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H B Pollard

Publications and source records attributed to H B Pollard.

At least 19 recordsLinked to original sources
PubMed1995

Effect of MPTP on dopaminergic neurons in the goldfish brain: a light and electron microscope study.

The neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) causes a Parkinsonian syndrome in the goldfish (Carassius auratus), characterized by transient bradykinesia, the accumulation of MPP+ in the brain, and a decrease in the forebrain and midbrain content of catecholamines (Pollard et al., FASEB J., 6 (1992) 3108-3116). Using light and electron microscopy, we studied the effect of MPTP on the distribution and ultrastructure of tyrosine hydroxylase (TH)-immunoreactive, dopaminergic neurons, and on the ultrastructure of other selected areas of the goldfish brain. Goldfish were treated with MPTP (50 mg/kg) in the absence or presence of L-deprenyl (10 mg/kg) or clorgyline (10 mg/kg). In the medial part of the central telencephalon, the nucleus telencephali, pars medialis, MPTP caused a decrease in the number of TH-immunoreactive neurons and distortions in their labelling pattern. Electron microscopic observations showed that MPTP caused swelling of cell processes, changes in neuronal nuclear profiles, dilation of endoplasmic reticulum, intracellular vacuolization and membrane distortions, and degeneration of neuronal fibers in this brain area. MPTP also caused a small reduction and some diffuseness in the labelling of dopaminergic neurons in several diencephalic periventricular nuclei. Moreover, MPTP induced cell swelling and degeneration in the subependymal cell layers along the forebrain ventricles. In all areas, L-deprenyl appeared to partially prevent the MPTP-induced degenerative changes. We conclude that in the goldfish MPTP causes marked histochemical changes in selected dopaminergic brain systems coincident with the Parkinson-like locomotor and neurochemical deficits.

Animals↗
PubMed1995

A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation.

Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulates 36Cl- efflux from pancreatic CFPAC-1 cells which bear the delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36Cl- efflux from cells expressing the CFTR (delta F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR (delta F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36Cl- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3-dimethylxanthine),3,4-dehydro-CPX,3-F-CPX,3-1-CPX, and KW-3902 (8-noradamantyl-1,3-dipropylxanthine).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Stimulation by alkylxanthines of chloride efflux in CFPAC-1 cells does not involve A1 adenosine receptors.

A series of 8-substituted derivatives of 1,3,7-alkylxanthines was synthesized as potential activators of chloride efflux from a human epithelial cell line (CFPAC) expressing the cystic fibrosis transmembrane regulator (CFTR) delta F508 mutation. Their interactions with rat brain A1 and A2a receptors were also studied in radioligand binding experiments. Substitution was varied at the xanthine 1-, 3-, 7- and 8-positions. 1,3-Dipropyl-8-cyclopentylxanthine (CPX) stimulated Cl- efflux in the 10(-8) M range, with a maximal effect reaching 200% of control and diminishing at higher concentrations. The potent adenosine antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]- 1,3-dipropylxanthine, nonselective at human A1 and A2a receptors, was inactive in Cl- efflux. 1,3-Diallyl-8-cyclohexylxanthine (DAX) was highly efficacious in stimulating chloride efflux with levels reaching > 300% of control, although micromolar concentrations were required. 1,3,7-Trimethyl-8-(3-chlorostyryl)xanthine, an A2a-selective adenosine antagonist, was only weakly active. Caffeine, which acts as an nonselective adenosine antagonist in the range of 10(-5) M, was active in Cl- efflux in the low nanomolar range but with low efficacy. Thus, among the xanthine derivatives of diverse structure, there was no correlation between potency in Cl- efflux and adenosine antagonism. Poly(A)+ RNA isolated from CFPAC-1 cells showed no hybridization to a human A1 receptor cDNA probe, excluding this receptor as a mediator of CPX-elicited Cl- efflux. Thus, this action of xanthines in stimulating Cl- efflux in CFPAC cells, which express a defective CFTR, represents a novel site of action apparently unrelated to adenosine receptors.

