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1.
Virology ; 348(1): 233-41, 2006 Apr 25.
Article in English | MEDLINE | ID: mdl-16448680

ABSTRACT

The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U(L)4, U(L)29, U(L)30, and U(L)31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.


Subject(s)
Gene Expression Profiling , Herpesvirus 1, Human/genetics , Herpesvirus 2, Human/genetics , Oligonucleotide Array Sequence Analysis , Transcription, Genetic , Animals , Biological Transport/genetics , Blotting, Northern , Herpesvirus 1, Human/growth & development , Herpesvirus 2, Human/growth & development , Kinetics , Mice , NIH 3T3 Cells , Nucleic Acid Hybridization , RNA, Messenger/analysis , RNA, Viral/analysis , Time Factors , Viral Proteins/genetics
2.
Methods Mol Biol ; 292: 423-48, 2005.
Article in English | MEDLINE | ID: mdl-15507724

ABSTRACT

The design and construction of a long (75-mer) oligonucleotide-based DNA microarray for herpes simplex virus type 2 transcripts is described. This array is utilized to generate an analysis of HSV-2 transcript abundance as a function of conditions of infection of human cells, and global patterns of HSV-2 transcript abundance are compared with those for HSV-1. General similarities in patterns along with notable differences in specific details are noted. These results reveal a marked conservation in the program of gene activity between phenotypically diverged strains.


Subject(s)
Herpes Genitalis/metabolism , Herpesvirus 2, Human/genetics , Oligonucleotide Array Sequence Analysis/methods , Chromosome Mapping , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Herpesvirus 2, Human/metabolism , Humans , RNA, Messenger/classification , RNA, Messenger/metabolism , Transcription, Genetic
3.
Virology ; 329(1): 142-56, 2004 Nov 10.
Article in English | MEDLINE | ID: mdl-15476882

ABSTRACT

To investigate the impact of stress kinase p38 activation on HSV-1 transcription, we performed a global transcript profile analysis of viral mRNA using an oligonucleotide-based DNA microarray. RNA was isolated from Vero cells infected with the KOS strain of HSV-1 in the presence or absence of SB203580, a pyridinyl imidazole inhibitor of p38. Under conditions that eliminated ATF2 activation but had no effect on c-Jun, and reduced virus yield by 85-90%, no effect on accumulation of viral IE, DE, or L transcripts was observed by array analysis or selected Northern blot analysis at 2, 4, and 6 h post infection. Results of array data from cells infected with the ICP27 mutant d27-1 in the presence or absence of SB203580 only reflected the known restricted transcription phenotype of the ICP27 mutant. This result is consistent with a role for p38 activation on virus replication lying downstream of the essential role of ICP27 in DE and perhaps late transcription regulation. No effect of SB203580 on transcription was detected after infection with the ICP0 mutant 7134, at 0.5 or 5.0 PFU/cell, though decreases in the rate of accumulation of all kinetic classes of mRNA could be detected, relative to wt virus. These results indicate that inhibiting p38 activity in Vero cells, while significantly reducing wt virus yield, demonstrated no obvious impact on the program of viral transcription.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Viral , Herpesvirus 1, Human/pathogenicity , Transcription, Genetic , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Animals , Chlorocebus aethiops , Enzyme Inhibitors/pharmacology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Imidazoles/pharmacology , Oligonucleotide Array Sequence Analysis , Pyridines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Viral/genetics , RNA, Viral/metabolism , Vero Cells
4.
J Virol ; 78(10): 5038-44, 2004 May.
Article in English | MEDLINE | ID: mdl-15113885

ABSTRACT

The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17DeltaPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.


