Search PubMed⌕ Search

Biomedical subjects

D B Bylund

Publications and source records attributed to D B Bylund.

At least 55 records · Page 3Linked to original sources
PubMed1993

Radioligand binding methods: practical guide and tips.

Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.

Animals↗
PubMed1992

Subtypes of alpha 1- and alpha 2-adrenergic receptors.

The adrenergic receptors are members of the superfamily of G protein-coupled receptors. There are three major types of adrenergic receptors: alpha 1, alpha 2, and beta. Each of these three major types can be divided into three subtypes. Within the alpha 1-adrenergic receptors, alpha 1A and alpha 1B subtypes have been defined pharmacologically on the basis of reversible antagonists, such as WB4101 and phentolamine, and the irreversible antagonist chloroethylclonidine. In at least some tissues the mechanism of action of the alpha 1A subtype is related to activation of a calcium channel, whereas the alpha 1B receptor exerts its effect through the second messenger inositol trisphosphate. Both of these receptor subtypes as well as a third, the alpha 1C, have been identified by molecular cloning. Three pharmacological subtypes of the alpha 2-adrenergic receptor have also been identified. Prototypic tissues and cell lines in continuous culture have been developed for each of these subtypes, which facilitated their study. The definition of the alpha 2 subtypes has been based on radioligand binding data and more limited functional data. All three subtypes have been shown to inhibit the activation of adenylate cyclase and thus reduce the levels of cAMP. Three alpha 2-adrenergic receptor subtypes have been identified by molecular cloning in both the human and rat species. There is reasonable agreement between the pharmacological identified subtypes and those identified by molecular cloning.

Adrenergic alpha-Antagonists↗
PubMed1992

Synthesis and binding to beta-adrenergic receptors of p-aminobenzyl analogues of practolol and atenolol.

The p-aminobenzyl analogues (8a and 8b, respectively) of the cardioselective beta-adrenergic receptor antagonists practolol and atenolol were prepared from the corresponding phenoxymethyloxiranes in 30 and 13% yields, respectively. The dissociation constants for the beta-adrenergic receptor were measured in membrane preparations of rat heart and lung. In membranes from the heart (which contain mostly beta 1-adrenergic receptors), the affinities of the derivatives and parent compounds were similar. By contrast, in membranes from the lung (which contain mostly beta 2-adrenergic receptors), the derivatives were more potent than the parent compounds. Thus, the cardioselectivities of the p-aminobenzyl analogues 8a and 8b were about one-sixth those of the respective parents.

Adrenergic beta-Antagonists↗
PubMed1992

Differential localization of alpha 2-adrenergic receptor subtypes in brain.

The pharmacological identification and characterization of subtypes of alpha 2-adrenergic receptors have been confirmed by molecular biological investigations. Using receptor autoradiographic techniques, it has been possible to show regions of the brain where alpha 2 agonist binding ([3H]para-aminoclonidine) is preferentially labeling the presumed guaninenucleotide-sensitive, high-affinity conformations of the alpha 2 receptor. Careful examination of autoradiograms generated using the tritiated antagonists yohimbine, idazoxan, and rauwolscine also indicates some disparity in the regions occupied by these radiolabeled ligands. Inhibition of [3H]rauwolscine binding with the subtype selective compounds, ARC-239, or oxymetazoline demonstrates that there are discrete regions of the brain where one receptor subtype predominates over the other. These studies indicate that previous investigations utilizing the agonist para-aminoclonidine as the ligand for obtaining labeling of alpha 2 receptors have overlooked some regions of binding due to the subtype selectivity of this ligand. A more complete localization of alpha 2-adrenergic receptors can be obtained using the tritiated antagonist rauwolscine, and the differential distribution of at least two subtypes of the alpha 2 receptor can be obtained by selective inhibition of this binding.

Adrenergic alpha-Agonists↗
PubMed1992

Alpha-2A is the predominant alpha-2 adrenergic receptor subtype in human spinal cord.

alpha-2 Adrenergic receptors can be subdivided into four subtypes based on their pharmacologic properties. The subtype of alpha-2 adrenergic receptor present in human spinal cord has not been reported previously. The affinities of nine alpha-2 subtype-selective drugs for the alpha-2 adrenergic receptor in human spinal cord homogenates were determined using [3H]rauwolscine and [3H]RX821002. These drug affinities (pKi values) were highly correlated with those obtained in a tissue or cell line containing only the alpha-2A adrenergic subtype (correlation coefficient of 0.99 and 0.98 for human platelet and HT29 cells, respectively). In contrast, the correlation of pKi values for the human spinal cord with tissues or cell lines containing other adrenergic receptor subtypes was poor. The correlation coefficients for alpha-2B (neonatal rat lung), alpha-2C (OK cell), and alpha-2D (bovine pineal gland) were 0.15, 0.68, and 0.81, respectively. These data suggest that the predominant alpha-2 adrenergic subtype present in human spinal cord is the alpha-2A subtype. Both [3H]rauwolscine and [3H]RX821002 appeared to label a single class of binding sites. The alpha-2 adrenergic receptor density was significantly greater in the sacral region of the cord as compared to either the lumbar or thoracic regions.

