Molecular basis of retinoid action: The role of retinoic acid receptors in retinoic acid-induced tissue transglutaminase expression

Retinoic acid has profound effects on the cellular growth and differentiation of a variety of cells. However, the molecular basis of retinoic acid action has, until recently, not been well understood. The identification of retinoic acid receptors which bear a high degree of homology to members of the steroid receptor super-family has dramatically altered our understanding of the biology of retinoids. The focus of this dissertation has been toward identification of retinoic acid binding proteins responsible for the effects of this molecule on gene expression.^ We have characterized in detail the retinoic acid-dependent induction of tissue transglutaminase gene expression in a myeloid cell line, human promyelocytic leukemia cells (HL-60 cells). Using cDNA probes specific for tissue transglutaminase, we have determined that the retinoic acid induced increase in enzyme level is due to an increase in the level of tissue transglutaminase mRNA. We have used this model as a probe to investigate the molecular basis of retinoid regulated gene expression.^ This thesis demonstrates that retinoic acid receptors are expressed in cells which induce tissue transglutaminase expression in response to retinoic acid. In Hl-60 cells retinoic acid-induced transglutaminase expression is associated with saturable nuclear retonic acid binding. Transcripts for both the alpha and beta forms of the retinoic acid receptors can be detected in these cells. Pretreatment of HL-60 cells with agents that potentiate retinoic acid-induced transglutaminase expression also modestly induced the alpha form of the retinoic acid receptor. Studies in macrophages and umbilical vein endothelial cells have also associated expression of the beta form of the retinoic acid with retinoic acid induced tissue transglutaminase expression.^ To investigate directly if retinoic acid receptors regulate retinoic acid-induced tissue transglutaminase expression we developed a series of stably transfected Balb-c 3T3 cells expressing different levels of the beta or gamma form of the retinoic acid receptor. These studies indicated that either the beta or gamma receptor can stimulate endogenous tissue transglutaminase expression in response to retinoic acid. These are among the first studies in the steroid field to describe regulation of an endogenous gene by a transfected receptor. ^

Multiple GABA receptors in rabbit retina

The task of encoding and processing complex sensory input requires many types of transsynaptic signals. This requirement is served in part by an extensive group of neurotransmitter substances which may include thirty or more different compounds. At the next level of information processing, the existence of multiple receptors for a given neurotransmitter appears to be a widely used mechanism to generate multiple responses to a given first messenger (Snyder and Goodman, 1980). Despite the wealth of published data on GABA receptors, the existence of more than one GABA receptor was in doubt until the mid 1980's. Presently there is still disagreement on the number of types of GABA receptors, estimates for which range from two to four (DeFeudis, 1983; Johnston, 1985). Part of the problem in evaluating data concerning multiple receptor types is the lack of information on the number of gene products and their subsequent supramolecular organization in different neurons. In order to evaluate the question concerning the diversity of GABA receptors in the nervous system, we must rely on indirect information derived from a wide variety of experimental techniques. These include pharmacological binding studies to membrane fractions, electrophysiological studies, localization studies, purification studies, and functional assays. Almost all parts of the central and peripheral nervous system use GABA as a neurotransmitter, and these experimental techniques have therefore been applied to many different parts of the nervous system for the analysis of GABA receptor characteristics. We are left with a large amount of data from a wide variety of techniques derived from many parts of the nervous system. When this project was initiated in 1983, there were only a handful of pharmacological tools to assess the question of multiple GABA receptors. The approach adopted was to focus on a single model system, using a variety of experimental techniques, in order to evaluate the existence of multiple forms of GABA receptors. Using the in vitro rabbit retina, a combination of pharmacological binding studies, functional release studies and partial purification studies were undertaken to examine the GABA receptor composition of this tissue. Three types of GABA receptors were observed: Al receptors coupled to benzodiazepine and barbiturate modulation, and A2 or uncoupled GABA-A receptors, and GABA-B receptors. These results are evaluated and discussed in light of recent findings by others concerning the number and subtypes of GABA receptors in the nervous system. ^

