bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Adrenoceptors
Authors: Bylund David B.
Published: June 2004

Norepinephrine and epinephrine are important neurotransmitters in both the peripheral and the central nervous systems. The actions of these neurotransmitters are mediated by adrenoceptors. Three major types of adrenoceptors have been identified on the basis of pharmacological data: alpha1, alpha2, and beta. Within each major type, at least three receptor subtypes have been identified. Three basic types of radioligand binding experiments are provided in this unit: (1) saturation binding from which the affinity (Kd) of the radioligand for the adrenoceptor and the binding site density (Bmax) can be determined; (2) inhibition experiments from which the affinity (Ki) of a competing, unlabeled compound for the receptor can be determined; and (3) kinetic assays from which the forward and reverse rate constants of the binding process can be determined. Three support protocols are provided covering the preparation of membranes containing the receptor from tissue or cells in culture, calculation of Kd and Bmax from saturation experiments, and calculation of Ki from inhibition experiments.
Characterization of Dopamine Receptors
Authors: Levant Beth
Published: June 2004

Dopamine receptors are the principal targets of drugs used for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson's disease. This unit details protocols for radioligand binding assays for various subtypes of dopamine receptor. The scope is limited primarily to the most widely used, well‐established protocols for binding assays, which employ commercially available tritiated ligands. Protocols are given for radioligand binding assays for D1‐like and D2‐like receptors. A protocol for the putatively selective labeling of D3 sites is also presented. Although the basic protocols are used to assay dopamine receptors in membrane homogenates, support protocols describe the preparation of these homogenates from organs and from cultured cells. The calculation of the radioligand's molar concentration is also included.
Characterization of Neuronal Nicotinic Acetylcholine Receptors
Authors: Sullivan James P. , Anderson David J.
Published: June 2004

Neuronal nicotinic acetylcholine receptors are members of the ligand‐gated ion channel superfamily of receptors. The potential of nAChRs as targets of therapeutic intervention has been revealed, in part, by recent studies showing that a diversity of nAChR subunits exist in brain and other tissues, and that discrete subunit combinations may be involved in mediating specific neurophysiological functions and behaviors. To date, eleven gene products representing nAChRs have been identified. This unit examines the binding properties of the a4 b2 receptor in rat brain or in transformed cell lines expressing the recombinant receptor.
Characterization of P1 (Adenosine) Purinoceptors
Authors: Jarvis Michael F.
Published: June 2004

The purine nucleoside adenosine (ADO) is an important modulator of cellular function in mammalian tissues. ADO modulates neuronal excitability through an interaction with different cell surface receptor subtypes that are heterogeneously distributed in the mammalian central nervous system, as well as in many peripheral tissues. Four different ADO receptor subtypes have been cloned and characterized. Described in this unit are three radioligand binding assays for pharmacological characterization of the high‐affinity ADO receptor subtypes A1, A2A, and A3 receptors. Pharmacological characterization of the low‐affinity A2B receptor using radioligand binding has been limited by the paucity of potent and selective ligands for this subtype. Since receptor localization is an important criterion for differentiation of receptor subtypes, a support protocol that describes the methodology for the localization of ADO receptors in rat brain tissue using autoradiography is also included.
Characterization of Angiotensin II Receptors
Authors: Smith Ronald D. , McCall Dale E. , Timmermans Pieter B.M.W.M.
Published: June 2004

Angiotensin II (Ang II) is the primary mediator of the renin angiotensin system and acts primarily as a circulating hormone, but may also act as a paracrine or autocrine factor. Ang II binds specifically to G protein‐coupled receptors and triggers a number of characteristic cellular responses, including vascular smooth muscle contraction and adrenal cellular aldosterone secretion. There are multiple receptor subtypes for Ang II; however, the distinction between binding sites versus receptors with their associated functional manifestations has been controversial. There are two principal human receptor subtypes, designated AT1 and AT2, that are distinguished by the selective affinity of the nonpeptide antagonists losartan and PD123177. In rodents, additional isoforms of the Ang II receptor have been cloned (e.g., AT1A and AT1B), although their functional distinctions are undetermined. The focus of this unit is the identification of Ang II binding sites and receptor subtypes. Radioligand competition binding assays are provided for determining the affinity of Ang II for receptors on adrenal cortical microsomes, whole adrenal membranes, or CHO cells transfected with the AT1A receptor.
Characterization of Cholecystokinin (CCK) Receptors
Authors: Miller Laurence J.
Published: June 2004

Cholecystokinin (CCK) and gastrin are structurally related peptide hormones synthesized and secreted by endocrine cells in the gastric antrum, the proximal small intestine, and the central and enteric nervous systems. A broad spectrum of cells express receptors and represent physiologic targets for these peptides. The major physiologic targets for gastrin in the oxyntic gastric mucosa, including the parietal cells and select enterochromaffin cells, while CCK has a broader range of targets, with most involved in nutrient assimilation and appetite control, and the regulation of anxiety. This unit describes binding assays for the determination of receptor number, ligand affinity, and inhibition constants for both CCKA and CCKB receptors, in addition to procedures detailing the preparation of tissues from various sources.
Characterization of Corticotropin‐Releasing Factor (CRF) Receptors
Authors: Grigoriadis Dimitri E. , Lorang Marge T. , Duggan Nicola , De Souza Errol B.
Published: June 2004

