David K. Grandy - US grants

The goal of this proposal is to apply a new technology employing a novel silicon biosensing device to the study of neurotransmitter receptors. The biosensor to be used is a prototype silicon based microphysiometer which potentiometrically measures extracellular pH changes associated metabolic activity resulting from receptor activation. The microphysiometer performs nondestructive measurements of a cells metabolic activity in real time. We will study the catabolic events associated with dopamine D1 and D2 receptor activation. This will be carried out using stable transfectants expressing known levels of cloned dopamine receptors linked to well defined second messenger and effector responses. We will correlate various effector system activity with the amplitude and t1/2 of the metabolic records obtained with the microphysiometer. By relating the kinetics of the metabolic response with effector activity in a variety of well understood cellular environments we will assign a metabolic signature to Particular receptor/effector combinations. The techniques developed and information learned in this study of D1 and D2 receptors will subsequently be brought to bear on our studies of new G protein-coupled receptors. We will take advantage of the ability of the silicon microphysiometer to detect a broad range of cellular events to rapidly screen an assortment of cloned but as yet uncharacterized receptors. Using the microphysiometer we should be able to screen an assortment of stable transfectants expressing novel receptors with a large number of pharmacological agents. The kinetics of the microphysiometric measurements should suggest details about the effector mechanisms associated with the new receptors. All information concerning ligand specificity and second messenger involvement obtained with the silicon microphysiometer will be corroborated by conventional methods to document the accuracy of the method.

The dopamine system is a major neurotransmitter system in the mammalian brain. It regulates numerous physiological responses and is implicated in the pathophysiology of several disorders, in particular drug addiction, Parkinson's disease and schizophrenia. Until recently, dopamine was thought to interact with only two receptors, the D1 and D2 dopamine receptors. This two receptors-concept began to change when it was shown, two years ago, that there exist two different forms of D2 receptor and when, last year, a distinct and unexpected dopamine receptor was cloned, the D3 receptor. This concept is going to evolve even further with our discovery of another new dopamine receptor, the D4 receptor. The D4 receptor is especially interesting because of its potential involvement in the pathogenesis of schizophrenia. Until now, it was the D2 receptor which was specifically involved in the etiology of schizophrenia, since most neuroleptics are D2 receptor antagonists. However several observations about the D4 receptor suggest that it might have a predominant role in that disease. First, preliminary studies show that the D4 receptor is present in brain tissues expected to be involved in the etiology of schizophrenia. Second, its pharmacological profile indicates that it is also the target of common neuroleptics. Third, most interestingly, it has the highest affinity of all the dopamine receptors for clozapine, a particular antipsychotic drug which does not produce the extrapyramidal side effects that plague most neuroleptics directed at the D2 receptor. By using the D4 receptor clone, we propose to investigate several aspects of this hypothesis. First, in situ hybridization and immunocytochemical techniques will be used to locate neurons expressing D4 receptors, a task that our preliminary Northern blot analysis could not achieve, and determine whether the D4 receptor is present in the neuronal pathways expected to be important in the etiology of schizophrenia. Second, the D4 receptor's abilities to induce second messenger systems will be tested in a variety of cellular environments. These experiments will determine which intracellular changes are caused by D4 receptor stimulation and might differentiate it from the D2 receptor at the biological level. The hallmark of the D4 receptor is its ability to recognize clozapine. In contrast to D2 receptor, D4 receptor blockade seems to not produce locomotor side effects. It is therefore important to determine which D4- specific structural features allow for clozapine binding. These will be defined by site-directed mutagenesis. Finally, the diversity of the responses mediated by the dopamine system suggests the possible existence of other dopamine receptors. By using a low-stringency screening approach, we have already been able to isolate a gene fragment which codes for a putative D4-related receptor. In addition, the possible existence of D4-related receptors will be checked in a battery of tissues. Our last aim is to characterize these putative D4-related receptors by defining their pharmacological profile, biological activities and tissue distribution.