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

THE EFFECT OF GABAMIMETICS ON NEUROTRANSMITTER RECEPTOR BINDING AND FUNCTION IN RAT BRAIN

Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system and alterations in central GABAergic transmission may contribute to the symptoms of a number of neurological and psychiatric disorders. Because of this relationship, numerous laboratories are attempting to develop agents which will selectively enhance GABA neurotransmission in brain. Due to these efforts, several promising compounds have recently been discovered. Should these drugs prove to be clinically effective, they will be used to treat chronic neuropsychiatric disabilities and, therefore, will be administered for long periods of time. Accordingly, the present investigation was undertaken to determine the neurochemical consequences of chronic activation of brain GABA systems in order to better define the therapeutic potential and possible side-effect liability of GABAmimetic compounds.^ Chronic (15 day) administration to rats of low doses of amino-oxyacetic acid (AOAA, 10 mg/kg, once daily), isonicotinic acid hydrazide (20 mg/kg, b.i.d.), two non-specific inhibitors of GABA-T, the enzyme which catabolizes GABA in brain, or (gamma)-acetylenic GABA (10 mg/kg, b.i.d.) a catalytic inhibitor of this enzyme, resulted in a significant elevation of brain and CSF GABA content throughout the course of treatment. In addition, chronic administration of these drugs, as well as the direct acting GABA receptor agonists THIP (8 mg/kg, b.i.d.) or kojic amine (18 mg/kg, b.i.d.) resulted in a significant increase in dopamine receptor number and a significant decrease in GABA receptor number in the corpus striatum of treated animals as determined by standard in vitro receptor binding techniques. Changes in the GABA receptor were limited to the corpus striatum and occurred more rapidly than did alterations in the dopamine receptor. The finding that dopamine-mediated stereotypic behavior was enhanced in animals treated chronically with AOAA suggested that the receptor binding changes noted in vitro have some functional consequence in vitro.^ Coadministration of atropine (a muscarinic cholinergic receptor antagonist) blocked the GABA-T inhibitor-induced increase in striatal dopamine receptors but was without effect on receptor alterations seen following chronic administration of direct acting GABA receptor agonists. Atropine administration failed to influence the drug-induced decreases in striatal GABA receptors.^ Other findings included the discovery that synaptosomal high affinity ('3)H-choline uptake, an index of cholinergic neuronal activity, was significantly increased in the corpus striatum of animals treated acutely, but not chronically, with GABAmimetics.^ It is suggested that the dopamine receptor supersensitivity observed in the corpus striatum of animals following long-term treatment with GABAmimetics is a result of the chronic inhibition of the nigrostriatal dopamine system by these drugs. Changes in the GABA receptor, on the other hand, are more likely due to a homospecific regulation of these receptors. An hypothesis based on the different sites of action of GABA-T inhibitors vis-a-vis the direct acting GABA receptor agonists is proposed to account for the differential effect of atropine on the response to these drugs.^ The results of this investigation provide new insights into the functional interrelationships that exist in the basal ganglia and suggest that chronic treatment with GABAmimetics may produce extrapyramidal side-effects in man. In addition, the constellation of neurochemical changes observed following administration of these drugs may be a useful guide for determining the GABAmimetic properties of neuropharmacological agents. ^

Sheep macrophages express at least two distinct receptors for IgG which have similar affinity for homologous IgG1 and IgG2.

