Biomedical subjects

D T Beattie

Publications and source records attributed to D T Beattie.

36 records · Page 2Linked to original sources

PubMed1995

Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1.

Cholera vaccine candidate Peru-15 was derived from a Vibrio cholerae O1 El Tor Inaba strain by deleting the cholera toxin genetic element, introducing the gene encoding cholera toxin B subunit into recA, and screening for nonmotility. In a controlled study, Peru-15 (2 x 10(8) cfu) was administered to 11 volunteers. No vaccinee developed diarrhea, and 10 of 11 had > 4-fold rises in vibriocidal antibody titers. One month later, 5 vaccinees and 5 control volunteers were challenged with wild type V. cholerae O1. Four of 5 controls developed diarrhea (mean, 1.9 L). Two Peru-15 vaccinees developed diarrhea, 1 with < 0.3 L and 1 with approximately 1.0 L; this latter volunteer had not developed a significant vibriocidal immune response to vaccination. Peru-15 shows promise as a single-dose, oral cholera vaccine that is safe, immunogenic, and protective.

Administration, Oral↗

PubMed1995

The pharmacology of GR203040, a novel, potent and selective non-peptide tachykinin NK1 receptor antagonist.

1. The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), a novel, highly potent and selective non-peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2. GR203040 potently inhibited [3H]-substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea-pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5). GR203040 had little affinity (pIC50 < 6.0) at all non-NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH-SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca(2+)-evoked contractions of rat isolated portal vein (pKn = 4.1). The enantiomer of GR203040, GR205608 (2R, 3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3. In gerbil ex vivo binding experiments, GR203040 produced a dose-dependent inhibition of the binding of [3H]-substance P to cerebral cortical membranes (ED50 = 15 micrograms kg-1 s.c. and 0.42 mg kg-1 p.o.). At 10 micrograms kg-1 s.c., the inhibition of [3H]-substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg-1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4. In guinea-pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration-effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5. In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose-ratio of 10) = 1.1 micrograms kg-1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 micrograms kg-1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6. In anaesthetized rats, GR203040 (3 and 10 mg kg-1, i.v.) produced a dose-dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7. It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain.

Animals↗

PubMed1995

Heterologous antigen expression in Vibrio cholerae vector strains.

Live attenuated vector strains of Vibrio cholerae were derived from Peru-2, a Peruvian El Tor Inaba strain deleted for the cholera toxin genetic element and attRS1 sequences, which was developed as a live, oral vaccine strain. A promoterless gene encoding the Shiga-like toxin I B subunit (slt-IB) was inserted in the V. cholerae virulence gene irgA by in vivo marker exchange, such that slt-IB was under transcriptional control of the iron-regulated irgA promoter. slt-IB was also placed under transcriptional control of the V. cholerae heat shock promoter, htpGp, and introduced into either the irgA or lacZ locus, or both loci, on the chromosome of Peru-2, generating JRB10, JRB11, or JRB12, respectively. A new technique was used to perform allelic exchange with lacZ. This method uses plasmid p6891MCS, a pBR327 derivative containing cloned V. cholerae lacZ, to insert markers of interest into the V. cholerae chromosome. Recombinants can be detected by simple color screening and antibiotic selection. In vitro measurements of Slt-IB produced by the vector strains suggested that expression of Slt-IB from the irgA and htpG promoters was synergistic and that two copies of the gene for Slt-IB increased expression over a single copy. The V. cholerae vectors colonized the gastrointestinal mucosa of rabbits after oral immunization, as demonstrated by very high serum antibody responses to V. cholerae antigens. Comparison of the serologic responses to the B subunit of cholera toxin (CtxB) following orogastric inoculation either with the wild-type C6709 or with Peru-10, a strain containing ctxB regulated by htpGp, suggested that both the cholera toxin and heat shock promoters were active in vivo, provoking comparable immunologic responses. Orogastric inoculation of rabbits with vector strains evoked serum immunoglobulin G (IgG) responses to Slt-IB in two of the four strains tested; all four strains produced biliary IgA responses. No correlation was observed between the type of promoter expressing slt-IB and the level of serum IgG or biliary IgA response, but the vector strain containing two copies of the gene for slt-IB evoked greater serum IgG responses than strains containing a single copy, consistent with the increased expression of Slt-IB from this strain observed in vitro. A comparison of the serum and biliary antibody responses to Slt-IB expressed from htpGp versus CtxB expressed from the same promoter suggested that CtxB is a more effective orally delivered immunogen.

