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Biomedical subjects

T J Murphy

Publications and source records attributed to T J Murphy.

At least 19 recordsLinked to original sources
PubMed1992

A recombinant rat vascular AT1 receptor confers growth properties to angiotensin II in Chinese hamster ovary cells.

A rat vascular AT1 receptor cDNA has been stably expressed into Chinese Hamster Ovary cells and the resulting recombinant AT1a receptor has been functionally characterized. This receptor binds 125I Sar1-angiotensin II with an affinity of 0.9 nM and the displacement of this ligand by a series of peptidic and nonpeptidic analogs is shown. Binding of angiotensin II to this receptor causes a rapid increase in inositol phosphate production, whereas this effect is not observed in nontransfected cells. Des-aspartyl1 angiotensin II and at a lesser extent angiotensin I are also able to produce an increase in inositol phosphates. More importantly, the actions of angiotensin II on cell division were clearly demonstrated in this model, since angiotensin II is able to stimulate DNA synthesis by 400% and double the cell population of the transfected cells in 36 hours in the absence of any other growth factor, whereas no effect is observed in nontransfected cells.

Angiotensin II↗
PubMed1992

Molecular cloning of AT1 angiotensin receptors.

Angiotensin II is the principal effector molecule of the renin-angiotensin system. Its effects are mediated by cell surface proteins termed AT receptors. On the basis of radioligand binding studies, these have been pharmacologically subdivided into two classes, termed AT1 receptors and AT2 binding sites (Chiu AT, et al, Biochem Biophys Res Commun 1989;165:196-203). AT1 receptors appear to mediate the major cardiovascular effects of angiotensin II, whereas no known physiological properties appear to be coupled to AT2 binding sites (Wong PC, et al, J Pharmacol Exp Ther 1990;255:584-592). To gain further insight into the function of AT1 receptors we have isolated rat cDNA's and genes encoding two distinct but highly similar isoforms of AT1 receptors, termed AT1a and AT1b receptors. Two cDNA's encoding the vascular AT1a receptor were isolated by an expression cloning strategy from a cDNA library prepared from vascular smooth muscle cells. The properties of the clones isolated by this approach are consistent with known pharmacological, biochemical signaling, and tissue distribution properties of AT1 receptors. Using this cDNA as a probe, a second isoform of rat AT1 receptor was isolated from a genomic library. This receptor, termed the AT1b receptor, is 95% identical in amino acid sequence and is pharmacologically indistinguishable from the AT1a receptor. However, the tissue-specific expression pattern of the AT1b gene differs significantly from that for the AT1a receptor.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.

The constitutive endothelial cell nitric oxide synthase (NOS) importantly regulates vascular homeostasis. To gain understanding of this enzyme, a pEF BOS cDNA library of 5 x 10(5) clones was prepared from bovine aortic endothelial cells (BAEC) and screened with a 2.8-kb cDNA BamHI fragment of rat brain NOS. Clone pBOS13 was found to express NO synthase activity when transfected into COS-7 cells. Sequence analysis revealed sequences compatible with binding domains for calcium/calmodulin, flavin mononucleotide, flavin adenine nucleotide and NADPH. The deduced amino acid sequence revealed a protein with a relative mol mass of 133,286, which is 58% homologous to the rat cerebellar NOS and 51% homologous to the mouse macrophage NOS. The amino-terminal portion of the protein exhibits several characteristics peculiar to the endothelial cell NOS. These include a proline-rich region and several potential sites for proline-directed phosphorylation as well as a potential substrate site for acyl transferase. Northern hybridization to mRNA from cultured BAEC revealed an abundant 4.8-kb message, which was not increased by coincubation with tumor necrosis factor alpha, but was markedly increased by exposure to shear stress for 24 h. The unique features of the endothelial cell NO synthase, particularly in the amino terminal portion of the molecule, may provide for novel regulatory influences of enzyme activity and localization.

Amino Acid Oxidoreductases↗
PubMed1991

Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor.