Ovine bone marrow-derived macrophages (BMM) may express several IgG receptor (Fc gamma receptor; FcR) subsets. To study this, model particles (opsonized erythrocytes; EA), which are selectively handled by certain FcR subsets of human macrophages were used in cross-inhibition studies and found to react in a similar manner with FcR subsets of sheep macrophages. In experiments with monoclonal antibodies against subsets of human FcR, human erythrocytes (E) treated with human anti-D-IgG (anti-D-EAhu) and sheep E treated with bovine IgG1 (Bo1-EAs) were handled selectively by human macrophage FcRI and FcRII, respectively. Rabbit-IgG-coated sheep E (Rb-EAs) were recognized by FcRI, FcRII and possibly also by FcRIII of human macrophages. Anti-D-EAhu, Bo1-EAs and Rb-EAs were also ingested by sheep BMM. Competitive inhibition tests, using various homologous and heterologous IgG isotypes as fluid phase inhibitors and the particles used as FcR-specific tools in man (anti-D-EAhu and Bo1-EAs), revealed a heterogeneity of FcR also in sheep BMM. Thus, ingestion of anti-D-EAhu by ovine BMM was inhibited by low concentrations of competitor IgG from rabbit or man in the fluid phase, but not at all by bovine IgG1, whereas ingestion of Bo1-EAs was inhibited by bovine IgG1. This suggested that anti-D-EAhu were recognized by a FcR subset distinct from that recognizing bovine-IgG1. It was concluded that sheep BMM express functional analogs of human macrophage FcRI and FcRII and that Bo1-EAs and anti-D-EAhu are handled by distinct subsets of BMM FcR. All EAhu tested (EAhu treated with anti-D, sheep IgG1 or sheep IgG2) were ingested to a lower degree than EAs. This inefficient phagocytosis could be enhanced by treatment of EAhu with antiglobulin from the rabbit, suggesting that it is caused by a low degree of activity of opsonizing antibodies rather than special properties of the erythrocytes themselves. Several lines of evidence suggested that both FcR subsets of ovine BMM recognize both ovine IgG1 and IgG2. In contrast, bovine IgG1 reacts with one FcR subset and bovine IgG2 interacts inefficiently with all FcR of ovine BMM.

Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance

Abstract Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

A novel open channel blocker of GABA-A receptors

GABA-A receptors are chloride ion channels composed of five subunits, mediating fast synaptic and tonic inhibition in the mammalian brain. 19 different subunit isoforms have been identified, with the major receptor type in mammalian adult brain consisting of α1, β2, and γ2 subunits. GABA-A receptors are the target of numerous sedating and anxiolytic drugs such as benzodiazepines. The currently known endogenous ligands are GABA, neurosteroids and the endocannabinoid 2- arachidonoyl glycerol (2-AG). The pharmacological properties of this chloride ion channel strictly depend on receptor subunit composition and arrangement. GABA-A receptors bind and are inhibited by epileptogenic agents such as picrotoxin, and cyclodiene insecticides such as dieldrin. We screened aromatic monovalent anions with five-fold symmetry for inhibition of GABA-A receptors. One of the anions, PCCPinhibited currents elicited by GABA with comparable potency as picrotoxin. This inhibition showed all characteristics of an open channel block. The GABA-A receptor ion channel is lined by residues from the M2 membrane-spanning segment. To identify important residues of the pore involved in the interaction with the blocking molecules PCCP-, a mutation scan was performed in combination with subsequent analysis of the expressed mutant proteins using electrophysiological techniques. In a second project we characterised a light-switchable modulator of GABA-A receptors based on propofol. It was my responsibility to investigate the switching kinetics in patch clamp experiments. After its discovery in 1980, propofol has become the most widely used intravenous general anaesthetic. It is commonly accepted that the anaesthesia induced by this unusually lipophilic drug mostly results from potentiation of GABA induced currents. While GABA-A receptors respond to a variety of ligands, they are normally not sensitive towards light. This light sensitivity could be indirectly achieved by using modulators that can be optically switched between an active and an inactive form. We tested an azobenzene derivative of propofol where an aryldiazene unit is directly coupled to the pharmacophore. This molecule was termed azopropofol (AP2). The effect of AP2 on Cl- currents was investigated with electrophysiological techniques using α1β2γ2 GABA-A receptors expressed in Xenopus oocytes and HEK-cells. In the third project we wanted to investigate the functional role of GABA-A receptors in the liver, and their possible involvement in cell proliferation. GABA-A receptors are also found in a wide range of peripheral tissues, including parts of the peripheral nervous system and non-neural tissues such as smooth muscle, the female reproductive system, liver and several cancer tissues. However their precise function in non neuronal or cancerous cells is still unknown. For this purpose we investigated expression, localization and function of the hepatocytes GABA-A receptors in model cell lines and healthy and cancerous hepatocytes.