In addition to its importance as a neurotransmitter, dopamine has profound effects on heart rate, blood pressure, renal blood flow, sodium absorption and water retention. In the clinical setting dopamine is used to treat patients suffering from shock or impaired renal function. At low doses the renal effects of dopamine predominate and are mediated by two receptor subtypes: DA1 and DA2. Little is known about renal DA2 receptor physiology. In contrast, DA1 dopamine receptors mediate natriuresis and diuresis in the tubules of the renal cortex and vasodilation of the mesenteric and renal vascular beds. DA1 receptors in proximal convoluted tubule (PCT) epithelial cells couple to the stimulation of cAMP and diacylglycerol (DAG) production. Increases in the concentration of these two second messengers activate protein kinases A and C, respectively. Two targets of these kinases are the luminal Na+/H+ exchanger (NHE) and the basolateral Na+-K+ATPase. Both the NHE and the ATPase are involved in sodium transport but it is the phosphorylation and subsequent inhibition of Na+/H+ exchanger activity that results in sodium excretion (natriuresis). Our results suggest that the dopamine D5 receptor that we recently cloned is identical to the renal PCT DA1 receptor subtype. We propose that dopamine D5 receptors in the renal PCT epithelia couple to second messenger systems that participate in the regulation of Na+/H+ exchanger activity. Therefore, any interference with the ability of D5/Da1 receptor's to regulate sodium transport may play an important role in the etiology of certain forms of essential hypertension. We propose to test several aspects of this hypothesis. Recently we demonstrated that activated dopamine D5 receptors stimulate cAMP production and the secretion of H+. This latter effect is sensitive to amiloride suggesting that a Na+/H+ exchanger (NHE) activity is involved. We propose to pursue the in vitro characterization of D5's coupling to adenylyl cyclase and phospholipase C. In addition, we have the opportunity to develop a very powerful in vitro system in which to dissect D5's regulation of NHE activity. Cell lines expressing each of the three recently cloned NHEs and the D5 receptor will be evaluated with respect to dopaminergic regulation of sodium transport. We also have evidence to suggest that recombinant vaccinia virus vectors can be used to generate polyclonal anti-receptor antiserum. We will use this technology to produce anti-D5 antiserum, a valuable reagent for the analysis of dopamine D5 receptor expression and post translational modification in normal and diseased renal cortex tissue. Abnormal renal responses to dopamine have also been reported in two well-documented rat models of inherited hypertension and we are now in a unique position to determine whether the genetic defect lies within the dopamine D5 receptor gene. finally we intend to generate transgenic mice that lack functional dopamine D5 receptors. The production of these "knockout" mice will provide a new and powerful mouse model system in which the role of dopamine D5 receptors in renal physiology in general, and sodium transport in particular, can be evaluated.

Three major classes of opioid receptors, mu(mu), delta (delta) and kappa (kappa), have been defined based on differences in their pharmacology, physiology and tissue distribution. When stimulated in vivo the opioid receptors activate a cascade of intracellular reactions involving adenylyl cyclase, calcium channels and potassium channels, which result in many of the classical effects of opiate intoxication including euphoria, analgesia and physical dependence. The molecular characterization of the opioid receptors has been slow due to several factors, perhaps the most important of which being that they are intrinsic membrane proteins which are difficult to solubilize in active form and they are expressed in relatively low amounts. Recently these difficulties were overcome by the expression cloning of a mouse delta opioid receptor subtype from the neuroblastoma X glioma cell line NG108- 15. The primary known as the G protein-coupled receptors. In light of these recent reports we re-examined the sequence of an orphan receptor clone which we had obtained by degenerate PCR. This cDNA clone, referred to as R21, encodes a novel G protein-coupled receptor which shares significant sequence identity with the recently published mouse delta opioid receptor. Based on the conservation of key amino acid residues and the overall homology between R21 and the mouse delta opioid receptor we predict that R21 is a member of the opioid receptor family. To test this hypothesis we propose to pharmacologically characterize the receptor encoded by R21 and investigate the affect its stimulation has on adenylyl cyclase and a voltage-dependent outwardly rectifying potassium conductance. Once pharmacologically defined the tissue regulated the rat R21 gene will be characterized. The human homologue of the R21 gene will also be characterized to identify markers that can be used i genetic linkage and association studies of HR21 and human disease. Eventually the mouse gene, MR21, will be target and knocked out. These transgenic mice will be a valuable model system in which to evaluate the receptor's role in processes ranging from synaptic transmission to behavior.