Angiotensin II is an important effector molecule controlling blood pressure and volume in the cardiovascular system. Its importance is manifested by the efficacy of angiotensin-converting enzyme inhibitors in the treatment of hypertension and congestive heart failure. Angiotensin II interacts with two pharmacologically distinct subtypes of cell-surface receptors, AT1 and AT2. AT1 receptors seem to mediate the major cardiovascular effects of angiotensin II. Here we report the isolation by expression cloning of a complementary DNA encoding a unique protein with the pharmacological specificity of a vascular AT1 receptor. Hydropathic modelling of the deduced protein suggests that it shares the seven-transmembrane-region motif with the G protein-coupled receptor superfamily. Knowledge of the AT1 receptor primary sequence should now permit structural analysis, definition of the angiotensin II receptor gene family and delineation of the contribution of AT receptors to the genetic component of hypertension.

Amino Acid Sequence↗
PubMed1991

Assessment of schizophrenic inpatients with the MCMI.

The Million Clinical Multiaxial Inventory (MCMI) Psychotic Thinking scale previously was found insensitive in the detection of schizophrenia. The MCMI also was shown to be susceptible to undetected "faking good." We hypothesized that the insensitivity of the MCMI Psychotic Thinking scale was due to an unwillingness of schizophrenic patients to report psychotic symptoms. The MCMI was administered to 258 male schizophrenic inpatients who were classified as symptom-reporters or nonreporters based upon whether they endorsed psychotic symptoms on a separate problem checklist. Willingness to report psychotic symptoms was a significant factor in MCMI Psychotic Thinking scale scores as well as many other MCMI scales.

Adult↗
PubMed1991

Characterization of the alpha-2C adrenergic receptor subtype in the opossum kidney and in the OK cell line.

The alpha-2 adrenergic receptors have been subdivided into two pharmacological subtypes known as alpha-2A and alpha-2B. The OK cell, a cell line derived from an opossum kidney, expresses an alpha-2 adrenergic receptor which has pharmacological characteristics different from both the alpha-2A and alpha-2B subtypes, and thus may be a third subtype. To test this hypothesis, we compared the affinities (Ki values from radioligand binding) of 49 drugs in the OK cell with their affinity for the alpha-2A (HT-29 cells) and alpha-2B (neonatal rat lung) adrenergic receptor subtypes. Eight drugs (spiroxarine, prazosin, WY 27127, L-657,743, ARC 239, akuammigine, rauwolscine and oxymetazoline) were identified whose Ki differed by 10-fold or more between the OK cells and HT29 cells. Five drugs (BAM 1303, raubasine, WB 4101, akuammigine and rauwolscine) differentiated, by at least 10-fold, between OK cells and neonatal rat lung. Correlations of pKi values between the OK cell and tissues or cell lines expressing either the alpha-2A or alpha-2B subtypes were poor, confirming that the OK cell receptor could not be classified as either alpha-2A or alpha-2B. In contradistinction, there was a good correlation between pKi values for the OK cell line and opossum kidney showing that they express the same subtype. In addition, ratios of subtype-selective drug Ki values were similar for the OK cell and opossum kidney, but different from other tissues or cell lines expressing either the alpha-2A or alpha-2B subtypes. We conclude that the OK cell line and opossum kidney express a novel subtype of alpha-2 adrenergic receptor which we term the alpha-2C subtype.

Animals↗
PubMed1990

Determination of iodine in oyster tissue by isotope dilution laser resonance ionization mass spectrometry.

The technique of laser resonance ionization mass spectrometry has been combined with isotope dilution analysis to determine iodine in oyster tissue. The long-lived radioisotope, 129I, was used to spike the samples. Samples were equilibrated with the 129I, wet ashed under controlled conditions, and iodine separated by coprecipitation with silver chloride. The analyte was dried as silver ammonium iodide upon a tantalum filament from which iodine was thermally desorbed in the resonance ionization mass spectrometry instrument. A single-color, two-photon resonant plus one-photon ionization scheme was used to form positive iodine ions. Long-lived iodine signals were achieved from 100 ng of iodine. The precision of 127I/129I measurement has been evaluated by replicate determinations of the spike, the spike calibration samples, and the oyster tissue samples and was 1.0%. Measurement precision among samples was 1.9% for the spike calibration and 1.4% for the oyster tissue. The concentration of iodine determined in SRM 1566a, Oyster Tissue, was 4.44 micrograms/g with an estimate of the overall uncertainty for the analysis of +/- 0.12 microgram/g.