The Antimalarial Drug Proguanil Is An Antagonist at 5-HT3 Receptors

Proguanil is an antimalarial prodrug that is metabolized to 4-chlorophenyl-1-biguanide (CPB) and the active metabolite cycloguanil (CG). These compounds are structurally related to meta-chlorophenyl biguanide (mCPBG), a 5-hydroxytryptamine 3 (5-HT3) receptor agonist. Here we examine the effects of proguanil and its metabolites on the electrophysiology and ligand-binding properties of human 5-HT3A receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. 5-HT3 receptor responses were reversibly inhibited by proguanil, with an IC50 of 1.81 μM. Competitive antagonism was shown by a lack of voltage-dependence, Schild plot (Kb = 1.70 μM), and radioligand competition (Ki = 2.61 μM) with the 5-HT3 receptor antagonist [3H]granisetron. Kinetic measurements (kon = 4.0 × 104 M−1 s−1; koff = 0.23 s−1) were consistent with a simple bimolecular reaction scheme with a Kb of 4.35 μM. The metabolites CG and CPB similarly inhibited 5-HT3 receptors as assessed by IC50 (1.48 and 4.36 μM, respectively), Schild plot (Kb = 2.97 and 11.4 μM), and radioligand competition (Ki = 4.89 and 0.41 μM). At higher concentrations, CPB was a partial agonist (EC50 = 14.1 μM; I/Imax = 0.013). These results demonstrate that proguanil competitively inhibits 5-HT3 receptors, with an IC50 that exceeds whole-blood concentrations following its oral administration. They may therefore be responsible for the occasional gastrointestinal side effects, nausea, and vomiting reported following its use. Clinical development of related compounds should therefore consider effects at 5-HT3 receptors as an early indication of possible unwanted gastrointestinal side effects.

The antimalarial drugs quinine, chloroquine and mefloquine are antagonists at 5-HT 3 receptors

Background and Purpose: The antimalarial compounds quinine, chloroquine and mefloquine affect the electrophysiological properties of Cys-loop receptors and have structural similarities to 5-HT3 receptor antagonists. They may therefore act at 5-HT3 receptors. Experimental Approach: The effects of quinine, chloroquine and mefloquine on electrophysiological and ligand binding properties of 5-HT3A receptors expressed in HEK 293 cells and Xenopus oocytes were examined. The compounds were also docked into models of the binding site. Key Results: 5-HT3 responses were blocked with IC50 values of 13.4 μM, 11.8 μM and 9.36 μM for quinine, chloroquine and mefloquine. Schild plots indicated quinine and chloroquine behaved competitively with pA2 values of 4.92 (KB=12.0 μM) and 4.97 (KB=16.4 μM). Mefloquine displayed weakly voltage-dependent, non-competitive inhibition consistent with channel block. On and off rates for quinine and chloroquine indicated a simple bimolecular reaction scheme. Quinine, chloroquine and mefloquine displaced [3H]granisetron with Ki values of 15.0, 24.2 and 35.7 μM. Docking of quinine into a homology model of the 5-HT3 receptor binding site located the tertiary ammonium between W183 and Y234, and the quinoline ring towards the membrane, stabilised by a hydrogen bond with E129. For chloroquine, the quinoline ring was positioned between W183 and Y234 and the tertiary ammonium stabilised by interactions with F226. Conclusions and Implications: This study shows that quinine and chloroquine competitively inhibit 5-HT3 receptors, while mefloquine inhibits predominantly non-competitively. Both quinine and chloroquine can be docked into a receptor binding site model, consistent with their structural homology to 5-HT3 receptor antagonists.