Corticotropin‐releasing factor (CRF) and its receptors play a major role in regulating an organism's response to physical, emotional and environmental stress. While the primary role of CRF is the regulation of adrenocorticotropin hormone (ACTH) secretion from the pituitary and modulation of the hypothalamic‐pituitary adrenal axis, CRF is also widely distributed in the central nervous system where it produces a broad spectrum of autonomic, electrophysiological and behavioral effects consistent with a neurotransmitter or neuromodulator role in the brain. Methods are provided for characterizing the receptor proteins through which CRF exerts its function. A well‐characterized radioligand receptor binding assay is provided that yields quantitative information about the affinity and density of receptors in a variety of tissues, while another procedure utilizes similar kinetic theories but, in contrast to the homogenized or whole cell suspension approach, makes use of slide‐mounted tissue sections.
Characterization of Wild‐Type Excitatory Amino Acid Ion Channel Receptors
Authors: Ferkany John W.
Published: June 2004

The ubiquitous distribution of EAA receptors results from the fact that EAA‐mediated neurotransmission is the most prevalent type of fast, chemically‐mediated communication in the brain and spinal cord. Indeed, EAA‐mediated neurotransmission is thought to participate in a host of physiological and pathological events including learning and memory, epilepsy, neurodegenerative conditions such as Alzhiemer's, Parkinson's and Huntington's Diseases, and the neurodegeneration associated with stroke and head trauma and neuropathic pain. Described in this unit are receptor binding assays for studying ion‐channel forming excitatory amino acid (EAA) receptors in the mammalian central nervous system. One of the three major types of ionotropic (channel‐forming) EAA receptors are the AMPA receptors, and the other two types are classified on the basis of the original agonists identified for these sites: kainic acid (kainate; KA) and N‐methyl‐D‐aspartic acid (NMDA). Thus, the three recognized ionotropic receptors are the AMPA, KA and NMDA recognition sites. Like GABA receptors, the AMPA and NMDA receptors have multiple regulatory sites.
Characterization of the Picrotoxin Site of GABAA Receptors
Authors: Mehta Ashok K. , Ticku Maharaj K.
Published: June 2004

This protocol describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics and competition assays (IC50, Ki determination) are included in the protocol.
[3H](+)MK801 Radioligand Binding Assay at the N‐Methyl‐D‐Aspartate Receptor
Authors: Reynolds Ian J.
Published: June 2004

The N‐methyl‐D‐aspartate subtype of glutamate receptor is unusual in that it requires two endogenous agonists for activation. Thus, in addition to glutamate, the amino acid glycine (or possibly D‐serine) is an essential co‐agonist. This unit presents a radioligand binding protocol that detects ligand activity at the NMDA receptor‐associated glycine site. This is a convenient approach that exploits the ability of NMDA receptor modulators to alter the kinetics of ligands that bind to the channel‐blocking site of the NMDA receptor. This protocol takes advantage of one of the most potent and specific ligands that bind to this receptor, in this case [3H](+)MK801. Importantly, this assay can detect and differentiate agonists and antagonists that bind to the glycine site. A protocol for the measurement of glycine site activity with [3H](+)MK801 binding is provided, along with support protocols that provide information to aid in the design of assays of agonists and antagonists of the glycine site, and data analysis.
Overview of Receptor Allosterism
Authors: Christopoulos Arthur
Published: June 2004

Studies of the behavior of many ligand‐gated ion channels have long provided evidence that more than one molecule of ligand was able to bind to each receptor complex, a phenomenon termed cooperativity. The conclusion that some receptors possessed more than one binding site for ligands thus invoked another phenomenon that was originally described in the field of enzymology, that is, the concept of allosteric binding sites. The biological activity of enzymes can be modified, either in a positive or negative fashion, by the binding of ligands to sites that are topographically distinct from the substrate‐binding site. These accessory binding sites are termed allosteric sites, in contrast to the substrate‐binding (active) site (the isosteric site). Thus, allosteric interactions arise because the binding of a ligand to the allosteric site induces a conformational change in the protein that modulates the binding of the substrate to the isosteric site, and vice versa. The biological activity of the enzyme arises from the subsequent (modified) properties of the substrate‐binding site, and not through a direct effect of the allosteric modulator itself. This overview unit discusses definitions of various terms used in the field of receptor allosterism, and explains in clear terms how allosteric transitions and interactions can affect receptor activation. In addition, the functions of allosteric modulators are discussed along with explanations of how these modulators can be attractive tools for drug development.
Characterization of 5‐HT1A,B and 5‐HT2A,C Serotonin Receptor Binding
Authors: Clarke William P. , Berg Kelly A. , Gould Georgianna , Frazer Alan
Published: June 2004