Adrenergic alpha-Antagonists↗
PubMed1992

Desensitization and down-regulation of the 5-hydroxytryptamine1B receptor in the opossum kidney cell line.

In the opossum kidney cell line the 5-hydroxytryptamine (serotonin; 5-HT)1B receptor is negatively coupled to adenylyl cyclase via a Gi protein. Preincubation of opossum kidney cell line cell monolayers with 5-HT resulted in 5-HT1B receptor-mediated desensitization expressed as a 4-fold rightward shift of the dose-response curve and a 10 to 29% decrease of maximal inhibition of forskolin-stimulated cyclic AMP production. These moderate decreases in potency and efficacy were concentration- and time-dependent. Maximal desensitization occurred with 3 hr of 5-HT preincubation. Preincubation with 5-HT caused no change in the potency or efficacy of alpha-2 adrenergic agonist-mediated inhibition of forskolin-stimulated cyclic AMP production. Therefore, the desensitization caused by 5-HT preincubation appears to be homologous. Down-regulation of the 5-HT1B receptor, assessed with the high affinity radioligand [125I]iodocyanopindolol, also occurred, and was concentration- and time-dependent. Maximum down-regulation of 40% occurred after 20 hr of exposure to 10 microM 5-HT. These results demonstrate that, like other receptors coupled to the inhibition of adenylyl cyclase, exposure of 5-HT1B receptors to an agonist causes desensitization of the functional response followed by down-regulation of the receptor.

Animals↗
PubMed1992

Pharmacological characteristics of alpha 2-adrenergic receptors: comparison of pharmacologically defined subtypes with subtypes identified by molecular cloning.

On the basis of extensive radioligand data and more limited functional data, three pharmacological subtypes of alpha 2-adrenergic receptors have been identified. More recently, three human genes or cDNAs for alpha 2-adrenergic receptors have been identified by molecular cloning. The relationship, however, among the pharmacologically defined subtypes and those identified by molecular cloning has not been clear. In order to resolve this issue, we have compared the pharmacological characteristics of the receptors identified by molecular cloning and expressed in COS-7 cells with the characteristics of the pharmacologically defined receptors in their respective prototypic tissue or cell line. The affinities (Ki values) of 12 subtype-selective alpha 2-adrenergic antagonists were determined for the alpha 2 receptor in the six preparations, by radioligand binding. Correlation analyses of the pKi values indicate that the alpha 2A subtype, as defined in the HT29 cell line, the alpha 2B receptor of the neonatal rat lung, and the alpha 2C subtype, as defined in an oppossum kidney cell line, correspond to the cloned human alpha 2-C10, alpha 2-C2, and alpha 2-C4 receptor subtypes, respectively.

Adrenergic alpha-Antagonists↗
PubMed1992

Synthesis and biodistribution of the alpha 2-adrenergic receptor antagonist (11C)WY26703. Use as a radioligand for positron emission tomography.

The purpose of these experiments was to label an alpha 2-adrenergic receptor ligand with a positron emitting isotope and then test this radioligand in vivo. No-carrier-added [11C]WY26703 was synthesized by methylation of its desmethyl precursor, WY27050 with [11C]H3I followed by purification with HPLC in 14% yield in a synthesis time of 35 min from EOB. Ki values for unlabeled WY26703, ranged from 0.52-1.55 nM in tissues that express a single alpha 2-adrenergic receptor subtype. Tail vein injections of [11C]WY26703 in mice revealed that the compound was distributed in the brain, heart, lungs, spleen, and kidneys. In the brains of rats treated with atipamezole, an alpha 2-adrenergic receptor antagonist, there was no decrease in [11C] accumulation indicating a lack of observable specific binding of the radioligand. When brain tissue was homogenized and filtered, however, atipamezole decreased [11C] activity by 53%. Therefore, [11C]WY26703 crosses the blood-brain barrier and specifically binds to alpha 2-adrenergic receptors with high affinity. Atipamezole treatment decreased only the area of the locus coeruleus [11C] value of the various regions of the brain. The affinity, however, of [11C]WY26703 does not appear to distinguish alpha 2-receptors from nonspecific binding sites. PET study of [11C]WY26703 in a Rhesus monkey showed that influx of [11C]WY26703 into the brain was high for the first few minutes but radioactivity then declined rapidly and did not retain in a specific brain region. This suggests that [11C]WY26703 may not be a useful ligand for imaging human alpha 2-adrenergic receptors by positron emission tomography.

