David B. Bylund - US grants

Alpha-2 adrenergic receptors mediate several important functions of the endogenous catecholamines in both the central and peripheral nervous systems. Several clinically important drugs appear to produce their pharmacologic effects through an interaction at alpha-2 receptors. There now exists considerable evidence indicating significant differences in the pharmacological characteristics and regulation of mammalian alpha-2 adrenergic receptors from various tissues and species. We suggest that differences in the pharmacological properties and in the regulation (mechanism of action) of alpha-2 adrenergic receptors may be due to differences in the structure of the alpha-2 adrenergic receptor complex. To test this hypothesis, it is proposed to compare in various tissues and species: (1) The rank order and relative potencies of various adrenergic drugs using both radioligand binding and functional assays; (2) The thermal stability and pH optima of radioligand binding; (3) The functional size of the receptor complex using target analysis (radiation inactivation); (4) The apparent molecular size determined by gel filtration and sucrose density gradient centrifugation of receptor preparations solubilized in the presence and absence of agonist; and (5) The apparent molecular weight from SDS gel electrophoresis of partially purified receptors labeled with a photoaffinity probe. These studies should help to resolve the present confusion in classification of alpha receptors and may lead to more selective alpha adrenergic drugs for clinical use.

Norepinephrine and epinephrine are important neurotransmitters in the central nervous system, and alpha-2 adrenergic receptors, which are coupled to the inhibition of adenylate cyclase, are widely distributed. Clinically, alpha-2 adrenergic agonists such as clonidine and guanabenz are used to treat hypertension as well as several other disorders. While clonidine is thought to act centrally to control blood pressure, its mechanism of action is not well understood. The therapeutic effects of alpha-2 adrenergic agonists often are evident only after chronic treatment, suggesting the involvement of adaptive changes. Thus, a better understanding of the regulation and the mechanism of action of alpha-2 adrenergic receptors, their coupling to and their regulation of adenylate cyclase has a clear potential for the development of better drug therapies. The overall goal of the proposed research is to understand the molecular basis for the effects of preincubation of cells with an alpha-2 adrenergic agonist on subsequent alpha-2 adrenergic receptor binding and the activity of the enzyme adenylate cyclase. There are two main effects to be studied. The first is the down-regulation of receptor number and desensitization of the cyclic AMP response. Second, we have recently observed that alpha-2 agonist pretreatment results in a 20-fold increase in forskolin stimulated cyclic AMP synthesis, which we call "sensitization". For both sets of studies, the model system to be used is the HT29 cell line. Each effect will be characterized and then its molecular mechanism investigated. For the first effect, the hypothesis to be tested is that the mechanism of alpha-2 adrenergic receptor desensitization: uncoupling; receptor internalization; and receptor loss. Our initial studies indicate that "sensitization" is not mediated through cyclic AMP. Thus, other mechanisms which have been suggested for alpha-2 adrenergic receptor action, as well as other second messenger systems, will be investigated. Additional experiments are aimed at investigating the possible molecular alterations in the adenylate cyclase system which cause the increased cyclic AMP production seen with sensitization.

Norepinephrine and epinephrine are important neurotransmitters in the central nervous system, and alpha-2 adrenergic receptors, which are coupled to the inhibition of adenylate cyclase, are widely distributed. Clinicallly, alpha-2 adrenergic agonists such as clonidine and guanabenz are used to treat hypertension as well as several other disorders. While clonidine is thought to act centrally to control blood pressure, its mechanism of action is not well understood. The therapeutic effects of alpha-2 adrenergic agonists often are ivident only after chronic treatment, suggesting the involvement of adaptive changes. Thus, a better understanding of the regulation and the mechanism of action of alpha-2 adrenergic receptors, their coupling to and their regulation of adenylate cyclase, has a clear potential for the development of better drug therapies. The overall goal of the proposed research is to understand the molecular basis for the effects of incubation and preincubation of cells with an alpha-2 adrenergic agonist on subsequent alpha-2 adrenergic receptor binding and the activity of the enzyme adenylate cyclase. There are three effects to be studied. The first is the down-regulation of receptor number and desensitization of the cyclic AMP response. The second is the sensitization of stimulated cyclic AMP production following preincubation with an alpha-2 agonist. There appear to be two types of sensitization, which may have different mechanisms. The third effect is the potentiation of forskolin-stimulated cyclic AMP production by concurrent incubation with (a high concentration of) an alpha-2 agonist. We propose to determine the mechanisms of these effects in three cell lines, each of which express one of the three putative alpha-2 adrenergic receptor subtypes, and then determine if any differences are cell type specific or receptor subtype specific (or perhaps a combination of both). Our overall approach is to first study the mechanism of these phenomena in the three cell lines which normally express each alpha-2 adrenergic receptor subtype, and then to study a cell line (which normally lacks the receptor) stably transfected separately with each of the subtypes.