Administration, Oral↗

PubMed1995

Recent developments in tachykinin NK1 receptor antagonists: prospects for the treatment of migraine headache.

The role of substance P and the influence of neurokinin 1 (NK1) receptor antagonists in the cranial circulation are described in the present review, particularly with respect to the mechanisms involved in the etiology of migraine headache. Substance P is distributed throughout the cranial vasculature, in the trigeminal sensory afferent nerve fibres, and its release can be demonstrated following activation of the trigeminovascular system in animals and humans. Following its release and NK1 receptor activation, dilatation and edema result, two events that are implicated in the pathogenesis of migraine headache. The recently developed selective NK1 receptor antagonists inhibit substance P mediated dilatation and plasma protein extravasation in the cranial circulation, suggesting that they may provide an effective and novel acute treatment for migraine.

Brain↗

PubMed1994

Use of genetic recombination as a reporter of gene expression.

An understanding of the patterns of gene expression in response to specific environmental signals can yield insight into a variety of complex biological systems such as microbial-host interactions, developmental cycles, cellular differentiation, ontogeny, etc. To extend the utility of the reporter gene fusion approach to such studies, we have constructed a gene expression reporter cassette that permits the generation of transcriptional fusions to tnpR encoding resolvase, a site-specific recombinase of the transposable element gamma delta. Induction of the transcriptional fusions results in production of resolvase, which in turn, catalyzes excision of a linked tetracycline-resistance reporter gene flanked by direct repeats of res, the DNA sequences at which resolvase functions. The loss of tetracycline resistance in descendant bacteria serves as a permanent and heritable marker of prior gene expression. This gene fusion approach will allow us to assay the induction of gene expression in as few as one cell. Additionally, gene expression can be assayed at a later time and/or different place from the inducing environment facilitating the study of gene expression in complex environments such as animal tissues.

Alkaline Phosphatase↗

PubMed1994

Development of a live, oral, attenuated vaccine against El Tor cholera.

Vibrio cholerae El Tor strains from Peru, Bangladesh, and Bahrain were attenuated by deletion of a genetic element that encodes virulence factors and RS1. The B subunit of ctx (ctxB) was reintroduced into the recA gene of the deletion mutants, rendering them unable to recombine with exogenous genetic elements and generating Peru-3, Bang-3, and Bah-3. Fifteen volunteers received one dose of various vaccine strains at 4 x 10(6) to 1 x 10(8) cfu. All strains colonized the gut. A > or = 4-fold rise in vibriocidal titer was observed in 14 volunteers, with titers of > or = 1600 in 13. Peru-3 was the least reactogenic, but 2 of 6 volunteers had loose stools. Peru-14, a filamentous motility-deficient mutant of Peru-3, was well tolerated and colonized 18 of 21 volunteers at doses of 2 x 10(6) to 1 x 10(9) cfu. Also, when 8 Peru-3 or Peru-5 vaccinees, 5 Peru-14 vaccinees, and 8 controls were challenged with 2 x 10(6) cfu V. cholerae El Tor Inaba (N16961), 11 vaccinees were protected compared with no controls. Peru-14 shows promise as a safe, effective, single-dose oral vaccine against El Tor cholera.

Adolescent↗

PubMed1994

The influence of the trigeminal ganglion on carotid blood flow in anaesthetized guinea-pigs.

1. The influence of the trigeminal ganglion on the carotid circulation has been investigated by measuring electrical stimulation-induced alterations in carotid arterial blood flow and resistance in anaesthetized guinea-pigs. The effects of several receptor antagonists were assessed to determine which neurotransmitters are involved in regulating carotid blood flow. 2. Arterial blood pressure and carotid vascular resistance were reduced by electrical stimulation (0.5 mA, 1 ms, 5 Hz, 60 s) of the trigeminal ganglion ipsilateral to the carotid artery from which flow was measured. No consistent effect of electrical stimulation on carotid blood flow was observed. However, when guinea-pigs were pretreated with guanethidine (30 mg kg-1, s.c., 24 h prior to experiments), stimulation produced little change in blood pressure, while carotid blood flow was increased and vascular resistance decreased, consistent with vasodilatation in the cranial circulation. Stimulation of the trigeminal ganglion contralateral to the carotid artery from which blood flow was measured, had little effect on either carotid blood flow or vascular resistance. 3. In animals pretreated with guanethidine, intravenous administration of the vasoactive intestinal polypeptide (VIP) receptor antagonist, [p-Cl-D-Phe6,Leu17]-VIP (50 micrograms kg-1) significantly attenuated the increase in carotid blood flow and decrease in carotid vascular resistance evoked by trigeminal ganglion stimulation. Responses evoked by trigeminal ganglion stimulation were, however, unaffected by intravenous injection of the tachykinin NK1 receptor antagonists, GR82334 (0.3 mg kg-1) and CP-99,994 (0.4 mg kg-1), calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37 (0.9 mg kg-1) and the ganglion blocking agent, hexamethonium (10 mg kg-1). 4. It is concluded that in the guanethidine-pretreated guinea-pig, electrical stimulation of the trigeminal ganglion increases carotid blood flow and produces an accompanying decrease in carotid vascular resistance, consistent with the dilatation of carotid blood vessels. The transmitter mediating this effect is most likely to be VIP.