DESCRIPTION: (Applicant's Abstract) In humans the use of heroin, cocaine and methamphetamine often becomes habit forming. This is thought to be due, in part, to their strong positive reinforcing properties. Similar behavior has also been observed in rats and mice who will repeatedly self-administer opiates or psychostimulants if given the opportunity. Efforts to identify the neuroanatomical substrates that involve an animal's repeated administration of an opiate or a psychostimulant have relied on lesioning of specific brain nuclei, microdialysis and neuro-pharmacological manipulations. The current interpretation in this field is that the mesocorticolimbic dopamine neurons are, to a large extent, responsible for mediating many of the positive reinforcing properties of abused drugs. Originating in the ventral tegmental area these dopamine neurons project primarily to the nucleus accumbens, frontal cortex, olfactory tubercle, amygdala, and septum. The interest in the ventral tegmental area's input to the nucleus accumbens is based on the profound effects that lesioning and neuropharmacological manipulation of dopamine levels in the nucleus accumbens have on an animal's behavioral response to opiates and psychostimulants. Although dopamine is known to interact with a small number of proteins in the nucleus accumbens, including members of the dopamine receptor family and the dopamine transporter, role that each of these molecules play in rewarding and drug reinforced behavior remains to be elucidated. Transgenic mice that have been genetically altered to lack a particular gene product provide a powerful means by which to assess the role of that product in vivo. During the preceding funding period we successfully produced three strains of mice that carry mutated D2 or D4 receptor genes. In this competing continuation application we propose to: (1) Breed and test our three mutant mouse strains (N5 D2-/-, N5 D4-/- and D2-/-D4-/-) for their behavioral, physiological and biochemical responses to opiates and psychostimulants; (2) Explore neuronal plasticity at the level of gene expression in our dopamine receptor-deficient mice and relate these findings to drug sensitization; and (3) Develop new mouse strains that carry inducible targeting vectors that will permit the conditional expression of a given dopamine receptor.

Three major classes of opioid receptors, mu, delta and kappa, have been defined based on differences in their pharmacology, physiology and tissue distribution. When stimulated in vivo the opioid receptors activate a variety of intracellular reactions that effect calcium channels, potassium channels and adenylyl cyclase activity resulting in many of the classical effects of opiate intoxication including euphoria, analgesia and physical dependence. The molecular characterization of the opioid receptors was slow until the recent expression cloning of a mouse delta opioid receptor was reported. Based on this sequence we and others developed cloning strategies that led to the isolation of both kappa and mu opioid receptor cDNAs. The ability to express each of the opioid receptors in tissue culture permits their detailed pharmacological and physiological study. In a previous application we proposed that R21, a novel receptor we had cloned, encoded an opioid receptor based on the conservation of key amino acids and its overall homology with a mouse delta opioid receptor. Having demonstrated that R21 encodes a rat kappa opioid receptor, we are now ready to proceed with the molecular studies of this receptor. In particular we propose to extend out physiological studies to include the coupling of human and rat kappa opioid receptors to calcium and potassium channels. At the anatomical level as a first step towards evaluating the effects of chronic opiate use on kappa receptor expression we will begin to characterize its distribution in human brain material. An immunomodulatory role for opioids has been demonstrated and recently we obtained evidence that kappa opioid receptor mRNA is expressed in a mouse thymoma cell line. This provides us with an excellent opportunity to explore the effects of opioid exposure on cells of the immune system. To better understand how kappa opioid receptor expression is controlled we will characterize both the human and rat genes and in addition will attempt to identify markers that can be used in genetic linkage and association studies. Eventually the mouse kappa opioid receptor gene will be targeted and knocked out. These animals will be a valuable model system in which to evaluate the kappa receptor's role in processes ranging from synaptic transmission to behavior. Finally, we have cloned a receptor whose sequence and anatomical distribution suggest that it is a member of the opioid receptor gene family. We propose to continue the characterization of this interesting receptor using DNA sequence analysis, in vitro mutagenesis and expression studies.