Animals↗
PubMed1989

Planning abilities in alcoholics, process and reactive schizophrenics, and normals.

This study investigated planning abilities in alcoholics, process and reactive schizophrenics, and normals. Significant differences among the groups were found on all three planning tasks. Normals and alcoholics showed significantly greater planning ability on the picture arrangement task than the process schizophrenics. On the maze task, normals attained significantly higher scores than the process schizophrenics. On a task in which subjects predicted future effects from a present-day trend, alcoholics showed significantly greater planning ability than normals and process schizophrenics. Reactive schizophrenics did not differ significantly from any of the other groups on any task. The greater planning ability of the alcoholics in comparison with the normals on one of the tasks may be due to sampling and situational differences. The results suggest that deficits in planning abilities are temporary for alcoholics.

Adaptation, Psychological↗
PubMed1989

Characterization of serotonin-1B receptors negatively coupled to adenylate cyclase in OK cells, a renal epithelial cell line from the opossum.

We have previously reported the presence of a 5-HT1 (serotonin)-like receptor coupled in an inhibitory manner to adenylate cyclase in the opossum kidney cell line, which is derived from the kidney of a North American opossum. Pharmacological data from binding and cyclic AMP production studies indicate that this receptor does not have characteristics of a 5-HT1A, 5-HT1C or 5-HT1D receptor, but is similar to 5-HT1B receptors found in rodent tissues. Many serotonergic drugs, including 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyrindinyl)1H-indol, 5-HT and methysergide, but not (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide or buspirone, were full agonists at this receptor as defined by the inhibition of bovine parathyroid hormone peptide fragment 1-34-stimulated cyclic AMP production in an intact cell assay. Several classical beta adrenergic antagonists including propranolol and cyanopindolol were also full agonists at this receptor. Radioligand binding studies using [125I](-)-iodocyanopindolol gave a Bmax of 88 fmol/mg of protein and a KD of 47 pM for saturation experiments carried out in the presence of GTP. In the absence of GTP, the binding data were significantly better fit by a two-site model with KD values of 10 and 345 pM. Inhibition binding experiments were consistent with the results of the cyclic AMP experiments. The identification of 5-HT1B receptors in a tissue derived from the opossum kidney suggests that these receptors may be distributed more widely than previously thought, inasmuch as other studies have found them only in neuronal tissues of rodents.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylate Cyclase Toxin↗
PubMed1988

Alpha-2A and alpha-2B adrenergic receptor subtypes: antagonist binding in tissues and cell lines containing only one subtype.

The affinities of 34 adrenergic antagonists for alpha-2 adrenergic receptors were determined from homogenate radioligand binding studies using [3H]yohimbine and [3H]rauwolscine. It has been suggested that alpha-2 adrenergic receptors can be subdivided into alpha-2A and alpha-2B subtypes. Oxymetazoline is selective for alpha-2A receptors, whereas prazosin is alpha-2B selective. Five different tissues were used, each of which has only one of the two subtypes: human platelet (alpha-2A), HT29 cell line (alpha-2A), human cerebral cortex (alpha-2A), neonatal rat lung (alpha-2B), and NG108-15 cell line (alpha-2B). The drug affinities were highly correlated when alpha-2A tissues were compared with alpha-2A tissues (r = 0.97 to 0.98) or when the two alpha-2B tissues were compared (r = 0.99). By contrast, comparison of an alpha-2A tissue with an alpha-2B tissue resulted in poor correlations (r = 0.77 to -0.87). Three new subtype selective drugs were identified among these drugs on the basis of at least a 10-fold greater affinity for one subtype. All three were selective for the alpha-2B subtype: ARC-239 (100-fold selective), chlorpromazine (18-fold selective), and 7-hydroxychlorpromazine (17-fold selective). These studies, by demonstrating distinct pharmacological profiles for the two alpha-2 adrenergic receptor subtypes in several different tissues, further support the existence and definition of these subtypes. The identification of a cell line for each subtype should be useful in the further study of alpha-2 adrenergic receptor subtypes.

Animals↗
PubMed1988

Oxymetazoline inhibits adenylate cyclase by activation of serotonin-1 receptors in the OK cell, an established renal epithelial cell line.