This unit describes assays for measuring the binding of radioligands to two major types of receptors for 5‐hydroxytryptamine (5‐HT or serotonin), 5‐HT1 and 5‐HT2 receptors, in homogenates of brain tissue or cloned into cells in culture. The specific receptor subtypes covered are 5‐HT1A, 5‐HT1B, 5‐HT2A, and 5‐HT2C. In addition, methodology for using quantitative autoradiography to measure radioligand binding to serotonin receptors in brain slices is described. Protocols are provided for characterization of both saturation and competition binding assays, and instructions for data analysis of these assays is also described. In addition, methodology is provided for the quantification (image analysis) of radioligand binding in brain tissue sections to determine receptor density, preparation of rat brain sections for quantitative autoradiography, and thionin staining of thaw‐mounted tissue sections to define certain brain regions.
Characterization of Chemokine Receptors
Authors: Scheer Alexander , Proudfoot Amanda E.I.
Published: June 2004

This unit describes the procedures for measuring binding of a radiolabeled chemokine to chemokine receptors in cells or cell membranes. The whole‐cell binding assay can be used for both purified leukocytes as well as transfected cell lines expressing chemokine receptors. Two basic protocols are described. The first is applicable to all cells expressing chemokine receptors, both primary cultures as well as recombinantly transfected cell lines expressing a single chemokine receptor. The second is used principally for transfected cell lines and measures chemokine binding to cell membranes using scintillation proximity assay (SPA) methodology, but does not require washing steps and thus is applicable to automated high‐throughput screening strategies. An alternative procedure is also described which also uses SPA methodology to measure GTP‐gamma‐S binding to chemokine receptors in cell membranes. The two basic protocols can be used to determine the binding of compounds to chemokine receptors, while the alternate protocol determines the effects of compounds on the chemokine‐stimulated activation of the receptor.
Characterization of Cannabinoid Receptors
Authors: Cox Richard F. , Rash Vincent
Published: June 2004

This unit describes the use of cannabinoid radioligands in competitive binding assays for determining affinity parameters (IC50, Ki) of unlabeled compounds at cloned CB1 and CB2 receptors expressed in cells.
Overview of Signal Transduction
Authors: Esbenshade Timothy A. , van Biesen Tim
Published: June 2004

Signal transduction involves translating extracellular stimuli into appropriate intracellular events that mediate the intended physiological response. The aim of this overview is to present some common features of cellular signaling pathways and to discuss how they interact to mediate responses to environmental stimuli. In particular, it focuses on several common aspects of signal transduction, including the regulation of signaling pathways by protein functional domain interactions as well as the intracellular proteins that mediate signal transduction. Among the proteins covered are those associated with G protein receptor signaling (G proteins, phospholipases, nucleotide cyclases, and phosphatases); protein kinases (tyrosine kinases, serine/threonine kinases) and their respective phosphatases; and, very briefly, several common ion channels (calcium and potassium).
Adenylate and Guanylate Cyclase Assays
Authors: Kelly John J. , Stevens Troy , Thompson W. Joseph
Published: June 2004

This unit presents two basic protocols to determine adenylate cyclase and guanylate cyclase activity in tissue and cell homogenates, permeabilized cells, or subcellular fractions. Two parts are presented for each method. First, the enzyme reaction that causes the formation of the labeled cyclic nucleotide is performed. Second, the separation of cyclic nucleotide products from unreacted nucleotide triphosphates and metabolites is performed using Dowex 50 resin and aluminum oxide chromatographies. In the case of guanylate cyclase, alternative separation protocols are also provided. Additionally, support protocols are provided that describe both the preparation of columns used in the assays and the preparation of tissue or cells to be assayed.
Overview of Phosphoinositide Hydrolysis
Authors: Kendall David
Published: June 2004

Phosphoinositide hydrolysis is a ubiquitous, multifunctional, intracellular signaling mechanism induced (via receptor activation) by a wide variety of signaling molecules such as hormones and neurotransmitters. Phosphoinositide hydrolysis generates intracellular second messengers that regulate cell function. This overview describes the major molecular players involved in phosphoinositide signaling along with experimental strategies for measuring phosphoinositide hydrolysis, including inositol radiolabeling methodologies, membrane phospholipase C assays, cytidine prelabeling, and mass measurements of phosphoinositide cycle products.
Nitric Oxide Synthase Assays
Authors: Zhang Jie
Published: June 2004

Nitric oxide (NO) is a widespread messenger molecule that regulates cardiovascular tone, immune responses, and neuronal function. This unit provides a simple protocol for measuring NO production based on the stoichiometric formation of the byproduct L‐citrulline by nitric oxide synthase (NOS) when NO is produced. Support protocols describe the preparation and purification of NOS from tissues or cultured cells, and for the purification of the radiolabeled L‐arginine substrate by thin‐layer chromatography (TLC).
Analysis of Early Gene Responses
Authors: Duman Ronald S.
Published: June 2004