DESCRIPTION (Applicant's Abstract): Opiates are powerful analgesic drugs. Unfortunately, their chronic use often results in addiction and tolerance, liabilities that seriously limit their clinical usefulness. Changes at the cellular and opiate receptor level have proven to be inadequate explanations of tolerance and dependcence. An exciting alternative hypothesis is that "anti-opioid" systems, neurotransmitters or neuropeptides that functionally oppose the effects of opiate receptor activation, contribute to tolerance and dependence as well as to variations in analgesic potency of opioids in some pathological pain states. We have recently demonstrated the orphanin FQ (OFQ, also referred to as nociceptin) is the endogenous ligand for the opioid-like G protein-coupled receptor LC132 and that it reverses morphine-induced analgesia, hypothermia and Straub tail. These anti-opioid actions of OFQ/N together with its close evolutionary kinship to endogenous opioid peptides suggesty that the OFQ/LC132 neurotransmitter system may play an important role in the homeostasis of opioid mechanisms. We propose three complimentary Specific Aims taht are designed to clarify the interactions between the opiate and the OFQ/LKC132 neuropeptide transmitter systems. The experiments included in Specific Aim (1) are designed to extend our knowledge about the OFQ/N neuropeptide system at the molecular level. Specifically we propose to quantitate changes in OFQ/N and its receptor that may occur in opiate-tolerant animals. We also outline experiments designed to characterize two new forms of the receptor that we discovered since ourr last subsmission. The last set of experiments included in Specific Aim described our ongoing efforts to produce a strain of mouse that lacks the OFQ/N receptor. These mice should prove to be an important animals model in which to evaluate the involvement of the OFQ/N receptor system in mediating opiate tolerance, dependence and reward. In Specific Aim (2) we have proposed studies that continue our characterization of OFQ/N's electrophysiological effects on well-defined neurons in the ventral tegmental area. This area of the is known to be important in reward behavior and its neurons are known to be altered in animals repeatedly exposeds to morphine. The experiments proposed in specific aim (3) involve the identification of some of the neuroanatomical substrates that mediate OFQ/N's ati-opioid activity. These microinjection studies will provide us with an elegant means by which to determine whetheer OFQ/N is able to produce a tolerance-like effect in drug-naive rats. These studies will advance our understanding OFQ/N's involvement in opioid analgesia as well as tolerance, and could ultmately lead to improved treatment of pain and addiction.

[unreadable] DESCRIPTION (provided by applicant): Attention Deficit Hyperactivity Disorder (ADHD) is a complex behavioral condition characterized, in part, by distractibility, impulsivity, hyperactivity, and abnormal novelty-seeking behavior. ADHD is currently estimated to affect 2.5 million children and adults nationwide. The molecular basis for ADHD is unknown but a wealth of clinical data supports the hypothesis that dysregulation of dopamine (DA) signaling in the central nervous system significantly contributes to its etiology. Although most individuals diagnosed with ADHD benefit from low doses of the psychostimulant Ritalin (r) (methylphenidate, MPD), its exact mode of action and long-term consequences of exposure are unclear. However, since MPD can elevate extracellular DA levels by interfering with DA transporter function, its cIinical benefit may involve an indirect stimulation of DA receptors (DARs). Of the five known DAR subtypes, recent family and twin studies have revealed an association between an allele of the human DA D4R gene (DRD4.7) and ADHD. Anatomically, D4Rs are expressed in brain regions thought to be relevant to ADHD. Furthermore, incipient congenic D4R-/-mice display locomotor supersensitivity to the psychostimulants cocaine and methamphetamine in addition to elevated striatal dopamine content and diminished novelty-seeking behavior. Taken together these observations suggested to us the hypothesis: DA D4Rs mediate MPD's effects in mice and humans. Since the selective pharmacological antagonism of D4Rs in vivo has yet to be convincingly demonstrated, we chose to begin testing our hypothesis in D4R-deficient mice and have found that congenic (N10 on C57B1/6J) D4R-/- mice display a dose-dependent locomotor supersensitivity to MPD. Encouraged by these findings we propose the following course of experimentation: In specific aim 1 we describe studies designed to