Subject(s)
Herpesvirus 1, Human/physiology , Keratitis, Herpetic/virology , Viral Proteins/physiology , Virus Latency , Animals , Cornea/virology , DNA, Viral/analysis , Disease Models, Animal , Genotype , MicroRNAs , Phenotype , Rabbits , Trigeminal Ganglion , Virus Replication
5.
BMC Infect Dis ; 2: 9, 2002 May 24.
Article in English | MEDLINE | ID: mdl-12052246

ABSTRACT

BACKGROUND: Dimethyl sulfoxide (DMSO) is frequently used at a concentration of up to 95% in the formulation of antiherpetic agents because of its properties as a skin penetration enhancer. Here, we have analyzed the effect of DMSO on several parameters of Herpes Simplex Virus replication. METHODS: Productive infection levels of HSV-1 were determined by plaque assay or by reporter gene activity, and its DNA replication was estimated by PCR. Transcript levels were evaluated with HSV-specific DNA micro-arrays. RESULTS: DMSO blocks productive infection in vitro in different cell types with a 50% inhibitory concentration (IC50) from 0.7 to 2% depending upon the multiplicity of infection. The concentration dependence exhibits a Hill coefficient greater than 1, indicating that DMSO blocks productive infection by acting at multiple different points (mechanisms of action) with positive cooperativity. Consistently, we identified at least three distinct temporal target mechanisms for inhibition of virus growth by DMSO. At late stages of infection, DMSO reduces virion infectivity, and markedly inhibits viral DNA replication. A third mode of action was revealed using an oligonucleotide-based DNA microarray system for HSV. These experiments showed that DMSO reduced the transcript levels of many HSV-1 genes; including several genes coding for proteins involved in forming and assembling the virion. Also, DMSO markedly inhibited some but not all early transcripts indicating a previously unknown mode for inhibiting the early phase of HSV transcription-replication cycle. CONCLUSION: These observations suggest that DMSO itself may have a role in the anti-herpetic activity of formulations utilizing it as a dispersant.


Subject(s)
DNA Replication/drug effects , Dimethyl Sulfoxide/pharmacology , Herpesvirus 1, Human/drug effects , Virus Replication/drug effects , Animals , Cells, Cultured , Chlorocebus aethiops , Dimethyl Sulfoxide/therapeutic use , Herpes Simplex/drug therapy , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/physiology , Humans , Oligonucleotide Array Sequence Analysis , Rabbits , Vero Cells
6.
Neurosci Lett ; 309(3): 177-80, 2001 Aug 31.
Article in English | MEDLINE | ID: mdl-11514070

ABSTRACT

Our aim was to investigate the neuromodulatory role of diadenosine tetraphosphate (Ap(4)A). Ap(4)A-binding sites were detected in striatum and hippocampus membranes using [(35)S]-ADP beta S as radioligand and Ap(4)A and epsilon-(Ap(4)A), di-ethenoadenosine tetraphosphate, as displacers. Effects of epsilon-(Ap(4)A) on extracellular glutamate levels were studied using intracerebral perfusion. Both areas contain high-affinity binding sites for [(35)S]-ADP beta S with K(d) values in the low nM range. [(35)S]-ADP beta S binding was displaced by Ap(4)A and epsilon-(Ap(4)A). At 1 and 10 microM doses, epsilon-(Ap(4)A) markedly decreased glutamate levels in the striatum. The possibility of Ap(4)A acting as an endogenous modulator of excitatory neurotransmission is discussed.


Subject(s)
Adenosine Diphosphate/analogs & derivatives , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Dinucleoside Phosphates/metabolism , Dinucleoside Phosphates/pharmacology , Glutamic Acid/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Diphosphate/metabolism , Animals , Dose-Response Relationship, Drug , Extracellular Space/drug effects , Extracellular Space/metabolism , Hippocampus/drug effects , Hippocampus/metabolism , Male , Neurotransmitter Agents/pharmacology , Rats , Rats, Wistar , Thionucleotides/metabolism
7.
Eur J Biochem ; 268(5): 1289-97, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11231280