Early response genes, also referred to as immediate early genes, are rapidly induced in response to a variety of extracellular stimuli, and have been used as markers of cellular activation. Expression of immediate early genes, such as c‐fos, can be used to determine the influence of specific drugs on receptor regulation of intracellular signaling pathways. This unit describes two techniques for the analysis of mRNA from immediate early genes. Northern blot analysis provides a method for size‐fractionation of native mRNA by gel electrophoresis and visualization of a specific mRNA in a pool of total RNA isolated from a tissue of interest. Thus, this analysis provides important information on the size and abundance of the mRNA of interest and on the specificity of the hybridization probe. However, this approach does not provide information on the cellular localization of the mRNA. Such data may be obtained by in situ hybridization of mRNA in tissue sections. In addition to the northern and in situ hybridization techniques, this unit provides two support protocols for radiolabeling either RNA or DNA probes along with information on the selection of an appropriate probe and hybridization conditions.
Assay of Receptor‐Stimulated Phosphoinositide Turnover
Authors: Kendall David A. , Alexander Stephen P.H.
Published: June 2004

The stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein‐coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [3H]myo‐inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal‐to‐noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion‐exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)‐trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [3H]myo‐inositol, chromatography columns, tissue slices, and the IP3‐binding protein.
HIV Protease Assays
Authors: Furfine Eric S.
Published: June 2004

The fluorescence‐based assay described in this unit is used not only to screen large numbers of compounds in a 96‐well format for the ability to inhibit human immunodeficiency virus‐1 (HIV) protease, but also to determine accurately the affinity of inhibitors for the enzyme. A method for performing an active site titration, using tight‐binding inhibitors, and determining the Km of a substrate is also provided. Because numerous potent inhibitors of HIV protease have been identified, information is provided on the analysis required to deal with these potent compounds and with fluorometric enzyme assay data in general.
Topoisomerase Assays
Authors: Nitiss John L.
Published: June 2004

Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of topoisomerases: the type I enzymes, which make single‐stranded cuts in DNA, and the type II enzymes, which cut and pass double‐stranded DNA. In this unit, an in vitro assay for topoisomerase I activity based on relaxation of supercoiled DNA by that enzyme is described. This is followed by an assay for topoisomerase II based on the decatenation of double‐stranded DNA. In conjunction with these assays, the preparation of mammalian cell extracts for assaying topoisomerase activity is described. Procedures are also detailed for the assay of topoisomerase covalent complexes in vivo and for measuring DNA cleavage caused by topoisomerase I and topoisomerase II in vitro. A related electrophoretic method for mapping topoisomerase II cleavage sites is finally included.
Protein Farnesyltransferase Assays
Authors: Zhang Fang L. , Bishop W. Robert
Published: June 2004

The enzyme protein farnesyltransferase (FPT) transfers a farnesyl group from the prenyl donor farnesyl diphosphate (FPP) to a cysteine residue on substrate proteins which contain a C‐terminal CaaX motif, where C is cysteine, a is an aliphatic amino acid, and X is methionine, serine, or glutamine. Known substrates for FPT include nuclear lamin B and the small GTP‐binding proteins H‐, K‐, and N‐Ras. Short peptides encompassing the CaaX motif of these proteins are also farnesylated by FPT. In this unit, two methods for assaying FPT activity and testing inhibitors are described. Both are based on measuring the transfer of [3H]farnesyl from FPP to a protein or peptide substrate. The first method, the TCA assay, uses native protein substrates of FPT (a support protocol details procedures for expressing and purifying histidine‐tagged H‐Ras in bacteria), and the prenylated product is collected by trichloroacetic acid (TCA) precipitation in the presence of carrier protein. A support protocol is also provided for preparing bovine brain membrane extract for use as the carrier. The second method employs scintillation proximity assay (SPA) technology in which a biotinylated peptide is used as a substrate, and streptavidin SPA beads are used to capture the farnesylated peptide. These procedures can be easily modified to measure prenylation of other protein substrates by FPT and related enzymes.
Protein Tyrosine Kinase Activity Assays
Authors: Hawes Brian E. , van Biesen Tim
Published: June 2004

Protein tyrosine kinases (PTKs) are ubiquitous enzymes that are integrally involved in the regulation of transformation mechanisms, normal and pathological growth, cell cycle regulation, immune responses, and a variety of intracellular signaling mechanisms. This rapidly growing family of enzymes is generally divided into two groups: receptor PTKs (with more than twelve distinct families) and nonreceptor PTKs (with more than nine distinct families). PTKs mediate the enzymatic transfer of the gamma phosphate of ATP to the phenolic groups on tyrosine residues to generate phosphate monoesters. In this unit, several assays are provided to measure the ability of PTKs to transphosphorylate protein and peptide substrates, and to autophosphorylate. Phosphorylation of exogenous substrates or autophosphorylation is detected using a 32P‐ or 33P‐phosphorylated protein. Alternatively, antibodies recognizing phosphorylated tyrosine residues can be used to quantify PTK activity. In some cases, antibodies are available for context‐specific phosphotyrosine residues, thereby enabling the detection of PTK‐specific substrate phosphorylation.
Monoamine Oxidase Assays
Authors: Tipton Keith F. , Davey Gavin , Motherway Martha
Published: June 2004