Anesthesia↗

PubMed1993

The influence of neurokinins and calcitonin gene-related peptide on cerebral blood flow in anaesthetized guinea-pigs.

The effects of systemically-administered human alpha calcitonin gene-related peptide (h.alpha CGRP), substance P and the selective neurokinin receptor agonists, GR73632 (NK1) and GR64349 (NK2) on cerebral blood flow (CBF) were studied in anaesthetized guinea-pigs using a laser-Doppler flowmeter. h.alpha CGRP (0.1 and 0.3 nmol/kg), substance P (0.03-1.0 nmol/kg), GR73632 (0.03-0.3 nmol/kg) and GR64349 (0.3 nmol/kg) each, following intra-carotid artery injection, reduced transiently (< 5 min) blood pressure and CBF. GR73632 (0.1 and 0.3 nmol/kg) and GR64349 (0.3 nmol/kg), but not h.alpha CGRP (0.01-0.3 nmol/kg) or substance P(0.01-1.0 nmol/kg), then produced a more prolonged increase in CBF, the peak effect occurring 10-15 min after injection. It is likely that this increase in CBF was due to their bronchoconstrictor activity, rather than a direct effect on the cerebrovasculature; arterial PaCO2 levels were increased and PaO2 decreased by both compounds. Following pretreatment with urea (5 M) to disrupt the blood brain barrier, h.alpha CGRP (0.1 nmol/kg) produced a significant increase in CBF (13 +/- 4%), implying that access to its receptors on the cerebrovascular smooth muscle had been achieved. Substance P (0.1 nmol/kg) remained inactive. The study has demonstrated that compounds acting on neuropeptide receptors have little direct influence on CBF following systemic administration. CGRP requires access to its receptors on the cerebrovascular smooth muscle, while selective NK1 and NK2 receptor agonists increase CBF, probably indirectly via their bronchoconstrictor activity. The lack of effect of substance P may be due to its rapid breakdown by peptidases, a property not shared by the selective neurokinin agonists.

Anesthesia↗

PubMed1993

Neurokinin-induced changes in pial artery diameter in the anaesthetized guinea-pig.

1. The effects of selective neurokinin agents on pial artery diameter, measured with an on-line image analyser, have been studied in anaesthetized guinea-pigs in order to characterize the neurokinin receptors present on pial arteries. 2. Perivascular injection of either substance P (0.01-1 microM) or the selective NK1 receptor agonists, substance P methyl ester (SPOMe, 0.01-1 microM) and GR73632 (0.1 microM), increased pial artery diameter. 3. In contrast, the selective NK2 receptor agonist, GR64349 (1 microM), produced a small vasoconstriction while the NK3 receptor-selective agonist, senktide (1 microM) was inactive. 4. Co-administration of GR82334 (1 microM), a selective NK1 receptor antagonist, inhibited the vasodilatation produced by SPOMe (0.1 microM) but not that caused by calcitonin gene-related peptide (CGRP, 0.01 microM). 5. The results are consistent with an involvement of NK1 receptors in the neurokinin-induced increase in guinea-pig pial artery diameter.

Animals↗

PubMed1993

Repressor binding to a regulatory site in the DNA coding sequence is sufficient to confer transcriptional regulation of the vir-repressed genes (vrg genes) in Bordetella pertussis.

