Hardy W. Chan

The pharmacology and distribution of human 5‐hydroxytryptamine2B (5‐HT2b) receptor gene products: comparison with 5‐HT2a and 5‐HT2c receptors

1 Full length clones of the human 5‐HT2B receptor were isolated from human liver, kidney and pancreas. The cloned human 5‐HT2B receptors had a high degree of homology (∼80%) with the rat and mouse 5‐HT2B receptors. 2 PCR amplification was used to determine the tissue distribution of human 5‐HT2B receptor mRNA. mRNA encoding the 5‐HT2B receptor was expressed with greatest abundance in human liver and kidney. Lower levels of expression were detected in cerebral cortex, whole brain, pancreas and spleen. Expression was not detected in heart. 3 Northern blot analysis confirmed the presence of 5‐HT2B receptor mRNA (a 2.4 kB sized band) in pancreas, liver and kidney. An additional 3.2 kB sized band of hybridization was detected in liver and kidney. This raises the possibility of a splice variant of the receptor or the presence of an additional homologous receptor. 4 The human 5‐HT2B receptor was expressed in Cos‐7 cells and its ligand binding characteristics were compared to similarly expressed human 5‐HT2A and 5‐HT2C receptors. The ligand specificity of the human 5‐HT2B receptor (5‐HT > ritanserin > SB 204741 > spiperone) was distinct from that of the human 5‐HT2A (ritanserin > spiperone > 5‐HT > SB 204741) and 5‐HT2C (ritanserin > 5‐HT > spiperone = SB 204741) receptors. On the basis of a higher affinity for ketanserin and a lower affinity for yohimbine the human 5‐HT2B receptor also appeared to differ from the rat 5‐HT2B receptor. 5 These findings confirm the sequence of the human 5‐HT2B receptor and they demonstrate that the receptor has a widespread tissue distribution. In addition, these data suggest that there are differences in ligand affinities between different species homologues of the receptor. Finally, the finding of two distinct bands on the Northern blots of liver and kidney raises the possibility of splice variants or subtypes of 5‐HT2B receptors, within these tissues.

Degeneration of vascular muscle cells in cerebral amyloid angiopathy of Alzheimer disease

In cerebral amyloid angiopathy, the amyloid-beta (A beta) deposits lie primarily in the tunica media suggesting that smooth muscle cells play an important role in A beta deposition. To define this role, we conducted an immunocytochemical study of brain tissue from cases of Alzheimer disease with extensive cerebral amyloid angiopathy and cerebral hemorrhage. Antibodies specific to recombinant beta protein precursor (beta PP) and synthetic peptides homologous to various beta PP sequences from residue 18 to 689 of beta PP695 were used. Antibodies to actin, tropomyosin, alpha-actinin or desmin were used to label muscle cells. Antibodies to A beta sequences intensely recognized the extracellular amyloid deposit. Antibodies raised against beta PP sequences other than the A beta domain recognized smooth muscle cells. beta PP-immunoreactivity was reduced in regions of A beta deposits, since no muscle cells were recognized by cytoskeletal markers or observed ultrastructurally. In order to assess why A beta is deposited in the tunica media, we used biotin-labelled beta PP to determine if beta PP can be locally retained. We found beta PP bound to the tunica media of vessels but not other brain elements. These findings suggest A beta in blood vessels derives from degenerating beta PP-containing smooth muscle cells.

Cloning and expression of a 5-hydroxytryptamine7 receptor positively coupled to adenylyl cyclase.

Abstract: A cDNA clone (designated as GP2‐7) encoding a novel 5‐hydroxytryptamine (5‐HT) receptor was isolated from a guinea pig hippocampal library. The receptor shares amino acid homology within the hydrophobic domains with other cloned 5‐HT receptor subtypes (34–48%). The sequence of GP2‐7 is homologous to that described for a novel receptor previously cloned from a rat brain cDNA library and provisionally designated as 5‐HT7. mRNA for GP2‐7 was detected in cortical and limbic brain regions. Transiently expressed GP2‐7 showed high‐affinity binding to [3H]5‐HT (pKi = 9.0) with the following rank order of affinities: 5‐carboxyamidotryptamine (5‐CT) > 5‐HT = 5‐methoxytryptamine (5‐MeOT) > methiothepin > 8‐hydroxy‐2‐(dipropylamino)tetralin (8‐OH‐DPAT) > spiperone ≫ sumatriptan. Adenylyl cyclase activity in CHO‐K1 cells transiently transfected with GP2‐7 was stimulated by several analogues of 5‐HT with the following order of potency: 5‐CT > 5‐HT = 5‐MeOT > dipropyl‐5‐CT > 8‐OH‐DPAT. Methiothepin and spiperone were potent antagonists. Preliminary analysis suggests that GP2‐7 closely resembles a receptor in the guinea pig hippocampus that exhibits a high affinity toward 5‐CT.