Monoamine oxidase catalyzes the oxidative deamination of primary aliphatic and aromatic amines and some secondary and tertiary amines, including the hormone and neurotransmitter amines epinephrine, dopamine, norepinephrine and serotonin. The two isoenzymes, MAO‐A and MAO‐B, differ according to substrate and inhibitor specificities. For example, selective inhibitors of MAO‐A have been shown to be effective antidepressants whereas some MAO‐B inhibitors have been reported to be beneficial in the treatment of Parkinson's and perhaps Alzheimer's diseases. Thus, it is important to have accurate procedures for determining the activities of each MAO isoenzyme in tissues that may contain both. In this unit, several MAO assay procedures are described, along with support protocols for equilibrating ion‐exchange resin, preparing aldehyde dehydrogenase, preparing dose‐response curves for determining the relative proportions of isoenzyme activities, determining concentrations of each isoenzyme, assessing new compounds as putative MAO inhibitors, and determining protein concentration of membrane‐bound proteins.
In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B
Authors: Lubben Thomas , Clampit Jill , Stashko Michael , Trevillyan James , Jirousek Michael R.
Published: June 2004

Many hormone or growth factor receptors signal via the activation of protein‐tyrosine kinases and phosphatases. Alteration of the phosphorylation state of tyrosine residues in certain proteins can directly regulate enzyme activity or cause formation of protein complexes necessary for transducing intracellular signals. Genetic and biochemical evidence also implicates protein‐tyrosine phosphatases in several disease processes, including negative regulation of insulin receptor signaling at the level of the insulin receptor and perhaps in signaling at the IRS‐1 level. The expression of protein tyrosine phosphatase‐1B (PTP1B) is elevated in muscle and adipose tissue in insulin‐resistant states both in man and rodents suggesting that PTP1B may play a role in the insulin‐resistant state associated with diabetes and obesity. As described in this unit, PTP1B activity can be determined with the small molecule substrate, p‐nitrophenyl phosphate (pNPP), in which the cleavage of the phosphate results in production of p‐nitrophenol (pNP) and an increase in absorbance at 405 nm. Alternatively, PTP1B activity can be measured as described using model phosphotyrosyl‐containing peptide substrates in which the release of free phosphate from the peptide is determined using a malachite green colorimetric assay.
Measurement of Glutamate Carboxypeptidase II (NAALADase) Enzyme Activity by the Hydrolysis of [3H]‐N‐Acetylaspartylglutamate (NAAG)
Authors: Tiffany Carol W. , Slusher Barbara S.
Published: June 2004

The peptide N‐Acetylaspartylglutamate (NAAG) is hydrolyzed by N‐Acetylated‐alpha‐linked‐acidic dipeptidase (NAALADase, glutamate carboxypeptidase II) into N‐Acetylated aspartate (NAA) and glutamate. Hydrolysis can be measured as described in this unit by employing radiolabeled NAAG (NAA‐[3H]glu) as the substrate. The occurrence of NAALADase activity in a wide range of tissues has implications for a variety of physiological purposes. The assay described here is useful for the analysis of NAALADase activity and its inhibition in brain synaptosomal preparations, tissue homogenates and tissue culture cell pellets.
Overview of Receptor Interactions of Agonists and Antagonists
Authors: Kenakin Terry
Published: June 2004

Experiments with isolated tissues have been crucial in defining interactions between drugs and receptors, and in formulating drug receptor theory. Described in this unit are procedures which can be used to define properties of drugs and receptors that transcend tissue hosts. This allows the extrapolation of such data to other tissue hosts and also to human therapeutic potential. The common currency of such studies is the dose‐response curve. In this unit, two views of agonism are discussed: classical receptor occupancy theory and the operational model. The models yield similar equations for the calculation of drug receptor constants in isolated tissues. The techniques for estimating molecular constants for drug/receptor pairs are identical within the context of either the occupancy or the operational theory; only the interpretation differs. Both theoretical frameworks are widely used in current pharmacological literature, and therefore techniques for measuring drug receptor constants utilize both models in this unit. Experiments with isolated tissues have been crucial in defining interactions between drugs and receptors, and in formulating drug receptor‐theory.
Isolated Blood Vessel Assays
Authors: Kenakin Terry
Published: June 2004

One of the most versatile and accessible types of isolated tissue used in pharmacology is the isolated blood vessel. Arteries and veins contain a wide range of receptor types mediating contraction, relaxation, and endothelial function. The protocols in this unit describe methods for measuring agonist response as changes in blood vessel tension.
α‐Adrenoceptor Assays
Authors: Hancock Arthur A.
Published: June 2004

α‐adrenoceptors are pivotal in controlling cardiovascular function both within the central nervous system and at the periphery.
β‐Adrenoceptor Assays
Authors: Kenakin Terry , Lenhard James M. , Paulik Mark A.
Published: June 2004

This unit describes the most commonly used isolated‐tissue and cellular preparations for studying the β‐adrenoceptor subtypes.
Opioid Receptor Assays
Authors: Berzetei‐Gurske Ilona
Published: June 2004

Opioid receptors mediate the pharmacologic actions of morphine‐like analgesics, a class of drugs that are important clinical agents which are also widely abused socially as a result of their euphorogenic and addictive properties. Cloning of the opioid receptors has confirmed the original postulate that there are only three classes of opioid receptors: µ, δ, and κ. The most commonly used isolated tissue preparations for measuring activity at these receptors are the guinea pig ileum and the mouse vas deferens. This unit describes preparation of these tissues to measure agonist and antagonist properties of test compounds at the µ‐ and κ‐opioid receptors in guinea pig ileum and the d‐opioid receptor in mouse vas deferens.
Tachykinin Receptor Assays
Authors: Patacchini Riccardo , Maggi Carlo Alberto
Published: June 2004