Serotonin (5HT) is an important neurotransmitter in the central nervous system and is thought to be involved in a variety of mental disorders. The recent proliferation of 5HT receptor subtypes provides a unique opportunity to study the involvement of these subtypes in mental disorders and to develop subtype-specific drugs for the treatment of these disorders. The long-term objective of this application is to understand the regulation of the various 5HT, receptor subtypes on two levels. The first is regulation at the level of the receptor protein in the membrane and its relationship to adenylate cyclase; the second is the genetic regulation of the expression of the receptor. We propose to use the 5HT(1B) receptor in the OK cell line as a model system to study the 5HT(1) receptors in the CNS. Homologous down-regulation and desensitization of the 5HT(1B) receptor in the OK cell line will be studied by a variety of assays including radioligand binding and cyclic AMP production assays. We propose to clone the 5HT(1B) receptor from the OK cell and the 1B and/or 1D receptor from rat brain. These clones will be used as tools in the proposed studies of the regulation of the receptor system. We also plan to define the tissue distribution and level of expression of 5HT(1) receptors in the rat brain by Northern blot analysis and in situ hybridization.

DESCRIPTION (provided by applicant): The up-regulation or down-regulation of neurotransmitter receptors has become one of the major, proposed mechanisms for a variety of therapeutic drugs. Drug-induced regulation of central catecholamine receptors has been extensively studied in adults, but little is known about their regulation in young animals. It is particularly important to understand the nature of drug-induced regulation of adrenergic receptors in young animals for two reasons. First, adrenergic agents are used clinically in children and adolescents. Our preliminary data show that the effects of adrenergic agents on the young adrenergic system are qualitatively different from their effects on the adult. Second, because the adrenergic system is late developing, and is still changing during periadolescent development, perturbations in this system are more likely to have greater, qualitatively different, and longer-lasting effects than on a mature system. The central hypothesis of the proposed studies is that CNS alpha-2 and beta adrenergic receptors are differentially regulated in periadolescent as compared to adult rats, as reflected by responses to two drugs that effect the noradrenergic and serotonin systems (the selective re-uptake inhibitors desipramine and fluvoxamine). The first specific hypothesis, that central adrenergic receptors will be up-regulated in periadolescent animals rather than down-regulated as observed in adult animals, will be tested using quantitative autoradiography to determine alpha-2 and beta adrenergic receptor density. The second hypothesis, that the functional coupling between the adrenergic receptors and their G proteins will be increased in the young animals as compared to adults, will be tested using cyclic AMP accumulation and GTPyS binding assays. The third hypothesis, that the functional responses to agonist stimulation will be increased in periadolescent animals as consequences of these alterations in regulation and coupling, will be studied using regulation of immediate early gene expression and agonist induced-hypothermia. These studies in rats will help provide the necessary foundation for understanding the regulation of the adrenergic receptor systems in young animals leading to an eventual understanding of the effects of chronic drug treatment in children.

DESCRIPTION (provided by applicant): Major Depressive Disorder (clinical depression) is a severe and potentially incapacitating mental illness that is common in children and adolescents, with an estimated lifetime prevalence of 15 -20 % in this population. An important difference between clinical depression in children and adolescents, as compared to adults, is its response to antidepressant drugs. Tricyclic antidepressants have not been shown to be effective in treatment of child and adolescent clinical depression. Although antidepressant drugs have numerous neurochemical actions, the therapeutic mechanisms of action of antidepressant drugs in relieving depression remain unknown. In non-depressed persons antidepressant drugs are not euphoriant or stimulant. Therefore, investigations related to which of the many neurochemical effects of antidepressant drugs are functionally related to their therapeutic efficacy in relieving depression requires research using a behavioral animal model of clinical depression. To better understand the neurobiology underlying the differences between children and adolescents, and adults in the response to pharmacological treatment of clinical depression, animal models of childhood and adolescent depression are needed. Currently, there are no established juvenile animal models of clinical depression. Although the models developed in adult animals can serve as a starting point, they must be adapted and validated in juvenile animals due the many differences between juvenile and adult animals. The overall goal of this proposal is to assess the validity and usefulness of two well-established rat animal models of adult clinical depression as models of childhood and adolescent clinical depression. Specifically, we propose to assess the usefulness of the forced-swim test and of learned helplessness as animal models for clinical depression in juvenile rats. The key questions which are addressed by this proposal are: 1) Do juvenile rats respond to antidepressant drugs with decreased immobility in the forced swim? 2) Do juvenile rats develop learned helplessness after inescapable stress in both the acute and persistent paradigms, as demonstrated by shuttlebox testing? 3) Do juvenile rats respond to antidepressant drugs in the both acute and persistent learned helplessness paradigms with decreased escape latencies in shuttlebox testing? Ultimately, the models may facilitate a better understanding of the underlying neurobiology of clinical depression, and serve as predictive measures of antidepressant efficacy in children and adolescents.

DESCRIPTION (provided by applicant): The proposed Short Course in Integrative and Organ Systems Pharmacology is designed to provide graduate students, postdocs, junior faculty and industrial scientists with lectures, demonstrations and hands on experiences with whole animal. This has become an issue of increasing importance as more and more pharmacology training programs have become exclusively cellular and molecular in nature. The course to be offered over two weeks in the summer will focus on cardiovascular and neuropharmacology. Lectures will be provided on the functioning of IACUCs, the humane treatment of animals and regulations appropriate to this, background material on cardiovascular physiology, pharmacology and neuropharmacology. Instrumented animals will be used (dogs, rabbits) for whole animal cardiovascular experiments and various techniques will be used for examining nociception and cognition in rodents. Isolated vascular material and hearts will be used for organ system studies. The drugs used in these experiments will be the same as those used in the instrumented animals. There will also be 1.5 days set aside for elective, hands on, focused experiences selected by the students. An optional third week internship will be available for interested students.