establish whether acute and/or chronic MPD exposure influences novelty-seeking and impulsivity behaviors in juvenile and adolescent mice lacking D4Rs. In aim 2 we address the question: What are the effects of acute and chronic MPD exposure on gene expression in ADHD-relevant brain regions of wild-type and D4R-deficient juvenile and adolescent mice? Finally, in aim 3, we propose to generate three novel strains of knock-in mice that express either the human allele associated with ADHD (DRD4.7), a "normal" human D4R allele (D4.4), or Green Fluorescent Protein-tagged (GFP) murine D4Rs to more reliably localize the receptor protein in mouse brain tissue. It is our expectation that upon the successful completion of these aims, a better understanding of the complex relationship between MPD exposure, D4R stimulation, gene expression, and rodent behaviors relevant to ADHD will emerge [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this CUTTING EDGE BASIC RESEARCH AWARD (CEBRA) application, submitted in response to PAR-09-222, is to determine the extent to which the G protein-coupled trace amine-associated receptor 1 (TAAR1) contributes to intravenous (i.v.) methamphetamine (METH) self-administration (SA). METH consumption is declining nation-wide yet significant use persists in specific demographics of the United States and around the world in spite of the overwhelmingly negative personal, familial, societal, and legal consequences associated with its use and abuse. For more than 50 years psychostimulant research has attempted to identify the biological substrates mediating METH's abuse potential. In spite of clarifying some of the biological mechanisms underlying METH's actions (e.g. interference with monoamine oxidase activity as well as dopamine, norepinephrine and vesicular monoamine transporter functions) there is still no widely accepted pharmacologic approach to medically managing the METH abstinence syndrome or preventing relapse to METH abuse. The lack of progress in this area suggests important biological molecules that contribute to METH's abuse liability other than enzymes and transporters remain to be discovered;a view supported by research involving genetically engineered mice. Recently we reported recombinant TAAR1, a G1s-coupled G protein-coupled receptor, is directly activated in vitro by nanomolar concentrations of METH to stimulate cAMP production. Furthermore, as no TAAR1 antagonist is commercially available we recently designed and synthesized a novel composition of matter, ET-92, that acts as a TAAR1-antagonist in vitro. These findings suggest to us TAAR1 might be a novel mediator of METH's actions in vivo and ET-92 could be a lead in developing a medication that interferes with behaviors relevant to METH abuse. However, an essential step in establishing the involvement of TAAR1 in METH abuse and the potential of ET-92 as an anti- METH lead compound is to evaluate them both in established animal models of drug taking behavior. We think our proposal is appropriate for the CEBRA mechanism because the hypothesis to be tested is unconventional: i.v. METH SA is mediated by TAAR1. To test our hypothesis we propose 2 specific aims: (1) Determine the extent to which TAAR1 mediates i.v. METH SA in adult wild type and TAAR1-deficient mice of both sexes and (2) Determine whether the novel TAAR1 antagonist ET-92 interferes with the acquisition, maintenance, extinction and/or reinstatement of i.v. METH SA in adult wild type mice of both sexes. We anticipate the successful completion of these aims will significantly influence the field of psychostimulant research by: (1) establishing a novel mechanism of METH's action that has long eluded the field and (2) by sparking interest in novel TAAR1-selective compounds as potential leads in the development of medications for preventing relapse to METH abuse in humans. PUBLIC HEALTH RELEVANCE: The abuse of methamphetamine (METH) has reached near epidemic proportions in many communities in the United States and its use continues to grow worldwide with devastating consequences for users, their families and society. Although behavioral modification strategies can prolong abstinence in some they do not prevent relapse to abuse in most individuals. At the present time no pharmacological treatment is available to prevent relapse to METH abuse. It is expected that the successful completion of the proposed research aims will result in a better understanding of the role the G protein-coupled trace amine-associated receptor 1 (TAAR1) plays in relapse to METH self- administration and in the process establish whether TAAR1 as an important new target for anti-METH medication development.