The tachykinins are a family of peptides (including Substance P, neurokinin A and neurokinin B) that mediate a wide variety of biological activities, and smooth muscle contraction following stimulation of the tachykinin receptors has been well characterized, making isolated smooth muscle is a preferred tissue in which to study tachykinin receptors and their ligands. Three receptor subtypes have been isolated and cloned, and the protocols in this unit provide precise instruction on ligand‐binding assays using isolated tissues bearing the various subtypes (NK1, NK2 or NK3). A plethora of representative data illustrate expected results, so the investigator can compare and validate data obtained from test compounds.
Dopamine 2 (DA‐2) Receptor Assays Using Sympathetic Nerve Terminals
Authors: Long John P.
Published: June 2004

In vitro experiments using isolated atria with and without superfusion can be used to quantify the dopamine‐2 (DA‐2) receptor stimulating activity of compounds. This receptor mechanism decreases the amount of norepinephrine released from sympathetic nerve terminals by neuronal impulses. DA‐2 receptor agonists (stimulants) are very active and induce several physiological changes, such as bradycardia, hypotension, lower intraocular pressure, etc. In this unit, tissue preparation and dopamine receptor assays are described for isolated cat atria with field stimulation or with superfusion, and isolated guinea pig atria with field stimulation.
5‐Hydroxytryptamine Receptor Assays
Authors: Figueroa Katherine W. , Morton Magda F. , Prendergast Clodagh E. , Shankley Nigel P. , Martin Graeme R.
Published: June 2004

This unit describes in vitro bioassays for studying 5‐hydroxytryptamine receptors in isolated intact animal tissues. In total, nine in vitro bioassays are described for 5‐HT1A, 5‐HT1B, 5‐HT1D, 5‐HT2A, 5‐HT2B, 5‐HT2C, 5‐HT3, 5‐HT4, and 5‐HT7 receptors in isolated intact guinea pig, rat and rabbit tissues. The 5‐HT1A, 5‐HT3, and 5‐HT7 assays use ileum preparations, whereas, the 5‐HT1B, 5‐HT1D, 5‐HT2A and 5‐HT2C receptor assays are all based on vascular preparations. The 5‐HT2B receptor is best studied in stomach fundic muscle and the 5‐HT4 receptor in esophageal tissue. To date, no functional isolated organ bioassays are available for the 5‐ht1e, 5‐ht1f, 5‐ht5 or 5‐HT6 receptors. This unit describes in vitro bioassays for studying 5‐hydroxytryptamine receptors in isolated intact animal tissues
Models of Pulmonary Disease: Acute and Chronic Allergic Asthma in the Monkey and Acute and Chronic Viral Pulmonitis in the Mouse
Authors: Wegner Craig D. , Shaughnessy Thomas , Mussatto Donna
Published: June 2004

Three models/protocols designed to mimic an inflammation process characteristic of a specific lung disease are described in this unit. Included are a single allergen inhalation in monkeys to model acute asthmatic episodes, repeated allergen inhalations in monkeys to model chronic asthma, and a lower‐respiratory viral infection in mice to model acute and chronic viral alveolitis and bronchitis. In each case, alterations in lung functions believed to correlate with symptoms of the respective disease are measured along with pulmonary inflammation.
Gastrointestinal Models: Intestinal Transit and Ulcerogenic Activity in the Rat
Authors: Lacroix Pierre , Guillaume Philippe
Published: June 2004

Slowing or acceleration of intestinal transit is readily observed in laboratory animals following administration of a number of drugs such as those interfering with the autonomic nervous system. Because serious side effects of these drugs include constipation or diarrhea, it is important to assess such effects when developing new drugs. Additionally, acute mucosal erosions are readily observed in the stomachs of laboratory animals following oral administration of ulcerogens such as nonsteroidal anti‐inflammatory drugs (NSAIDs). A serious side‐effect of these drugs in man is development or reactivation of gastroduodenal ulcers. This unit presents protocols to detect the effect of compounds on intestinal transit in the small intestine in the rats and mice, and to detect the ulcerogenic potential of compounds in the rat.
Models of Inflammation: Carrageenan Air Pouch in the Rat
Authors: Whiteley Phyllis E. , Dalrymple Stacie A.
Published: June 2004

The rat air pouch is an in vivo model used to study acute inflammation. The air pouch is formed by subcutaneous injection of air into the intracapsular area of the back of the rat. The pouch is composed of a lining of cells that consists primarily of macrophages and fibroblasts. Injection of a carrageenan solution into the pouch produces an inflammatory reaction that is characterized by an infiltration of cells, an increase in exudate, and a marked production of biochemical mediators such as prostaglandins, leukotrienes, and cytokines. This unit describes a method to elicit a cellular influx and inflammatory exudate in the six‐day‐old air pouch in the rat. Potential anti‐inflammatory drugs can be tested by measuring the reduction in carrageenan‐induced inflammation.
Models of Pain: Hot‐Plate and Formalin Test in Rodents
Authors: Bannon Anthony W.
Published: June 2004