Adrenergic beta-Antagonists↗
PubMed1991

Identification and characterization of alpha 2D-adrenergic receptors in bovine pineal gland.

The vertebrate mechanisms for generating circadian rhythms, regulating the synthesis of melatonin, and modulating the activities of pineal indole-synthesizing enzymes differ significantly among species. The synthesis of melatonin in mammalian pineal glands is stimulated by beta 1-adrenergic receptor agonists and is potentiated by alpha 1-adrenergic receptor agonists, whereas in the avian pineal gland alpha 2-adrenergic receptor agonists inhibit its synthesis. By using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, we have identified for the first time alpha 2-adrenergic receptor sites in a mammalian pineal gland. [3H] Rauwolscine bound in a saturable manner to a single class of receptors, with an equilibrium dissociation constant (KD) of 1.4 nM and a density (Bmax) of 71 fmol/mg of protein, in crude synaptic membrane preparations from bovine pineal gland. Competition studies carried out with various adrenergic antagonists supported the conclusion that [3H]rauwolscine binding sites were alpha 2-adrenergic receptors. The bovine pineal alpha 2-adrenergic receptor appears to represent a pharmacological subtype distinct from the three currently proposed subtypes, i.e., alpha 2A found in a human colonic adenocarcinoma cell line (HT29 cell), alpha 2B found in rat lung, and alpha 2C found in an opossum kidney cell line (OK cell). However, the pharmacologic profile of the pineal alpha 2 receptor resembles that found in the rat submaxillary gland. We suggest that the bovine pineal receptor may represent a fourth pharmacological subtype, which would be designated as alpha 2D.

Animals↗
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↗
PubMed1991

Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms.

The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipase A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.

Adenylate Cyclase Toxin↗
PubMed1990

Effects of alpha 2-adrenergic agonist preincubation on subsequent forskolin-stimulated adenylate cyclase activity and [3H]forskolin binding in membranes from HT29 cells.

alpha 2-Adrenergic agonist preincubation resulted in a leftward shift in the subsequent concentration-response curve to forskolin-stimulated adenylate cyclase activity in membranes from HT29 cells, a human colon adenocarcinoma cell line. This effect was much less pronounced than the effect seen in the intact cell cyclic AMP production assays. Removal of GTP from the assay caused a further slight leftward shift in the concentration-response curve. In [3H]forskolin binding experiments, alpha 2-adrenergic agonist preincubation caused a doubling of the maximal number of binding sites (80 vs 31 fmol/mg protein) compared to control. The addition of MgCl2 and NaF to the assay buffer increased control binding 5-fold. With agonist preincubation, there was a further increase in binding in the presence of MgCl2 and NaF which was not significantly different from the appropriate control. Pertussis toxin pretreatment blocked both the leftward shift in the forskolin concentration-response curve and the increase in maximal number of binding sites, indicating that a pertussis toxin sensitive protein is involved in these changes. Activation of cyclic AMP production in the intact cell by cholera toxin followed by norepinephrine preincubation and then stimulation by forskolin resulted in a degree of sensitization similar to that seen in the membrane adenylate cyclase and binding assays. Pertussis toxin also blocked this sensitization. It appears that if the cyclase system is highly activated, then the degree of sensitization is similar in the membrane and intact cell assay.

Adenylate Cyclase Toxin↗
PubMed1990

Effects of light and an alpha-2-adrenergic agonist on serotonin N-acetyltransferase activity in chick pineal gland.

Pineal serotonin N-acetyltransferase (NAT) is the enzyme that catalyzes the production of N-acetylserotonin from serotonin and is the rate limiting step in the biosynthesis of melatonin in the chick pineal gland. Chick pineal NAT activity is decreased by light and by noradrenergic agents that act at the alpha-2-adrenergic receptor. Light-induced inhibition of nocturnal NAT activity can be demonstrated by exposing 4-day-old chicks to light, or by exposing pineal gland explants cultured in vitro either to light or to UK 14,304 (an alpha-2-adrenergic agonist). The inhibition by either light or UK 14,304 is preceded by a transient, but consistent rise in NAT activity. The paradoxical, transient light-induced rise in enzyme activity is mimicked by UK 14,304 which suggests a similarity in mechanisms of light and adrenergic signal transduction associated with the regulation of NAT activity.