Five TnphoA fusions to vir-repressed genes (vrg genes) have been identified in the respiratory pathogen Bordetella pertussis. A comparison of vrg DNA sequences suggests a consensus DNA element within the coding regions of four of five vrg genes. To determine the role of this DNA sequence in vrg regulation, a nucleotide substitution mutation in the conserved region of vrg-6 was isolated. This mutant showed constitutively high levels of expression in the absence of antigenic modulators, MgSO4 and nicotinic acid, suggesting that this DNA element may be a control site for vrg repression. Moreover, Northern (RNA) analysis and transcriptional fusion analysis suggest that vrg genes are regulated at the transcriptional level. To determine whether sequences in the coding region were sufficient to respond to antigenic modulation, a vrg-6::TnphoA promoter deletion plasmid that contained a heterologous promoter driving the expression of vrg-6 coding sequences from the vrg-6 translation start site to the TnphoA fusion junction was constructed. This heterologous construct responded to modulators in a vir-dependent fashion, indicating that sequences upstream of the coding sequence are not required for antigenic modulation. Southwestern (DNA-protein) analysis and mutational studies suggest that the vrg consensus DNA sequence is specifically recognized by a 34-kDa vir-activated gene (vag) product, whose binding results in down-regulation of vrg transcript levels. We conclude, at least for the vrg::TnphoA fusion strains, that a site on the DNA that corresponds to a consensus sequence located in the vrg coding region is sufficient to confer the transcriptional regulation (repression) of vrg genes when B. pertussis strains are grown under nonmodulating conditions.

Amino Acid Sequence↗

PubMed1992

A vir-repressed gene of Bordetella pertussis is required for virulence.

Coordinate regulation of gene expression in Bordetella pertussis is controlled by the products of the vir locus, BvgA and BvgS. In the presence of modulating signals such as MgSO4 and nicotinic acid, expression of vir-activated genes (vag) is reduced, while expression of vir-repressed genes (vrg) is maximal. We have cloned one of these vir-repressed genes, vrg-6, in Escherichia coli. DNA sequencing has shown that vrg-6 is contained on a single EcoRI restriction endonuclease fragment and is predicted to code for a protein of 105 amino acids with a molecular weight of 11,441. The predicted protein product appears to have two domains, one consisting of seven hydrophobic proline-rich pentameric repeats and the other consisting of five alkaline trimeric repeats. Southern blot analysis has revealed vrg-6-homologous sequences in the chromosomes of Bordetella bronchiseptica and Bordetella parapertussis, but, unlike Bordetella pertussis, these species do not express vrg-6-homologous RNA when grown under modulating conditions. In order to assess the role of vrg gene products in B. pertussis pathogenesis, two 18323 derivatives which harbor TnphoA insertions in vrg genes were analyzed in a mouse model of respiratory infection. Strain SK6, which carries a vrg-6::TnphoA mutation, failed to induce lymphocytosis and was significantly less able to colonize lungs and trachea than its parent strain 18323 or than SK18, which harbors a TnphoA fusion in the vrg-18 locus. This is the first evidence that a vir-repressed gene may play an important role in the virulence of B. pertussis and the pathogenesis of whooping cough.

Amino Acid Sequence↗

PubMed1992

Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene.

The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals. Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature. We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci. Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events. The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B. pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions. The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature. Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active. In B. pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects. In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations.

Alleles↗

PubMed1991

Novel class of amino acid antagonists at non-N-methyl-D-aspartic acid excitatory amino acid receptors. Synthesis, in vitro and in vivo pharmacology, and neuroprotection.