ABSTRACT

Xenopus laevis oocytes exhibit ectoenzymatic activity able to hydrolytically cleave extracellular diadenosine polyphosphates (Ap(n)A). The basic properties of this ectoenzyme were investigated using as substrates di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(4)-tetraphospate [epsilon-(Ap(4)A)] and di-(1,N(6)-ethenoadenosine) 5',5"'-P(1),P(5)-pentaphospate [epsilon-(Ap(5)A)], fluorogenic derivatives of Ap(4)A and Ap(5)A, respectively. epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are hydrolysed by folliculated oocytes according to hyperbolic kinetics with K(m) values of 13.4 and 12.0 microM and Vmax values of 4.8 and 5.5 pmol per oocyte per min, respectively. The ectoenzyme is activated by Ca(2+) and Mg(2+), reaches maximal activity at pH 8--9 and is inhibited by suramin. Defolliculated oocytes also hydrolyse both substrates with similar K(m) values but V(max) values are approximately doubled with respect to folliculated controls. Chromatographic analysis indicates that extracellular epsilon-(Ap(4)A) and epsilon-(Ap(5)A) are first cleaved into 1,N(6)-ethenoAMP (epsilon-AMP) + 1,N(6)-ethenoATP (epsilon-ATP) and epsilon-AMP + 1,N(6)-ethenoadenosine tetraphosphate (epsilon-Ap(4)), respectively, which are catabolized to 1,N(6)-ethenoadenosine (epsilon-Ado) as the end product by folliculated oocytes. Denuded oocytes, however, show a drastically reduced rate of epsilon-Ado production, epsilon-AMP being the main end-product of extracellular epsilon-(Ap(n)A) catabolism. Results indicate that, whereas the Ap(n)A-cleaving ectoenzyme appears to be located mainly in the oocyte, ectoenzymes involved in the dephosphorylation of mononucleotide moieties are located mainly in the follicular cell layer.


Subject(s)
Adenosine/metabolism , Dinucleoside Phosphates/metabolism , Oocytes/enzymology , Xenopus laevis/metabolism , Adenosine/analogs & derivatives , Animals , Calcium/pharmacology , Cations, Divalent/pharmacology , Chromatography, High Pressure Liquid , Fluorescence , Hydrogen-Ion Concentration , Hydrolysis/drug effects , Kinetics , Magnesium/pharmacology , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Suramin/pharmacology
8.
Virology ; 258(1): 141-51, 1999 May 25.
Article in English | MEDLINE | ID: mdl-10329576

ABSTRACT

Several polysulfonate compounds have been shown to have the potential to inhibit the replication of herpesviruses by blocking binding and penetration of the host cell. We analyzed the actions of the polysulfonate compound suramin on the replication of herpes simplex virus type 1 (HSV-1) and compared them with the actions of heparin. We used the expression of a reporter gene (beta-galactosidase) recombined into the latency-associated transcript region of the 17syn+ strain of HSV-1 to quickly evaluate productive cycle activity and have shown that it can be directly correlated with virus replication under the conditions used. We find that suramin, like heparin, blocks the binding of HSV-1 to the cell membrane. Also, suramin efficiently blocks the cell-to-cell spread of the virus; this effect has not been previously reported. Our control experiments demonstrate that heparin also has some effect on intercellular spread of HSV-1 but to a significantly lesser degree than does suramin. We suggest that suramin and related polysulfonate compounds have potential for developing of antiherpes treatments.