Accumulation of the β amyloid precursor protein at sites of ischemic injury in rat brain

We used various antibodies to the beta amyloid precursor protein (APP) of Alzheimers disease to study changes in the cellular distribution of APP in experimental ischemic brain injury. In contrast to sham operated controls, rats with repeated reversible occlusions of one middle cerebral artery showed striking APP reactivity in astrocytic processes in perifocal regions and white matter tracts. Dystrophic axons and neurons with accumulated APP were also evident in the ipsilateral neocortex and hippocampus. Such changes were also apparent in rats subjected to partial forebrain ischemia by bilateral occlusion of the carotid arteries. Our studies suggest that focal ischemic insults or chronic hypoperfusion leads to increased accumulation of APP in surviving brain cells that may pertain to enhanced beta amyloid deposition in Alzheimers disease.

Soluble derivatives of the β amyloid protein precursor of Alzheimer's disease are labeled by antisera to the β amyloid protein

Abstract The amyloid deposited in Alzheimers disease (AD) is composed primarily of a 39–42 residue polypeptide (βAP) that is derived from a larger β amyloid protein precursor (βAPP). In previous studies, we and others identified full-length, membrane-associated forms of the βAPP and showed that these forms are processed into soluble derivatives that lack the carboxyl-terminus of the full-length forms. In this report, we demonstrate that the soluble ∼125 and ∼105 kDa forms of the βAPP found in human cerebrospinal fluid are specifically labeled by several different antisera to the βAP. This finding indicates that both soluble derivatives contain all or part of the βAP sequence, and it suggests that one or both of these forms may be the immediate precursor of the amyloid deposited in AD.

Molecular cloning, genomic characterization and expression of novel human α1A-adrenoceptor isoforms

We have isolated and characterized from human prostate novel splice variants of the human α1A‐adrenoceptor, several of which generate truncated products and one isoform, α1A‐4, which has the identical splice site as the three previously described isoforms. Long‐PCR on human genomic DNA showed that the α1A‐4 exon is located between those encoding the α1A‐1 and α1A‐3 variants. CHO‐K1 cells stably expressing α1A‐4 showed ligand binding properties similar to those of the other functional isoforms as well as agonist‐stimulated inositol phosphate accumulation. Quantitative PCR analyses revealed that α1A‐4 is the most abundant isoform expressed in the prostate with high levels also detected in liver and heart.

Accumulation of amyloid precursor fragment in Alzheimer plaques

Regenerative and degenerative neurites are components of classical senile plaques found in brain tissue of patients with Alzheimers disease (AD). Amyloid beta/A4-protein derived from its precursor, amyloid beta/A4-protein precursor (APP/ABPP), constitutes the major portion of the amyloid core of senile plaques. A large N-terminal portion of APP (approximately Mr 100,000) is released from cells, leaving a minor C-terminal portion (approximately Mr 15,000) behind. A series of antisera against various sequences of APP were prepared and used to study the localization of each sequence in brain tissue. Plaque neurites stained as intensely as neuronal cell bodies with three antisera against the N-terminal portion of APP (N-terminal to a.a. 225), whereas five other antisera directed against the other C-terminal portions of APP (a.a. 284 to C-terminal) and antisera against the Kunitz-type protease inhibitor portion of APP stained plaque neurites less intensely than neuronal cell bodies in the hippocampus. These results suggest that a major part of the APP present in the neuritic component of senile plaques is a fragment representing the N-terminal one-third of the molecule.