Animals↗
PubMed1995

Effect of protein synthesis inhibitors on synexin levels and secretory response in bovine adrenal medullary chromaffin cells.

The effects of the protein synthesis inhibitors actinomycin D and cycloheximide on the cellular content of the calcium binding protein synexin, and on the secretory response of cultured bovine adrenal medullary chromaffin cells were determined. Both protein synthesis inhibitors produced a slow decrease in the cellular synexin content. The synexin level was reduced by 50% after 133 h of incubation in the presence of 2 micrograms/ml actinomycin D or 5 micrograms/ml cycloheximide. However, this was partly due to an artefactual stabilization of synexin, since metabolic labelling of synexin with [35S]methionine showed that the half-time of degradation was only 40 h. The secretory response of chromaffin cells was quickly diminished in the presence of protein synthesis inhibitors. Catecholamine secretion induced by membrane depolarization or barium stimulation of intact cells, or by calcium stimulation of digitonin-permeabilized cells was decreased by 77-82% after 24 h of incubation in the presence of 5 micrograms/ml cycloheximide. These results suggest that, in addition to synexin, at least one or more proteins with a shorter half-time of degradation than synexin are involved in the secretory response of adrenal chromaffin cells.

Adrenal Medulla↗
PubMed1995

CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells.

The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.

Calcium↗
PubMed1994

beta-Amyloid Ca(2+)-channel hypothesis for neuronal death in Alzheimer disease.

The Alzheimer's Disease (AD) amyloid protein (A beta P[1-40]) forms cation selective channels when incorporated into planar lipid bilayers by fusion with liposomes containing the peptide. Since the peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, we also tested the possibility of direct incorporation of the soluble A beta P[1-40] into the membrane. We found the peptide can also form similar channels in acidic phospholipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome mediated incorporation, the A beta P[1-40]-channel in the solvent-free membrane patch exhibits multiple cation selectivity (Cs+ > Li+ > Ca2+ > or = K+), and sensitivity to tromethamine. The fact that equivalent A beta P[1-40] amyloid channels can be detected by two different methods thus provides additional validation of our original observation. Further studies with a beta P-channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective but loses its tromethamine sensitivity. By contrast, amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would disrupt cellular homeostasis. We therefore interpret these data as providing further support for our beta-amyloid Ca(2+)-channel hypothesis for neuronal death in Alzheimer's Disease.

Alzheimer Disease↗
PubMed1994

Human cytochrome b561: a revised hypothesis for conformation in membranes which reconciles sequence and functional information.

Cytochrome b561 is a major transmembrane protein of catecholamine and neuropeptide secretory vesicles. In this report, we describe the cloning and properties of a full-length cDNA encoding human neuroendocrine cytochrome b561 from a human caudate cDNA library and a human peripheral blood genomic library. The human cDNA contains two major transcription start sites and only one translation start site that codes for an apocytochrome b561, which is 22 amino acid residues smaller than the previously deduced amino acid sequence from bovine cDNA. This smaller version of cytochrome b561 may contain only five transmembrane segments rather than the previously proposed six segments. The new model is in agreement with our previous results on transmembrane topology of the gene product. Northern-blot analysis shows an expanded tissue distribution of cytochrome mRNA expression where previous immunological assays were negative. These results support the hypothesis that cytochrome b561 is a marker for peptidergic and adrenergic tissues.

Amino Acid Sequence↗
PubMed1994

Genomic organization and chromosomal localization of the mouse synexin gene.

