David Schwarz

Characterization of γ-Aminobutyric Acid Receptor GABAB(1e), a GABAB(1) Splice Variant Encoding a Truncated Receptor

We have identified a splice variant encoding only the extracellular ligand-binding domain of the γ-aminobutyric acid B (GABAB) receptor subunit GABAB(1a). This isoform, which we have named GABAB(1e), is detected in both rats and humans. While GABAB(1e) is a minor component of the total pool of GABAB(1) transcripts detected in the central nervous system, it is the primary isoform found in all peripheral tissues examined. When expressed in a heterologous system, the truncated receptor is both secreted and membrane associated. However, GABAB(1e) lacks the ability to bind the radiolabeled antagonist [3H]CGP 54626A, activate G-protein coupled inwardly rectifying potassium channels, or inhibit forskolin-induced cAMP production when it is expressed alone or together with GABAB(2). Interestingly, when co-expressed with GABAB(2), not only does the truncated receptor heterodimerize with GABAB(2), the association is of sufficient avidity to disrupt the normal GABAB(1a)/GABAB(2) association. Despite this strong interaction, GABAB(1e) fails to disrupt G-protein coupled inwardly rectifying potassium activation by the full-length heterodimer pair of GABAB(1a)/GABAB(2).

Identification of differentially expressed genes induced by transient ischemic stroke.

We have used a rat model of focal cerebral ischemia to investigate changes in gene expression that occur during stroke. To monitor these changes, we employed representational difference analysis-polymerase chain reaction (PCR). A total of 128 unique gene fragments were isolated, and we selected 13 of these for quantitative reverse transcriptase-PCR analysis. Of these 13 genes, we found seven that were differentially expressed. Four of these genes have not previously been implicated in stroke, and include neuronal activity regulated pentraxin (Narp), cysteine rich protein 61 (Cyr61), Bcl-2 binding protein BIS (Bcl-2-interacting death suppressor), and lectin-like ox-LDL receptor (LOX-1). We demonstrated differential expression of each gene by quantitative PCR analysis, and in the case of LOX-1, we further confirmed differential expression by in situ hybridization. LOX-1 expression is induced greater than ten fold at the core lesion site, and is essentially localized to the ipsilateral half of the brain. LOX-1 appears to be expressed in a non-neuronal cell type, and it does not appear to be expressed in vascular endothelial cells within the brain. This suggests that LOX-1 may serve a novel function in the brain.

Differential kinetics of hypocretins in the cerebrospinal fluid after intracerebroventricular administration in rats

Different potencies for hypocretin-1 and -2 in sleep-wake regulation and feeding after intracerebroventricular (ICV) administration have been reported. These differences were often explained by the selectivity of the two hypocretins for hypocretin receptor-1 and -2, but little attention has been paid to kinetics of hypocretin peptides. We investigated the kinetics of the ICV hypocretin-1 and -2 in rats. ICV hypocretin-1 (10 nmol) increased hypocretin-1 peptide level in the CSF by 800-fold from baseline with the elevation lasting over 4 h. In contrast, after ICV hypocretin-2 (10 nmol), no significant rise in the CSF was found. CSF hypocretin levels were significantly correlated with the biological activities of CSF hypocretin-1 and -2 using the Ca(2+) mobilization assay. Difference in the kinetics of hypocretins should be considered for interpreting ICV effects of hypocretins.

Cutting Edge: Diabetes-Associated Quantitative Trait Locus, Idd4, Is Responsible for the IL-12p40 Overexpression Defect in Nonobese Diabetic (NOD) Mice

APCs of the nonobese diabetic (NOD) mouse have a genetically programmed capacity to overexpress IL-12p40, a cytokine critical for development of pathogenic autoreactive Th1 cells. To determine whether a diabetes-associated NOD chromosomal locus (i.e., Idd) was responsible for this defect, LPS-stimulated macrophages from several recombinant congenic inbred mice with Idd loci on a C57BL/6 background or with different combinations of NOD and CBA genomic segments were screened for IL-12p40 production. Only macrophages from the congenic strains containing the Idd4 locus showed IL-12p40 overproduction/expression. Moreover, analysis of IL-12p40 sequence polymorphisms demonstrated that the Idd4 intervals in these strains contained the IL-12p40 allele of the NOD, although further analysis is required to determine whether the IL-12p40 allele itself is responsible for its overexpression. Thus, the non-MHC-associated Idd4 locus appears responsible for IL-12p40 overexpression, which may be a predisposing factor for type 1 diabetes in NOD mice.

Manipulation of small-molecule inhibitory kinetics modulates MCH-R1 function

The capacity of novel benzopyridazinone-based antagonists to inhibit MCH-R1 function, relative to their affinity for the receptor, has been investigated. Three compounds that differ by the addition of either a chlorine atom, or trifluoromethyl group, have nearly identical receptor affinities; however their abilities to inhibit receptor elicited signaling events, measured as a function of time, are dramatically altered. Both the chlorinated and trifluoromethyl modified compounds have a very slow on-rate to maximal functional inhibition relative to the unmodified base compound. A similar impact on inhibitory capacity can be achieved by modifying the side-chain composition at position 2.53 of the receptor; replacement of the native phenylalanine with alanine significantly reduces the amount of time required by the chlorinated compound to attain maximal functional inhibition. The primary attribute responsible for this alteration in inhibitory capacity appears to be the overall bulk of the amino acid at this position-substitution of the similarly sized amino acids leucine and tyrosine results in phenotypes that are indistinguishable from the wild type receptor. Finally, the impact of these differential inhibitory kinetics has been examined in cultured rat neurons by measuring the ability of the compounds to reverse MCH mediated inhibition of calcium currents. As observed using the cell expression models, the chlorinated compound has a diminished capacity to interfere with receptor function. Collectively, these data suggest that differential inhibitory on rates between a small-molecule antagonist and its target receptor can impact the ability of the compound to modify the biological response(s) elicited by the receptor.

Pharmacology of Hypocretin/Orexin Peptides and Small Molecules

Despite their relatively recent discovery in 1998, the hypocretins (also known as orexins) and their receptors are already the focus of several investigations as sites for therapeutic intervention in a number of endocrinological and neurological disorders. The rapidity with which the hypocretin system has been adopted as a high-interest target is mainly the result of an accumulation of compelling evidence from in vivo studies showing that the hypocretins regulate a number of aspects of physiology and behavior, especially those involved in sleep, arousal, and energy homeostasis. High-throughput screening efforts by a number of pharmaceutical companies have now identified novel small molecules that interact potently and specifically with the hypocretin receptors. Despite these considerable efforts, very little is known regarding the structures of the receptors, their endogenous ligands, the molecular basis of their interactions, or the signaling pathways they use. For example, no attempt has been made through receptor mutagenesis, or by any other means, to define the key interactions that occur between the receptors and the endogenous peptide ligands. Although such studies are likely to be ongoing within pharmaceutical companies with active hypocretin receptor drug discovery programs, the information available to the general scientific community remains very limited. This chapter reviews what is currently known about the molecular pharmacology of this system, focusing on the structures and activities of the peptides and some of the small molecules for which published biological data exist.