Subject(s)
Antiviral Agents/pharmacology , Herpesvirus 1, Human/drug effects , Membrane Fusion/drug effects , Suramin/pharmacology , Animals , Chlorocebus aethiops , DNA Replication , DNA, Viral/biosynthesis , Gene Expression , Genes, Reporter , Heparin/pharmacology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Herpesvirus 1, Human/physiology , Humans , Membrane Fusion/physiology , Time Factors , Vero Cells , Viral Plaque Assay , Virion/physiology , Virus Assembly , Virus Replication , beta-Galactosidase/genetics
9.
J Virol ; 71(9): 7039-47, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9261434

ABSTRACT

PCR analysis of herpes simplex virus (HSV) genome replication and productive-cycle transcription was used to examine the role of the cornea in the latency-associated transcript (LAT)-mediated reactivation of HSV type 1 (HSV-1) in the rabbit eye model. The reduced relative reactivation frequency of 17 delta Pst (a LAT- virus) compared to those of wild-type and LAT+ rescuants correlated with reduced levels of viral DNA and transcription in the cornea following epinephrine induction. The timing of virus appearance in the cornea was most consistent with tissue peripheral to the cornea itself mediating a LAT-sensitive step in the reactivation process. Specific results include the following. (i) While viral DNA was found in the corneas of rabbits latently infected with either the LAT+ or LAT- virus prior to and during the first 16 to 24 h following induction, more was found in animals infected with the LAT+ virus. (ii) A significant increase in levels of viral DNA occurred 20 to 168 h following induction. (iii) The average relative amount of viral DNA was lower at all time points following reactivation of animals infected with the LAT- virus. (iv) Expression of productive-cycle transcripts could be detected in corneas of some rabbits latently infected with either the LAT+ or LAT- virus, and the amount recovered and the timing of appearance differed during the reactivation of rabbits latently infected with the LAT+ or LAT- virus. (v) Despite the reduced recoveries of LAT- virus DNA and productive-cycle transcripts in reactivating corneas in vivo compared to those of their LAT+ counterparts, such differences were not detected in cultured keratinocytes or in experiments in which relatively high titers of virus were superinfected into the eyes of latently infected rabbits. (vi) A number of LAT(+)-virus-infected rabbits expressed LAT in corneas isolated from uninduced rabbits. When seen, its amount was significantly higher than that of a productive-cycle (VP5) transcript.


Subject(s)
Cornea/virology , Herpesvirus 1, Human/genetics , Transcription, Genetic , Virus Activation , Virus Replication , Adrenergic Agonists/pharmacology , Animals , Capsid/genetics , Capsid Proteins , Cells, Cultured , Chlorocebus aethiops , DNA Replication , DNA, Viral/biosynthesis , Disease Models, Animal , Epinephrine/pharmacology , Genome, Viral , Herpesvirus 1, Human/growth & development , Herpesvirus 1, Human/physiology , Humans , Keratinocytes/cytology , Keratinocytes/virology , Polymerase Chain Reaction , RNA, Viral/biosynthesis , Rabbits , Trigeminal Ganglion/virology , Vero Cells , Virus Latency
10.
Biochem J ; 307 ( Pt 3): 729-34, 1995 May 01.
Article in English | MEDLINE | ID: mdl-7741703

ABSTRACT

We have isolated the gene for a chick A1 adenosine receptor along with a cDNA that codes for the same adenosine receptor. The cDNA clone was isolated from both adipose tissue and heart cDNA libraries and encodes a 324-amino acid protein with 80% identity with mammalian A1 adenosine receptors. Transient expression of the cDNA in human embryonic kidney (HEK) 293 cells shows that it encodes a protein that binds [3H]CCPA (2-chloro-N6-[cyclopentyl-2,3,4,5-3H]cyclopentyladenosine, a specific agonist radioligand, with a KD of 5.6 +/- 2.4 nM. Cyclic AMP measurements in HEK 293 cells co-transfected with the chick cDNA and a cDNA for a luteinizing hormone/choriogonadotropin receptor shows that A1 adenosine receptor agonists antagonize the cyclic AMP-elevating effect of bovine luteinizing hormone. Two partial genomic clones were isolated. The first contains 5'-untranslated sequence including a putative promoter region which does not contain a TATA box, an intron and the first third of the coding sequence of the A1 adenosine receptor cDNA. The coding sequence of this partial genomic clone terminates at a second intron. The second partial genomic clone contains the rest of the coding sequence and the 3'-untranslated elements in a single exon. Thus the chick A1 adenosine receptor gene contains one intron in the 5'-untranslated region and a minimum of one intron in the coding sequence.