We have isolated and characterized the gene encoding mouse synexin, which consists of 14 exons and spans approximately 30 kbp of genomic DNA. The protein's unique N-terminal domain is encoded by six exons, and the C-terminal tetrad repeat, the site of the membrane-fusion and ion-channel domain, is encoded by seven exons. The first exon encodes the 5'-untranslated region. Analysis of synexin-gene expression in different mouse tissues shows that mRNA with exon 6 is only present in brain, heart and skeletal muscle. mRNA lacking exon 6 is expressed in all tissues we have examined. The initiation site for transcription was determined by primer-extension analysis and S1 nuclease mapping. Sequence analysis of the 1.3 kb 5'-flanking region revealed that the promoter has a TATA box located at position -25 and a number of potential promoter and regulatory elements. A CCAAT motif was not observed but CCATT is located in an appropriate position for the CCAAT motif upstream from the transcription-initiation start site. In addition, the 5'-flanking region contains two sets of palindromic sequences. Finally, we have determined that the functional synexin gene (Anx7) is located on mouse chromosome 14 and that a pseudogene (Anx7-ps1) is located on chromosome 10.

3T3 Cells↗
PubMed1994

Divergent structure of the human synexin (annexin VII) gene and assignment to chromosome 10.

The human synexin (annexin VII) gene occurs as a single copy at chromosome 10q21.1-21.2 and substantially deviates in size and in the location of splice junctions from the other two well-characterized members of the annexin gene family, lipocortin I (annexin I) and calpactin I (annexin II). The synexin gene contains 14 exons, including an alternatively spliced cassette exon, and spans approximately 34 kb of DNA. Only five of the fourteen splice junctions are conserved compared to other annexins, and the differences are particularly pronounced in the exons that encode the C-terminal third and fourth conserved repeats in the gene product. Although parallels between exons and protein domains were not apparent, we did observe clustering of splice junctions corresponding to either the unique N-terminal domain or the conserved C-terminal tetrad repeat domain, which is common to all annexins. Furthermore, a complete analysis of the 5' flanking region of the annexin VII gene revealed an entirely different set of cis-acting and enhancer elements compared to other annexin genes. We conclude that the annexin VII gene may have arisen by a divergence from the evolutionary pathway taken by both annexins I and II.

Amino Acid Sequence↗
PubMed1994

Agonist-stimulated release of von Willebrand factor and procoagulant factor VIII in rats with and without risk factors for stroke.

Lipopolysaccharide (LPS)-induced (i.v. or i.c.v., 1.8 mg/kg) release of von Willebrand factor (vWF) was examined in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. SHR rats released significantly (P < 0.05) more vWF than WKY rats in response to LPS. LPS also inhibited factor VIII procoagulant activity (FVIII:c) which may indicate an increase in thrombin activity. Cultured cerebrovascular endothelial cells (EC) derived from both SHR and WKY rats, as well as human umbilical vein EC (HUVEC) cultures constitutively released vWF. Treatment with agonists including LPS, thrombin and tumor necrosis factor-alpha (TNF alpha) did not affect the in vitro secretion of vWF by cerebrovascular EC cultures but significantly upregulated vWF release by HUVEC cultures. Preincubation of cerebrovascular EC cultures with interleukin-1 (IL-1) +/- TNF alpha or co-culturing in the presence of LPS-activated syngeneic monocytes had no effect on vWF secretion. The findings demonstrate that conditions of hypertension may affect endothelial cells and make them more responsive to agonist stimulation and thereby increase secretion of vWF, an important factor in hemostasis as well as thrombosis. The capacity of LPS to significantly affect the in vivo secretion of vWF in SHR and WKY rats but not cultured cerebrovascular EC indicates that observed elevations in plasma vWF were not derived from cerebrovascular EC. It is suggested that hypertension may function as a risk factor for thrombotic stroke by influencing factors involved in coagulation processes, such as vWF and factor VIII:c.

Animals↗
PubMed1994

A geometric sequence that accurately describes allowed multiple conductance levels of ion channels: the "three-halves (3/2) rule".