The isoxazole amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) (1), which is a highly selective agonist at the AMPA subtype of excitatory amino acid (EAA) receptors, has been used as a lead for the development of two novel EAA receptor antagonists. One of the compounds, 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA, 7), was synthesized via O-alkylation by ethyl chloroacetate of the amino acid protected AMPA derivative 4. The other compound, 2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)-methyl-5-methyl-3-+ ++oxoisoxazolin -4-yl]propionic acid (AMNH, 14) was synthesized with use of 4-(chloromethyl)-3-methoxy-5-methylisoxazole (8) as the starting material. The intermediate 4-(chloromethyl)-2-(3-methoxy-5-methylisoxazol-4-yl)methyl-5-me thylisoxazolin- 3-one (11) was converted into the acetamidomalonate (12), which was stepwise deprotected to give 14. Compounds 7 and 14 were stable in aqueous solution at pH values close to physiological pH. Neither 7 nor 14 showed detectable affinities for the receptor, ion channel, or modulatory sites of the N-methyl-D-aspartic acid (NMDA) receptor complex. Quantitative receptor autoradiographic and conventional binding techniques were used to study the affinities of 7 and 14 for non-NMDA receptor sites. Both compounds were inhibitors of the binding of [3H]AMPA (IC50 = 90 and 29 microM, respectively). Compounds 14 and 7 were both very weak inhibitors of the high-affinity binding of radioactive kainic acid [( 3H]KAIN). Compound 14, but not 7, was, however, shown to be an inhibitor of low-affinity [3H]KAIN binding (IC50 = 40 microM) as determined in the presence of 100 mM calcium chloride. In the rat cortical slice preparation, 7 was shown to antagonize excitation induced by 1 with some selectivity, whereas 14 proved to be a rather selective antagonist of KAIN-induced excitation. Both antagonists showed very weak effects on the excitatory effects of NMDA. Compound 7 was a poor antagonist of excitation by quisqualic acid (2), whereas 14 did not affect excitation by this nonselective AMPA receptor agonist. On cat spinal neurones, both 7 and 14 reduced excitations by 1 and KAIN, but, again, the excitatory effects of 2 were much less sensitive. Compound 14 and, in particular, 7 effectively protected rat striatal neurones against the neurotoxic effects of KAIN, whereas the toxic effects of 1 were reduced only by 7. Neither antagonist showed protection against the cell damage caused by intrastriatal injection of the NMDA agonist quinolinic acid.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Evidence that modulation requires sequences downstream of the promoters of two vir-repressed genes of Bordetella pertussis.

Gene expression in Bordetella pertussis is altered by environmental signals in a process called antigenic modulation. In the presence of modulating signals, expression of several known virulence factors and outer membrane proteins is coordinately reduced. From a bank of TnphoA fusions, we have identified five genes whose expression profiles are reciprocal of those of the major virulence determinants; that is, alkaline phosphatase activity is maximal during growth in the presence of the modulators nicotinic acid and MgSO4 (S. Knapp and J. J. Mekalanos, J. Bacteriol. 170:5059-5066, 1988). We have called these loci vir-repressed genes (vrg). Two of these gene fusions (vrg-6 and vrg-18) have been cloned in Escherichia coli, returned on low-copy-number plasmids to several strains of B. pertussis, and found to be regulated similarly to the fusions harbored on the chromosome. Deletions of the two vrg promoters were constructed and returned to B. pertussis. Regulation was maintained even when all but 24 nucleotides upstream of the vrg-18 initiation codon and 60 nucleotides upstream of the vrg-6 initiation codon were deleted, suggesting that cis-acting regulatory elements of these genes lie very near or within the coding region. We observed a 21-base palindromic sequence overlapping an 8-base direct repeat within the signal sequence coding region of vrg-6; insertion of a 6-bp linker in this region abolished regulation. These repetitive sequences are also at the site of greatest primary sequence identify between vrg-6 and vrg-18 and correspond to the signal sequence coding region. We propose models that involve recognition of this region by a vir-regulated gene product.

Alkaline Phosphatase↗

PubMed1990

Synthesis and single cell pharmacology of potential heterocyclic bioisosteres of the excitatory amino acid antagonist glutamic acid diethyl ester.

A series of heterocyclic analogues of glutamic acid diethyl ester (GDEE), an antagonist at central excitatory amino acid receptors, have been synthesized and tested biologically. (RS)-Ethyl alpha-amino-alpha-(3-ethoxyisoxazol-5-yl)acetate (7), (RS)-ethyl 2-amino-3-(3-ethoxy-5-methylisoxazol-4-yl)propionate (16) and closely related analogues were synthesized. Compound 7, a diethyl derivative of the naturally occurring excitatory amino acid ibotenic acid (IBO), was synthesized from 3-hydroxy-5-methylisoxazole (1) via 3-ethoxyisoxazol-5-ylacetic acid (5) and its ethyl ester. Nitrosation of this ester followed by catalytic reduction gave 7. The ethyl ester of IBO, 9, was synthesized in a similar manner from 3-benzyloxyisoxazol-5-ylacetic acid (8). Ethyl derivatives of the synthetic excitatory amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) were synthesized from 3-hydroxy-4,5-dimethylisoxazole (10) through a diethyl acetylaminomalonate derivative, which upon deprotection gave the 3-ethoxy derivative of AMPA (15). Esterification of 15 gave the diethyl derivative 16 and the ethyl ester of AMPA (18) as well as N-ethylated derivatives of AMPA, 21 and 22 were synthesized. The final products were tested microelectrophoretically. The derivatives 7, 9, 15, 16 and 18 were weak and non-selective excitatory amino acid antagonists, whereas 21 and 22 were found to be inactive.