Subject(s)
DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Receptors, Purinergic P1/genetics , Adenosine/analogs & derivatives , Adenosine/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Brain Chemistry , Chickens , Cloning, Molecular , Cyclic AMP/metabolism , DNA Probes , Dogs , Gene Amplification , Gene Expression , Genome , Introns , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Transcription, Genetic , Transfection
11.
Neurochem Res ; 17(12): 1235-9, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1461370

ABSTRACT

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of different selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60-80%) correspond to M2 muscarinic subtypes, and the rest is constituted by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.


Subject(s)
Adrenal Medulla/metabolism , Receptors, Muscarinic/metabolism , Animals , Atropine/pharmacology , Binding Sites , Binding, Competitive , Cattle , Cell Membrane/metabolism , Dicyclomine/pharmacology , Muscarine/antagonists & inhibitors , Piperidines/pharmacology , Pirenzepine/analogs & derivatives , Pirenzepine/pharmacology , Quinuclidinyl Benzilate/metabolism
12.
Brain Res ; 563(1-2): 163-70, 1991 Nov 01.
Article in English | MEDLINE | ID: mdl-1786528

ABSTRACT

An antiserum that recognizes a sequence from the putative third cytoplasmic loop of the m2 subtype of muscarinic receptors (mAchR) has been raised and used to map the cellular distribution of this subtype in rat olfactory bulb. The antiserum was obtained by injecting BALB/C mice with a BSA-conjugated synthetic peptide whose sequence corresponded to amino acids 240-259 of the porcine cardiac m2 mAChR gene. Antibodies recognized the synthetic peptide in ELISA screening and labelled a single band corresponding to the peak of [3H]PrBCM-labelled heart mAchRs in immunoblots. Immunostaining of olfactory bulb, a region of the brain enriched in this muscarinic receptor subtype, showed that the antibodies labelled cell bodies and multiple dendritic processes. Broad fluorescent labelling throughout cell bodies was consistent with binding to the cytoplasmic face of the surface membrane, in support of the predicted cytoplasmic loop structure. m2-Positive cells throughout the bulb were sparsely distributed in different layers representing small subpopulations of the cells in each region: glomeruli, 6%; external plexiform layer, 16%; inner plexiform and granule cell layer, 3%. The results show that antibodies against specific sequences of different muscarinic receptor subtypes can be used to localize subtypes in situ, that the m2 subtype within the rat olfactory bulb is broadly distributed, and that the m2 subtype can occur postsynaptically in this central nervous system (CNS) region. The mapping of m2-positive cells in olfactory bulb may be of particular interest because loss of this subtype and degeneration of the olfactory system have been observed in Alzheimer's disease.


Subject(s)
Cytoplasm/metabolism , Nerve Tissue Proteins/metabolism , Olfactory Bulb/metabolism , Peptides/immunology , Receptors, Muscarinic/metabolism , Animals , Antibodies/immunology , Blotting, Western , Carrier Proteins/metabolism , Cytoplasm/immunology , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Indicators and Reagents , Membranes/chemistry , Mice , Mice, Inbred BALB C , Myocardium/metabolism , Nerve Tissue Proteins/immunology , Olfactory Bulb/cytology , Olfactory Bulb/immunology , Peptides/metabolism , Receptors, Muscarinic/immunology , Swine
13.
Biochem Pharmacol ; 41(5): 735-42, 1991 Mar 01.
Article in English | MEDLINE | ID: mdl-1998528