Ion channels can express multiple conductance levels that are not integer multiples of some unitary conductance, and that interconvert among one another. We report here that for 26 different types of multiple conductance channels, all allowed conductance levels can be calculated accurately using the geometric sequence gn = g(o) (3/2)n, where gn is a conductance level and n is an integer > or = 0. We refer to this relationship as the "3/2 Rule," because the value of any term in the sequence of conductances (gn) can be calculated as 3/2 times the value of the preceding term (gn-1). The experimentally determined average value for "3/2" is 1.491 +/- 0.095 (sample size = 37, average +/- SD). We also verify the choice of a 3/2 ratio on the basis of error analysis over the range of ratio values between 1.1 and 2.0. In an independent analysis using Marquardt's algorithm, we further verified the 3/2 ratio and the assignment of specific conductances to specific terms in the geometric sequence. Thus, irrespective of the open time probability, the allowed conductance levels of these channels can be described accurately to within approximately 6%. We anticipate that the "3/2 Rule" will simplify description of multiple conductance channels in a wide variety of biological systems and provide an organizing principle for channel heterogeneity and differential effects of channel blockers.

Animals↗
PubMed1994

Theoretical models of the ion channel structure of amyloid beta-protein.

Theoretical methods are used to develop models for the ion channel structure of the membrane-bound amyloid beta-protein. This follows recent observations that the beta-protein forms cation-selective channels in lipid bilayers in vitro. Amyloid beta-protein is the main component of the extracellular plaques in the brain that are characteristic of Alzheimer's disease. Based on the amino acid sequence and the unique environment of the membrane, the secondary structure of the 40-residue beta-protein is predicted to form a beta-hairpin followed by a helix-turn-helix motif. The channel structures were-designed as aggregates of peptide subunits in identical conformations. Three types of models were developed that are distinguished by whether the pore is formed by the beta-hairpins, the middle helices, or by the more hydrophobic C-terminal helices. The latter two types can be converted back and forth by a simple conformational change, which would explain the variable conduction states observed for a single channel. It is also demonstrated how lipid headgroups could be incorporated into the pore lining, and thus affect the ion selectivity. The atomic-scale detail of the models make them useful for designing experiments to determine the real structure of the channel, and thus further the understanding of peptide channels in general. In addition, if beta-protein-induced channel activity is found to be the cause of cell death in Alzheimer's disease, then the models may be helpful in designing counteracting drugs.

Alzheimer Disease↗
PubMed1993

Giant multilevel cation channels formed by Alzheimer disease amyloid beta-protein [A beta P-(1-40)] in bilayer membranes.

We have recently shown that the Alzheimer disease 40-residue amyloid beta-protein [A beta P-(1-40)] can form cation-selective channels when incorporated into planar lipid bilayers by fusion of liposomes containing the peptide. Since A beta P-(1-40) comprises portions of the putative extracellular and membrane-spanning domains of the amyloid precursor protein (APP751), we suggested that the channel-forming property could be the underlying cause of amyloid neurotoxicity. The peptide has been proposed to occur in vivo in both membrane-bound and soluble forms, and we now report that soluble A beta P-(1-40) can also form similar channels in solvent-free lipid bilayers formed at the tip of a patch pipet, as well as in the planar lipid bilayer system. As in the case of liposome-mediated incorporation, the amyloid channel activity in the patch pipet exhibits multiple conductance levels between 40 and 400 pS, cation selectivity, and sensitivity to tromethamine (Tris). Further studies with A beta P channels incorporated into planar lipid bilayers from the liposome complex have also revealed that the channel activity can express spontaneous transitions to a much higher range of conductances between 400 and 4000 pS. Under these conditions, the amyloid channel continues to be cation selective. Amyloid channels were insensitive to nitrendipine at either conductance range. We calculate that if such channels were expressed in cells, the ensuing ion fluxes down their electrochemical potential gradients would be homeostatically dissipative. We therefore interpret these data as providing further support for the concept that cell death in Alzheimer disease may be due to amyloid ion-channel activity.