The nonselective alpha-adrenergic agonist oxymetazoline inhibits parathyroid hormone (PTH)-stimulated cAMP production in intact OK cells, an epithelial cell line derived from an American opossum kidney. This inhibition, however, is not blocked by alpha 2-adrenergic receptor antagonists. After excluding several alternate hypotheses to explain this anomalous activity of oxymetazoline, we hypothesized that oxymetazoline activates a receptor in OK cells that is negatively coupled to adenylate cyclase but distinct from the alpha 2-adrenergic receptor. Prior exposure of OK cells to pertussis toxin blocks the inhibitory response to oxymetazoline, suggesting involvement of a guanine nucleotide-binding regulatory protein. Screening various compounds for attenuation of PTH-stimulated adenylate cyclase showed that serotonin (5HT) is a potent and fully efficacious agonist. Desensitization of alpha 2-receptor-mediated inhibition of cAMP production by epinephrine did not alter the response to either 5HT or oxymetazoline, indicating that these compounds do not produce their effect by activating alpha 2-adrenergic receptors. The 5HT1 receptor-selective antagonist methiothepin, but not ketanserin (5HT2-selective) or ICS-205,930 (5HT3-selective), blocked the response to both 5HT and oxymetazoline. The potency of methiothepin for antagonizing oxymetazoline-induced inhibition of PTH-stimulated cAMP production was not significantly different from its potency for the 5HT-induced effect. These data indicate that OK cells express a 5HT1 receptor that is negatively coupled to adenylate cyclase and that oxymetazoline is an agonist at these receptors.

Adenylyl Cyclase Inhibitors↗
PubMed1988

Characterization of alpha-2 adrenergic receptors in the OK cell, an opossum kidney cell line.

We have characterized alpha-2 adrenergic receptors in OK cells, an opossum kidney-derived cell line. In membrane saturation binding experiments, [3H]rauwolscine (Kd = 74 pM) was 3-fold more potent than [3H]yohimbine (Kd = 230 pM). Each labeled a single class of binding sites with densities of 135 and 124 fmol/mg of protein for [3H]rauwolscine and [3H]yohimbine, respectively. Inhibition of [3H]rauwolscine and [3H]yohimbine binding by several alpha adrenergic agonists and antagonists demonstrated the radioligands labeled an alpha-2 type adrenergic receptor with a pharmacological profile similar to the alpha-2B receptor subtype. The rank order of potency for antagonist inhibition of binding was yohimbine greater than prazosin = phentolamine greater than chlorpromazine = corynanthine, whereas the rank order of agonist potency was oxymetazoline = clonidine greater than or equal to UK-14,304 greater than or equal to (-)-epinephrine greater than (-)-norepinephrine. The oxymetazoline, clonidine and antagonist inhibition curves were routinely monophasic and modeled best as a single class of binding sites. For the other agonists, inhibition binding curves were biphasic with approximately 35% of the binding sites existing in a high affinity state. These curves were shifted to the right in the presence of 0.1 mM GTP, and in general modeled as a single class of binding sites. UK-14,304, (-)-epinephrine, (-)-norepinephrine and oxymetazoline attenuated parathyroid hormone-stimulated cyclic AMP production by up to 70% in whole cell monolayers in a dose-dependent manner via a pertussis toxin-sensitive mechanism. With the exception of oxymetazoline, this inhibition could be reversed with alpha adrenergic antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1986

Lifestyle modification with heavy alcohol drinkers: effects of aerobic exercise and meditation.

To assess the effects of exercise and meditation on alcohol consumption in social drinkers, 60 male students, between the ages of 21 and 30, all classified as heavy social drinkers, were randomly assigned to one of three conditions: exercise (running), meditation, and a no-treatment control group. The study consisted of three distinct phases: pretreatment baseline (2 weeks), treatment intervention (8 weeks), and a follow-up period (6 weeks). Subjects in the running and meditation conditions met as a group during the treatment intervention phase, and all subjects self-monitored their daily consumption of alcohol throughout the study. The results showed that subjects in the exercise condition significantly reduced their alcohol consumption compared to the no-treatment control condition. The implications of these findings for treatment intervention, and the importance of subject compliance are discussed.

Adult↗