ABSTRACT

Muscarinic receptors are altered by sulfhydryl reagents. Arsenic compounds, which have been used as insecticides, exert their toxic effects by combining with sulfhydryl groups. We compared the action of arsenicals and other sulfhydryl reagents on the muscarinic receptor from invertebrate and vertebrate species (locust and rat). Disulfide-reducing reagents dithiothreitol (DTT) and British Anti-Lewisite (BAL), but not arsenicals, inhibited [3H]quinuclidinyl benzilate ([3H]QNB) binding. However, after disulfide reduction, arsenicals caused a further inhibition of muscarinic binding. The effect of DTT + arsenicals was largely irreversible. The locust receptors were more sensitive to the action of both disulfide reagents either in the absence or presence of arsenicals than the rat receptors. The sulfhydryl reagent p-chloromercuric benzoate (PCMB) was more effective at inhibiting the locust receptors than the rat receptors, but addition of arsenicals did not cause further inhibition in either the locust or rat receptors. In locust, DTT + cacodylate and DTT + arsenite caused a reduction in the number of sites without modifying the affinity of [3H]QNB binding. In rat, DTT + arsenite caused a decrease in the affinity, while DTT + cacodylate caused a decrease in the affinity of [3H]QNB binding and its number of sites. Competition experiments after DTT + cacodylate showed that the IC50 and the Hill coefficient (nH) remained unchanged in the locust. In the rat, the IC50 for atropine was increased without alteration in the nH, and both parameters were increased for carbachol. These results are explained assuming that the binding site of the locust receptor has a disulfide group similar to that of the mammalian receptor, but that the hydrophobic interactions within the binding site are weaker in the locust receptor. The higher sensitivity of the insect receptor to sulfhydryl reagents could be of interest for developing methods of pest control.


Subject(s)
Arsenicals/pharmacology , Disulfides/pharmacology , Receptors, Muscarinic/drug effects , Animals , Atropine/antagonists & inhibitors , Binding, Competitive , Cacodylic Acid/pharmacology , Carbachol/antagonists & inhibitors , Dithiothreitol/pharmacology , Grasshoppers , Oxidation-Reduction , Quinuclidinyl Benzilate/metabolism , Rats
14.
FASEB J ; 3(10): 2132-40, 1989 Aug.
Article in English | MEDLINE | ID: mdl-2546848

ABSTRACT

This article describes ways in which receptors, key components of signal propagation through a synapse, can mediate changes in that propagation. Changes occur at four levels: in the signal-transducing capability of a single receptor molecule, in the number of receptors per cell, in the subcellular placement of receptor molecules, and in the cytoarchitecture of receptor-rich regions. The ability of receptors to shift between different desired states is called plasticity, and such shifts can be long-lived as well as transient. In this article we focus on neuronal receptors, although key findings from a variety of cell systems are reported. Neuronal receptor plasticity may have a special role in the assembly as well as the adaptability of the nervous system.


Subject(s)
Neurons/physiology , Receptors, Cell Surface/physiology , Animals , Humans , Neurons/ultrastructure , Receptors, Cell Surface/genetics , Signal Transduction , Synapses/physiology
15.
Neurochem Res ; 14(8): 765-70, 1989 Aug.
Article in English | MEDLINE | ID: mdl-2812253

ABSTRACT

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10-500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (KD') of [3H]QNB binding to rat membranes from 0.13 +/- 0.01 nM in control to 0.20 +/- 0.02 nM; there was also an small but significant reduction in the number of binding sites Bmax. In locust, 500 mM ethanol reduced the Bmax of [3H]QNB binding from 590 +/- 30 in control to 320 +/- 40 pmol/g protein; no significant alteration in the KD was detected. The dissociation rate constant (koff) of [3H]QNB increased from 0.020 +/- 0.003 in controls to 0.031 +/- 0.004 (min-1) in the presence of 500 mM ethanol, the association rate constant (Kon) did not change significantly. In locust, 500 mM ethanol did not affect either Kon or Koff. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.