Action Potentials↗

PubMed1989

Baclofen antagonism by 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid in the cat spinal cord.

When administered microelectrophoretically, 4-amino-3-(5-methoxybenzo[b]furan-2-yl)butanoic acid (MBFG) reversibly reduced the presynaptic depression by (-)-baclofen of the monosynaptic excitation of spinal interneurones by impulses in muscle low-threshold afferent fibres of the cat as well as the postsynaptic depression by (-)-baclofen of the firing of these neurones. MBFG, as an antagonist of (-)-baclofen, may be useful in investigating the structure-activity relationships of central and peripheral baclofen receptors.

Animals↗

PubMed1988

Baclofen antagonism by 2-hydroxy-saclofen in the cat spinal cord.

When administered microelectrophoretically, a sulphonic acid derivative of baclofen, 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid, reversibly reduced the presynaptic reduction by (-)-baclofen of the monosynaptic excitation of spinal interneurones by impulses in low threshold primary afferent fibres of the cat as well as the postsynaptic depression by (-)-baclofen of the firing of these neurones. This compound, 2-hydroxy-saclofen, may be useful in assessing the physiological significance of central baclofen receptors.

Action Potentials↗

PubMed1986

Effects of calcium channel antagonists on action potential conduction and transmitter release in the guinea-pig vas deferens.

The effects of the Ca2+ channel antagonists amlodipine, cobalt, diltiazem, nifedipine and verapamil and the local anaesthetic lignocaine were investigated on action potential conduction in and on evoked transmitter release from sympathetic nerves in the guinea-pig isolated vas deferens. Transmitter release was investigated by measurement of evoked (trains of pulses at 1 and 2 Hz, 0.1-0.5 ms supramaximal voltage) excitatory junction potentials (e.j.ps) using microelectrodes; tension was recorded simultaneously; tritium [3H] overflow from vasa preincubated (37 degrees C, 30 min) in Krebs solution containing either [3H]-noradrenaline (NA, 25 microCi ml-1, 2 X 10(-6) M NA) or [3H]-adenosine (50 microCi ml-1, 1 X 10(-6) M adenosine). Amlodipine (0.5-2 X 10(-4) M), verapamil (0.5-2 X 10(-4) M), diltiazem (1-8 X 10(-4) M), lignocaine (0.1-2 X 10(-3) M) and cobalt (2-6 X 10(-2) M) in descending order of potency, but not nifedipine (1-5 X 10(-3) M), increased the latency and inhibited, then abolished, the amplitude and number of action potentials in a concentration-dependent manner. Amlodipine (0.5-1 X 10(-4) M), verapamil (1-2 X 10(-4) M), diltiazem (1-5 X 10(-4) M) and cobalt (1 X 10(-3) M), in descending order of potency, but not nifedipine (5 X 10(-4) M), inhibited then abolished evoked e.j.ps in a concentration-dependent manner. Cobalt inhibited e.j.ps at a lower concentration than that (2-6 X 10(-2) M) required to block action potential conduction. In unstimulated tissues, the resting [3H] overflow following preincubation with [3H]-NA consisted largely of 4-hydroxy 3-methoxymandelic acid (VMA), 4-hydroxy 3-methoxy phenylglycol (MOPEG), 3,4 dihydroxyphenylglycol (DOPEG) and NA; stimulated tissues (300 pulses at 20 Hz, 0.5 ms supramaximal voltage) released mainly NA. Verapamil (0.1-1 X 10(-4) M), amlodipine (0.05-1 X 10(-4) M) and nifedipine (1-5 X 10(-4) M), but not cobalt (2 X 10(-3) M), increased, significantly, the resting overflow of 3H comprising mainly DOPEG. Cobalt (2 X 10(-3) M) inhibited, significantly, the stimulation-evoked overflow of 3H. Verapamil (1 X 10(-4) M) had little effect on the resting overflow of 3H following preincubation with [3H]-adenosine. Diltiazem (5 X 10(-4) M) and cobalt (2 X 10(-3) M) both inhibited evoked 3H overflow. Nifedipine (5 X 10(-4) M) was ineffective. 6 The effectiveness of Ca2+ channel antagonists at pre- and postjunctional sites differ; the results are discussed in terms of the selectivity of these drugs for each site and their differential effects on transmitter release.

Action Potentials↗