Experimental models of pain can be separated into three basic categories: acute, persistent, and neuropathic. Acute pain is modeled by the hot‐plate test, persistent pain by the formalin test, and neuropathic pain by nerve ligation. The neurotransmitters and neuromodulators that mediate the pain response in each of these models can differ. As a result, different drugs may be effective in attenuating pain for each model. A basic understanding of the mechanisms involved in the pain response can help investigators choose the model that would be most appropriate for testing the analgesic properties of specific compounds or classes of compounds. This unit presents protocols for both an acute model of pain using the hot‐plate test, and a persistent model of pain using the formalin test. Each protocol describes the preparation of animals (mice or rats) for the test, administration of a compound being tested for its analgesic properties, and data collection.
Models of Affective Illness: Forced Swimming and Tail Suspension Tests in Rodents
Authors: Porsolt Roger D. , Brossard Geneviève , Hautbois Carine , Roux Sylvain
Published: June 2004

Rodents forced to swim in a narrow space from which there is no escape adopt, after an initial period of vigorous activity, a characteristic immobile posture, moving only when necessary to keep their heads above the water. The animals immobility was hypothesized to show they had learned that escape was impossible and had given up hope. Immobility was therefore given the name behavioral despair. It was subsequently found that immobility could be reduced by a wide range of clinically active antidepressant drugs. This simple behavioral procedure has since become a widely used test for screening novel antidepressants. Rodents forced to swim in a narrow space from which there is no escape adopt, after an initial period of vigorous activity.
Models of Urogenital Dysfunction: Assessment of Volume‐Evoked Micturition Reflex (VEMR) in the Conscious Rat by Cystometry
Authors: Nunn Philip , Williamson Ian , Newgreen Donald , Bushfield Mark
Published: June 2004

This unit describes a conscious rat model that allows the investigation of compounds administered by various routes (e.g., oral, intravenous and subcutaneous). This procedure can be used to examine the effects of agents on supraspinal, spinal and peripheral control of the micturition reflex, in addition to effects on parasympathetic neurotransmission and detrusor relaxation. One advantage to this procedure over anesthetized dog and rat models is the ability to determine pharmacological effects in the absence of possible interfering effects of anesthetics.
Models of Dementia: Delayed Alternation in Aged Rats
Authors: Roux Sylvian , Hubert Isabelle , Porsolt Roger D.
Published: June 2004

Operant delayed alternation tasks are useful tools for assessing age‐related memory deficits in rats. Decreased accuracy in these tasks seems to reflect mainly an inability to remember and utilize recent events (short‐term memory deficit). The task described in this unit requires the rat to learn a delayed alternation for food reward in a standard Skinner box, so it must retain spatial information over a short period of time. Aged rats exhibit clear short‐term memory impairments (decrease in response accuracy) in the task compared to young rats during learning and later, after extended training, also exhibit performance deficits. An advantage of studying these two phases is the possibility to examine either chronic effects (repeated drug administrations during acquisition) or acute effects (single treatments given before the test session during the stabilized performance phase). Operant delayed alternation tasks are useful tools for assessing age‐related memory deficits in rats
Models of Anxiety: Stress‐Induced Hyperthermia (SIH) in Group‐Housed Mice
Authors: Zethof Theo J.J. , van der Heyden Jan A.M. , Olivier Berend
Published: June 2004

This unit describes a procedure for quantifying anxiety in group‐housed mice by measuring their rectal temperature at 1‐min intervals. When rectal temperature is measured in this fashion, the temperature of the last mouse (T10) is higher than that of the first (T0). This difference in temperature is termed stress‐induced hyperthermia (SIH), and is thought to reflect an increased anxiety level in the animal. It is a robust, stable, and reproducible parameter. Under these conditions, the rectal temperature of the last mouse is 1(to 1.5(C higher than that of the first mouse. When mice are pretreated with anxiolytics (benzodiazepines, 5‐HT1A receptor agonists, ethanol), SIH decreases, making this a relatively simple procedure for screening drugs of this class. Moreover, the test reveals the intrinsic effects of a test compound on basal body temperature. An advantage of this assay is that it does not require time‐consuming training of the animals.
Overview of the Use of Transgenic Animals in CNS Drug Discovery
Authors: Nestler Eric J. , Olivier Berend
Published: June 2004

This overview highlights how transgenic mice, and related genetic manipulations, can contribute in important ways to the goal of developing novel treatments for neuropsychiatric disorders. A brief background of the field of drug discovery is covered and then new animal models of CNS disorders are discussed along with the topics of target validation, identification of novel targets, and functional genomics.
Models of Epilepsy: Electroshock and Chemical Induced Convulsions in the Mouse
Authors: Giardina William J.
Published: June 2004