Subject(s)
Ethanol/pharmacology , Grasshoppers/drug effects , Nerve Tissue/metabolism , Rats/metabolism , Receptors, Muscarinic/drug effects , Animals , Ganglia/metabolism , Grasshoppers/metabolism , Kinetics , Membranes/drug effects , Membranes/metabolism , Quinuclidinyl Benzilate/metabolism , Receptors, Muscarinic/metabolism , Species Specificity , Synaptosomes/metabolism
16.
Neurochem Res ; 13(6): 525-30, 1988 Jun.
Article in English | MEDLINE | ID: mdl-3405377

ABSTRACT

A rapid, reliable filtration method for [3H]oxotremorine binding to membranes of the cerebral cortex that allows the direct study of regulation by guanine nucleotides of muscarinic receptors was developed. [3H]Oxotremorine binds to cerebral cortex membranes with high affinity (KD, 1.9 nM) and low capacity (Bmax, 187 pmol/g protein). These sites, which represent only about 18% of those labeled with [3H]quinuclidinyl benzilate, constitute a population of GTP-sensitive binding sites. Association and dissociation binding experiments revealed a similar value of KD (2.3 nM). Displacement studies with 1-4000 nM oxotremorine showed the existence of a second binding site of low affinity (KD, 1.2 microM) and large capacity (Bmax, 1904 pmol/g protein). Gpp(NH)p, added in vitro, produced a striking inhibition of [3H]oxotremorine binding with an IC 50 of 0.3 microM. Saturation assays, in the presence of 0.5 microM Gpp(NH)p, revealed a non-competitive inhibition of the binding with little change in affinity. These results are discussed from the viewpoint of conflicting reports in the literature about guanine nucleotide regulation of muscarinic receptors in reconstituted systems and membranes from different tissues.


Subject(s)
Cerebral Cortex/metabolism , Guanosine Triphosphate/analogs & derivatives , Guanylyl Imidodiphosphate/pharmacology , Oxotremorine/metabolism , Receptors, Muscarinic/metabolism , Synaptosomes/metabolism , Animals , Cattle , Cerebral Cortex/drug effects , Kinetics , Quinuclidinyl Benzilate/metabolism , Rats , Receptors, Muscarinic/drug effects , Synaptosomes/drug effects
17.
Neurochem Res ; 12(1): 83-91, 1987 Jan.
Article in English | MEDLINE | ID: mdl-3574589

ABSTRACT

The reconstitution of solubilized bovine atrial cholinergic muscarinic receptor into liposomes made of exogenous lipids has been achieved by polyethyleneglycol precipitation. Of the different lipid mixtures used, soybean lecithins were shown to be the best on the basis of receptor recovery. The receptor reconstituted into soybean lecithins liposomes exhibited ligand binding properties very similar to those of the native receptor. The dissociation constant of [3H]-N-methyl-scopolamine ([3H]NMS) was 0.46 and 0.30 nM as determined by equilibrium and kinetics experiments respectively. The potency of a range of muscarinic ligands in displacing [3H]NMS binding was atropine greater than methyl-atropine greater than scopolamine greater than pirenzepine oxotremorine greater than gallamine greater than carbamylcholine greater than pilocarpine bethanechol. The Hill slopes of the displacement curves were near 1 for the antagonists and smaller than 1 for the agonists and for gallamine. The agonist binding may be modulated by guanine nucleotides. These results indicate that soybean lecithins fulfill the lipid requirements for the reconstitution of the atrial muscarinic receptor.