Adrenergic alpha-Agonists↗
PubMed1990

Platelet alpha 2-adrenergic receptor binding to 3H-yohimbine and personality variations in normals.

The authors examined platelet alpha 2-adrenergic receptor binding to 3H-yohimbine and several personality variables in 58 adult males in a campus community. Subjects with high receptor Bmax levels exhibited personality traits of less social inhibition and more sensation seeking and thrill seeking behavior, were more playful and autonomous, and came from healthier, more intact families. The results also suggested that high affinity states of platelet alpha 2-adrenergic receptors (low Kd) correlate with traits suggestive of stability, i.e., Autonomy, Dominance, Nurturance, Order, Succorance, and General Sensation Seeking Scale of Zuckerman, while low affinity states (high Kd) of platelet alpha 2-receptors correlate with psychopathological traits of Dependence, Exhibitionism, and Paranoia.

Adult↗
PubMed1990

Ontogeny of light-induced decrease of N-acetyltransferase activity in explanted chick pineal glands.

The ontogeny of chick pineal serotonin N-acetyltransferase (NAT) activity was investigated in explanted chick pineal glands at 4, 10 and 21 d of age. Nocturnal levels of the enzyme and the response of the enzyme to light exposure were determined in pineal glands maintained in short-term culture at each age. The results indicate that nocturnal NAT activity was increased in the glands from older birds. Nocturnal levels of NAT activity at the time of the initiation of the experiment were threefold greater in glands from 21-d-old birds as compared to that in glands from 4-day-old chicks. The response to light was similar in all three ages examined; light induced a significant decrease in NAT activity within 60 min in explanted glands from 4-d-old chicks and within 180 min in the glands from the 10- and 21-d-old chicks. A paradoxical transient increase in enzyme activity occurred immediately (within 5 min) following light exposure which was significant in the glands from the youngest chicks, and present, but more variable, in the older chicks. These data indicate that the nocturnal enzyme activity is greater in glands from older birds, but that light exposure of explanted glands initiates a transient rise followed by a decrease in NAT activity at all three ages.

Acetyltransferases↗
PubMed1990

Desensitization of the alpha-2 adrenergic receptor in HT29 and opossum kidney cell lines.

Preincubation of HT29 cells with an alpha-2 adrenergic agonist resulted in a parallel rightward shift in the subsequent dose-response curve to 5-bromo-6-[2-imidazoline-2-yl-amino] quinoxaline (an alpha-2 adrenergic agonist) in inhibiting vasoactive intestinal peptide-stimulated cyclic AMP production. This rightward shift in the dose-response curve, which was concentration and time dependent, was interpreted as desensitization of the alpha-2 adrenergic receptor-mediated inhibition of cyclic AMP production. The fact that no decrease in efficacy was observed appears to result from a receptor reserve. Agonist preincubation effects on subsequent p-aminoclonidine (an alpha-2 adrenergic partial agonist) inhibition and partial irreversible inactivation of receptors confirmed the presence of an alpha-2 adrenergic receptor reserve in HT29 cells. Desensitization appeared to have a heterologous component since inhibition of vasoactive intestinal peptide-stimulated cyclic AMP production by somatostatin was also attenuated. We also assessed the effect of alpha-2 agonist preincubation on subsequent 5-bromo-6-[2-imidazoline-2-yl-amino] quinoxaline inhibition of parathyroid hormone-stimulated cyclic AMP production in OK cells. As with HT29 cells, agonist preincubation resulted in a concentration- and time-dependent shift in the dose-response curve. In both cell lines, long-term preincubation with an alpha-2 adrenergic agonist resulted in a 40% decrease in subsequent [3H]yohimbine binding, indicating down-regulation of the alpha-2 adrenergic receptor.

Animals↗
PubMed1989

Platelet MAO activity and personality variations in normals.

The authors examined platelet MAO activity and several personality variables in 58 non-clinic-referred healthy adult males. The psychological tests administered consisted of the Minnesota Multiphasic Personality Inventory, the Personality Research Form-E, and the Zuckerman Sensation Seeking Scale. The findings replicated previous findings in the literature.

Adult↗