Epilepsy is a disorder of the central nervous system that is characterized by seizures. This unit describes protocols for identifying and differentiating the antiseizure or anticonvulsant activity of novel test substances. Variations of two basic test methods using mice are described: blockade of electroshock‐induced convulsions (the maximal electroshock test) and blockade of chemical‐induced convulsions (the pentylenetetrazole test). This unit also describes variations of the same methods that can be used to assess potential proconvulsant activity of test substances in safety evaluation studies.
Classic In Vivo Cancer Models: Three Examples of Mouse Models Used in Experimental Therapeutics
Authors: Kruczynski Anna , Hill Bridget T.
Published: June 2004

Transplantable animal tumors have been associated with the discovery of most clinically active anticancer agents. They are still useful today in conducting detailed evaluations of new candidate anticancer drugs. Three protocols relating to transplantable experimental tumors are described in this unit. Included are the intravenously‐implanted murine P388 leukemia, the subcutaneously‐implanted murine B16 melanoma and two examples of subcutaneously‐implanted human tumor xenografts, the LX‐1 (lung) and MX‐1 (breast) tumors.
Guinea Pig Models of Asthma
Authors: Mazzone Stuart B. , Canning Brendon J.
Published: June 2004

This unit describes methods for establishing guinea pig models of asthma. Detailed descriptions are provided so that investigators can establish preparations in which bronchoconstriction, airways hyperresponsiveness, cough and airways inflammation can be studied.
Social Recognition Task in the Rat
Authors: Lemaire Martine
Published: June 2004

Among the numerous tasks designed for assessing distinct memory processes, the social recognition task in the rat offers the opportunity to evaluate a form of short‐term working memory in the domain of social cognition, and its modification by pharmacological agents or physiopathological states, such as aging. Social cognition in humans is obviously of great importance and its deficits, e.g., during aging and Alzheimer's dementia, often have dramatic consequences for the patient and their environment. Two protocols are described in this unit that permit evaluation of positive and negative drug effects on social recognition memory in adult male rats and beneficial drug effects on age‐related social recognition amnesia in aged male rats.
Emetic Liability Testing in Ferrets
Authors: Osinski Mark A. , Seifert Terese R. , Shaughnessy Thomas K. , Gintant Gary A. , Cox Bryan F.
Published: June 2004

Evidence of a candidate drug's efficacy and safety is mandatory for successful drug registration by regulatory authorities. However, a third property, tolerability, often determines a drug's acceptance by the patient population. Gastrointestinal events often determine the maximum tolerated dose in Phase I clinical trials. If the plasma concentrations achieved at the maximum tolerable dose are below those required for efficacy, the drug will certainly fail. The identification of a compound's emetic/nauseogenic liability early in the discovery process can be critical to the ultimate success of the drug discovery project. Ferrets are small carnivores (∼1 kg) of the Mustelidae family that vomit in response to many pharmacological classes of drugs as well as to cytotoxic chemotherapeutics and radiation. This unit describes a simple method for evaluating the emetic and nauseogenic potential of drug candidates in ferrets.
Overview of Receptor Cloning
Authors: Nash Norman
Published: June 2004

Receptors are generally categorized into four major superfamilies according to their structural and genetic similarities: steroid receptors, growth factor receptors, ionotropic receptors (i.e., ligand‐gated ion channels), and metabotropic receptors (i.e., G protein‐coupled receptors). This unit describes the methodology and critical issues for cloning receptors by several methods. Although cloning by protein purification was the first major method, it is now less frequently employed. The early, more tedious procedures broadened knowledge about receptors and established a strong foundation that has enabled the utilization of the newer approaches discussed in this unit, including: homology cloning by screening with oligonucleotides, DNA fragments or the products of direct polymerase chain reaction (PCR); expression cloning using function, ligand binding, and antibody recognition; and differential display techniques.
Receptor Reporter Systems
Authors: Burns David J.
Published: June 2004

Receptor‐linked reporter systems allow the study of pharmacological properties of known receptors, identification of new receptor subtypes, examination of the specific signal transduction pathways linked to receptor activation, and development of functional assays. Reporter systems provide high‐throughput screening methods that allow researchers to expeditiously identify and characterize compounds that modulate the activities of therapeutically relevant receptors or influence events downstream of receptor activation. This unit provides two representative protocols for coupling cell‐surface receptors to reporter molecules that signal receptor activation. The first is a luciferase‐based assay and the second uses green fluorescent protein (GFP). This unit provides a comparison of several reporter assays as well as reporter systems that rely on chloramphenicol acetyltransferase activity.
Expression of Cloned Receptors in Mammalian Cell Lines
Authors: Gopalakrishnan Murali , Molinari Eduardo J.
Published: June 2004

The expression of cloned receptor and ion‐channel proteins is a very useful tool for studying the physiological and pharmacological properties of receptors. The process of introducing a gene of interest into eukaryotic cells by biochemical or physical methods is termed transfection. After transfection, some of the DNA is transported into the nucleus, where it is transcribed. The resulting mRNA is transferred into the cytoplasm and translated into protein. The transfection method described in this unit is a versatile, plasmid‐based, liposome‐mediated gene‐transfer technique known as lipofection. This technique has gained wide acceptance because of its simplicity, suitability for a wide range of cell types, reasonable transfection efficiencies, and reproducibility. This unit also includes a procedure for assessing transfection efficiency via cotransfection with a reporter gene (lacZ) and assay for coexpression.