Subject(s)
Liposomes/metabolism , Myocardium/metabolism , Receptors, Muscarinic/metabolism , Animals , Binding, Competitive , Carbachol/metabolism , Cattle , Guanylyl Imidodiphosphate/pharmacology , Heart Atria/metabolism , Kinetics , N-Methylscopolamine , Phosphatidylcholines , Receptors, Muscarinic/drug effects , Scopolamine Derivatives/metabolism , Solubility , Glycine max/analysis
18.
Toxicol Appl Pharmacol ; 84(1): 192-5, 1986 Jun 15.
Article in English | MEDLINE | ID: mdl-3715864

ABSTRACT

In different brain regions of the rat we studied the effect of chronic feeding with the organochlorine insecticides p,p'-DDT and gamma-HCH on the cholinergic muscarinic receptors. Using [3H]quinuclidinyl benzylate binding to membranes from cerebral cortex, medulla pons, diencephalon, and cerebellum it was found that the two insecticides produced a decrease in the number of muscarinic receptor sites in cerebellum; while gamma-HCH also reduced these receptors in diencephalon. In both cases no changes in receptor affinity were observed. It is suggested that the chronic treatment with these organochlorine insecticides may cause an alteration in cholinergic transmission leading to a down regulation of the muscarinic receptor in certain brain regions.


Subject(s)
Brain Chemistry/drug effects , Receptors, Muscarinic/drug effects , Animals , Female , Insecticides/metabolism , Quinuclidinyl Benzilate/metabolism , Rats , Receptors, Muscarinic/analysis , Tritium
19.
Neurochem Res ; 11(5): 745-52, 1986 May.
Article in English | MEDLINE | ID: mdl-3724969

ABSTRACT

The injection of Bordetella pertussis, inactivated by merthiolate, causes a 2-fold increase in the IC50 of carbamylcholine (carbachol) in displacing [3H];-L(-) quinuclidinyl benzilate binding ([3H]QNB) to the receptor. In control animals, 50 microM Gpp(NH)p causes a 6-fold decrease in the affinity of carbachol binding, whereas after vaccination the reduction is only 1.6-fold. After pertussis treatment there is no alteration in the affinity and number of [3H]QNB binding sites of to the muscarinic receptor.


Subject(s)
Guanosine Triphosphate/analogs & derivatives , Guanylyl Imidodiphosphate/pharmacology , Myocardium/metabolism , Pertussis Vaccine/pharmacology , Receptors, Muscarinic/metabolism , Animals , Carbachol/metabolism , Cell Membrane/metabolism , Female , Male , Quinuclidinyl Benzilate/metabolism , Rats , Receptors, Muscarinic/drug effects
20.
Neurochem Res ; 11(3): 437-51, 1986 Mar.
Article in English | MEDLINE | ID: mdl-3703109

ABSTRACT

Membranes isolated from bovine atria were labeled with [3H]quinuclidinyl benzylate (3H-QNB), in control conditions and after 0.02% Triton X-100. This treatment inactivated about 20% of muscarinic receptor sites without loss of protein. The remaining 80% sites showed no changes in affinity, as determined by equilibrium or kinetic binding. Competition experiments with carbachol showed no differences in IC50 and Hill number between the control and detergent-membranes, suggesting that the different populations of agonist binding sites are inactivated in equal proportions by the detergent. In binding experiments, done in the presence of carbachol and guanine nucleotides, the detergent treated membranes were slightly more sensitive to the enhancing action of the nucleotide. The inhibition caused by ammonium ions was also more marked in the Triton X-100 treated membranes. The decay of binding with thermal inactivation was faster in the detergent treated membranes and this effect was enhanced in the presence of ammonium ions. These results may be interpreted as an indication that the receptors, remaining after the mild Triton X-100 treatment, are equally sensitive to the inactivation. We suggest that, while maintaining the heterogeneity of sites, the detergent produces a perturbation that could affect the molecular interactions between the receptor and other components of the membrane.


Subject(s)
Ammonia/pharmacology , Detergents/pharmacology , Guanosine Triphosphate/pharmacology , Heart Atria/metabolism , Polyethylene Glycols/pharmacology , Receptors, Muscarinic/drug effects , Surface-Active Agents/pharmacology , Animals , Cattle , Cell Membrane/drug effects , Cell Membrane/metabolism , Hot Temperature , Kinetics , Octoxynol , Quinuclidinyl Benzilate/metabolism , Receptors, Muscarinic/analysis
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