Amyloid beta-Peptides↗
PubMed1993

A new hypothesis for the mechanism of amyloid toxicity, based on the calcium channel activity of amyloid beta protein (A beta P) in phospholipid bilayer membranes.

Amyloid beta protein (A beta P) is the 40-42 residue polypeptide implicated in the pathogenesis of Alzheimer's disease (AD). We have reconstituted this peptide into phosphatidylserine liposomes and then fused the liposomes with a planar lipid bilayer. When incorporated into this bilayer, the A beta P forms cation selective channels capable of transporting calcium and some monovalent cations including cesium, lithium, potassium, and sodium. The channels behave in an ohmic fashion and single channels can be shown to exhibit multiple subconductance states. Hitherto, A beta P has been presumed to be neurotoxic, although direct demonstration of toxicity has proved elusive. On the basis of the present data we suggest that the ion channel activity of the polypeptide may be the basis of its neurotoxic effects.

Alum Compounds↗
PubMed1993

L-deprenyl confers specific protection against MPTP-induced Parkinson's disease-like movement disorder in the goldfish.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to the goldfish causes a reversible, Parkinson's disease-like syndrome which includes loss of noradrenaline and dopamine from the brain, accumulation of the toxic metabolite 1-methyl-4-phenylpyridinium species (MPP+), and substantial reduction in movement. L-Deprenyl, a selective monoamine oxidase-B inhibitor, protects the goldfish from loss of movement, but clorgyline, a selective monoamine oxidase-A inhibitor, has no such protective action. L-Deprenyl and clorgyline primarily inhibit goldfish brain monoamine oxidase-B and monoamine oxidase-A, respectively. The mechanism by which MPTP causes reduced movement in goldfish is to cause an increase in resting time. Otherwise normal average velocity occurred during periods of movement. L-Deprenyl protection results in entirely 'normal' levels of resting time and average velocity during times of movement. Equivalent observations regarding l-deprenyl and clorgyline have been made in primate models of MPTP toxicity, and l-deprenyl is used for treatment of Parkinson's disease in humans. Therefore it is suggested that the evolutionarily equivalent subcortical circuitry and neural density of the goldfish brain may provide a useful model upon which to search for drugs relevant to human Parkinson's disease.

Animals↗
PubMed1993

Mouse synexin (annexin VII) polymorphisms and a phylogenetic comparison with other synexins.

Two sets of cDNAs encoding mouse synexin were isolated from a liver cDNA library and sequenced. The coding regions of synexin clones show 99% identity. By contrast, the two mouse synexin cDNAs differ in a number of ways in both 5' and 3' non-coding regions. The two sets of cDNA encode a polypeptide of 463 amino acid residues which has a deduced molecular mass of 50 kDa. The amino acid sequence of mouse synexin shows a high degree of similarity to both the unique N-terminal domain and the highly conserved C-terminal domain of previously cloned human synexin. Northern-blot analysis using mouse liver polyadenylated RNA revealed two transcripts of 1.8 kb and 2.6 kb, corresponding to group I and group II respectively. Further hybridization analysis using specific sequences from each set of clones showed that the two sizes of mRNAs differ in the length of the 3' non-coding region which corresponded to the cDNAs. Both mouse liver synexin and recombinant mouse synexin expressed in Escherichia coli reacted after Western-blot analysis with a goat antibody against bovine synexin. Only in the larger group-II cDNAs do we find point mutations leading to amino acid replacements of Ser to Ala at residue 145 in the unique N-terminal domain, and of Ala to Gly at residue 304 in the transition zone between repeats II and III. We conclude from a comparison of mouse, human and Dictyostelium synexins that changes occur predominantly in the hydrophobic N-terminal domain, or, in the C-terminal region at the ends of some predicted alpha-helices, on the hydrophobic face of the amphipathic C-helices, and within a lengthy non-helical domain connecting major repeats II and III.

Amino Acid Sequence↗