/ml. The decrease in serum TSH was followed by decreases in serum 3,3',5-triiodothyroxine and thyroxine 4 hr after caffeine administration. Theophylline and theobromine had effects similar to those of caffeine on hormone levels. Caffeine did not significantly affect hormone secretion when incubated directly with rat pituitaries in vitro. Administration of antisomatostatin antiserum to rats blocked the inhibitory effects of caffeine on serum GH levels, suggesting that caffeine inhibits GH and TSH secretion by releasing hypothalamic somatostatin.

Animals↗
PubMed1979

Digoxin in the elderly: pharmacokinetic consequences of old age.

A study of single-dose digoxin kinetics was performed in 6 young and 7 elderly patients. The rate of absorption, determined by the time to peak concentration after an oral dose, was more rapid in the younger group. The extent of absorption, as measured by comparison of the area under the plasma concentration/time curve after oral and intravenous administration, was similar in both groups. Mean plasma half-life was longer and individual values were more variable in the elderly patients. The absolute apparent volume of distribution was reduced in the older patients and correlated inversely with age. When corrected for weight, however, the difference in the apparent volume of distribution did not approach significance. Both absolute and weight-corrected values for plasma clearance of digoxin were reduced in the elderly subjects.

Administration, Oral↗
PubMed1977

Tangier disease (alpha-lipoprotein deficiency).

A patient with asymptomatic hypocholesterolemia, mild hyperbilirubinemia, and splenomegaly was found, on lipoprotein analysis, to have Tangier disease (alpha-lipoprotein deficiency). He represents the first patient with the disease in the northeastern Unit States. Although free of clinical evidence of atherosclerosis at age 38 years, the patient has widespread tissue cholesterol ester deposition and a stron family history of atherosclerosis. Tangier disease may be much underdiagnosed; it should be suspected in every patient with hypocholesterolemia.

Adult↗