Human β nerve growth factor obtained from a baculovirus expression system has potent in vitro and in vivo neurotrophic activity

A baculovirus expression vector, which contains the coding sequences for human prepro (beta) nerve growth factor under control of the viral polyhedrin promoter, was constructed. Upon infection of insect cells with the recombinant virus, mature human beta nerve growth factor (rhNGF) was released into the culture fluid. The mature rhNGF was biologically active since rat pheochromocytoma (PC12) and human neuroblastoma (SH-SY5Y) cells were induced to extend neurites upon treatment with this material. This activity was abolished by treating with antiserum prepared against mature mouse beta NGF (mNGF). When compared with mNGF, rhNGF more rapidly elicited the differentiation response in both PC12 and SH-SY5Y cells. In an in vivo assay of cholinergic cell survival, rhNGF was nearly as potent as mNGF in protecting cholinergic neurons from degeneration following a fimbria-fornix lesion. These results show that the baculovirus expression system provides quantities of biologically potent human beta NGF suitable for a comprehensive program of research to ascertain beta NGFs potential as a therapeutic agent for the treatment of Alzheimers disease.

Amyloid precursor protein (APP) in the striatum in Alzheimer's disease: an immunohistochemical study.

Increasing recongnition of diffuse plaques has raised questions about the differences between diffuse and neuritic plaques, particularly in regard to the role of amyloid precursor protein (APP) processing in their formation. To address this issue, corpus striatum (containing almost exclusively diffuse plaques) and cerebral cortex (containing an admixture of plaque types) from patients with Alzheimers disease (AD) were examined immunihistochemically with antibodies to damin-specific sites of APP (N-terminal, C-terminal, ßA4-related, isoform-specific, and other epitopes). Striatal plaques labeled strongly with ßA4 antibodies as did cortical plaques in AD and the occasional diffuse plaques in cortex from nondemented elderly controls. Weak labelling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against controls. Weak labeling of some cortical neuritic plaques but not diffuse plaques was observed with antibodies directed against other APP epitopes. Electron microscopy of diffuse plaque-rich striatum in AD cases revealed onlly rare degenerating neurites without apparent fibrillar amyloid; no changes were noted in the plaque-free striatum of controls. These results suggest that antibodies to ßA4 recognize not only fibrillar amyloid of neuritic plaques but also antigenic determinants of diffuse plaques which lack fibrillar amyloid. Furthermore, the finding that antibodies to non-A4 domains of APP labeled onlyl cortical but not striatal plaques suggests that APP processing mechanisms in cortical and striatal tissues may differ.

Synthetic HIV-2 protease cleaves the GAG precursor of HIV-1 with the same specificity as HIV-1 protease

A 99-amino acid protein having the deduced sequence of the protease from human immunodeficiency virus type 2 (HIV-2) was synthesized by the solid phase method and tested for specificity. The folded peptide catalyzes specific processing of a recombinant 43-kDa GAG precursor protein (F-16) of HIV-1. Although the protease of HIV-2 shares only 48% amino acid identity with that of HIV-1, the HIV-2 enzyme exhibits the same specificity toward the HIV-1 GAG precursor. Fragments of 34, 32, 24, 10, and 9 kDa were generated from F-16 GAG incubated with the protease. N-terminal amino acid sequence analysis of proteolytic fragments indicate that cleavage sites recognized by HIV-2 protease are identical to those of HIV-1 protease. The verified cleavage sites in F-16 GAG appear to be processed independently, as indicated by the formation of the intermediate fragments P32 and P34 in nearly equal ratios. The site nearest the amino terminus is quite conserved between the two viral GAG proteins (...VSQNY-PIVQN...in HIV-1,...KGGNY-PVQHV...in HIV-2). In contrast, the putative second site (...IPFAA-AQQKG...) of HIV-2 GAG shares minimal sequence identity with site 2 of HIV-1 GAG (...SATIM-MQRGN...). These sequence variations in the substrates suggest higher order structural features that may influence recognition by the proteases. Pepstatin A inhibits HIV-2 protease, whereas 1,10-phenanthroline and phenylmethylsulfonylfluoride do not; these results are in agreement with the finding that proteases of HIV and other retroviruses are aspartyl proteases.