A-Current down-modulated by sigma receptor in frog pituitary melanotrope cells through a G protein-dependent pathway.
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Gramicidin perforated patch-clamp recordings were used to study the effects of two sigma 1 receptor ligands, (+)-N-cyclopropylmethyl-N-methyl-1, 4-diphenyl-1-ethyl-but-3-en-1-ylamine hydrochloride (JO 1784) and (+)-pentazocine, on the transient outward potassium current (IA) in cultured frog melanotrope cells. (+)-Pentazocine reversibly decreased the current amplitude in a dose-dependent manner. The effects of (+)-pentazocine were mimicked by JO 1784 and were markedly reduced by the sigma 1 receptor antagonist, N, N-dipropyl-2-[4-methoxy-3-2(2-phenylethoxy)phenyl]-ethylamine monohydrochloride (NE 100). Inactivation rate of IA was best fitted with a double exponential function, yielding time constants of 23.7 and 112.5 ms. (+)-Pentazocine (20 microM) accelerated the current decay, decreasing the time constants to 10.7 and 59 ms, respectively. Current-voltage experiments revealed that (+)-pentazocine (20 microM) did neither modify the open-state I/V curves nor the voltage dependence of IA. However, (+)-pentazocine (20 microM) shifted the steady-state inactivation curve toward more negative potentials and increased the time constant of the time-dependent removal of inactivation. In whole-cell experiments, internal dialysis of guanosine-5'-O-(3-thiophosphate) (100 microM) irreversibly prolonged the response to (+)-pentazocine. In addition, cholera toxin pretreatment (1 microgram. ml-1; 12 h) suppressed the inhibition of IA by (+)-pentazocine (20 microM). It is concluded that in frog melanotrope cells, a cholera toxin-sensitive, G protein-dependent inhibition of IA through a sigma 1 receptor activation, at least partially, underlies the excitatory effect of sigma ligands. (+info)
Antagonist pharmacology of metabotropic glutamate receptors coupled to phospholipase D activation in adult rat hippocampus: focus on (2R,1'S,2'R,3'S)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine versus 3, 5-dihydroxyphenylglycine.
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Metabotropic glutamate (mGlu) receptors coupled to phospholipase D (PLD) appear to be distinct from any known mGlu receptor subtype linked to phospholipase C or adenylyl cyclase. The availability of antagonists is necessary for understanding the role of these receptors in the central nervous system, but selective ligands have not yet been identified. In a previous report, we observed that 3, 5-dihydroxyphenylglycine (3,5-DHPG) inhibits the PLD response induced by (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate in adult rat hippocampal slices. We now show that the antagonist action of 3, 5-DHPG (IC50 = 70 microM) was noncompetitive in nature and nonselective, because the drug was also able to reduce PLD activation elicited by 100 microM norepinephrine and 1 mM histamine. In the search for a selective and more potent antagonist, we examined the effects of sixteen stereoisomers of 2-(2'-carboxy-3'-phenylcyclopropyl)glycine (PCCG) on the PLD-specific transphosphatidylation reaction resulting in the formation of [3H]phosphatidylethanol. The (2R,1'S,2'R,3'S)-PCCG stereoisomer (PCCG-13) antagonized the formation of [3H]phosphatidylethanol induced by 100 microM (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylate in a dose-dependent manner and with a much lower IC50 value (25 nM) compared with 3,5-DHPG. In addition, increasing concentrations of PCCG-13 were able to shift to the right the agonist dose-response curve but had no effect when tested on other receptors coupled to PLD. The potent, selective, and competitive antagonist PCCG-13 may represent an important tool for elucidating the role of PLD-coupled mGlu receptors in adult hippocampus. (+info)
Dual mechanism for presynaptic modulation by axonal metabotropic glutamate receptor at the mouse mossy fibre-CA3 synapse.
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1. To investigate mechanisms responsible for the presynaptic inhibitory action mediated by the axonal group II metabotropic glutamate receptor (mGluR) at the mossy fibre-CA3 synapse, we used a quantitative fluorescence measurement of presynaptic Ca2+ in mouse hippocampal slices. 2. Bath application of the group II mGluR-specific agonist (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV, 1 microM) reversibly suppressed the presynaptic Ca2+ influx (to 55.2 +/- 4.6 % of control, n = 5) as well as field EPSPs recorded simultaneously (to 3.1 +/- 2.0%). Presynaptic fibre volley was not affected by 1 microM DCG-IV. 3. A quantitative analysis of the inhibition of presynaptic Ca2+ influx and field EPSP suggested that DCG-IV suppressed the field EPSP to a greater extent than would be expected if the suppression were solely due to a decrease in the presynaptic Ca2+ influx. 4. DCG-IV at 1 microM suppressed the mean frequency (to 73.8 +/- 3.9% of control, n = 11), but not the mean amplitude (to 97.0 +/- 3.5%), of miniature EPSCs recorded from CA3 neurones using the whole-cell patch-clamp technique. 5. These results suggest that group II mGluR-mediated suppression is due both to a reduction of presynaptic Ca2+ influx and downregulation of the subsequent exocytotic machinery. (+info)
Linkers designed to intercalate the double helix greatly facilitate DNA alkylation by triplex-forming oligonucleotides carrying a cyclopropapyrroloindole reactive moiety.
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Triplex-forming oligonucleotides (TFOs) bind sequence-specifically in the major groove of double-stranded DNA. Cyclopropapyrroloindole (CPI), the electrophilic moiety that comprises the reactive subunit of the antibiotic CC-1065, gives hybridization-triggered alkylation at the N-3 position of adenines when bound in the minor groove of double-stranded DNA. In order to attain TFO-directed targeting of CPI, we designed and tested linkers to 'thread' DNA from the major groove-bound TFO to the minor groove binding site of CPI. Placement of an aromatic ring in the linker significantly enhanced the site-directed reaction, possibly due to a 'threading' mechanism where the aromatic ring is intercalated. All of the linkers containing aromatic rings provided efficient alkylation of the duplex target. The linker containing an acridine ring system, the strongest intercalator in the series, gave a small but clearly detectable amount of non-TFO-specific alkylation. An equivalent-length linker without an aromatic ring was very inefficient in DNA target alkylation. (+info)
The sigma ligand, igmesine, inhibits cholera toxin and Escherichia coli enterotoxin induced jejunal secretion in the rat.
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BACKGROUND: Cholera toxin, and Escherichia coli heat labile (LT) and heat stable (STa) enterotoxins induce small intestinal secretion in part by activating enteric nerves. Igmesine is a novel sigma receptor ligand that inhibits neurally mediated secretion. AIMS: To assess the antisecretory potential of igmesine in cholera toxin, LT, and STa induced water and electrolyte secretion using an in vivo rat model of jejunal perfusion. METHODS: After pretreatment with igmesine, 0.03-10 mg/kg intravenously, jejunal segments of anaesthetised, adult male Wistar rats were incubated with cholera toxin (25 microg), LT (25 microg), or saline. Jejunal perfusion with a plasma electrolyte solution containing a non-absorbable marker was undertaken. In some cases 200 microg/l STa was added to the perfusate. After equilibration, net water and electrolyte movement was determined. In additional experiments rats received igmesine, intravenously or intrajejunally, after exposure to cholera toxin. RESULTS: Cholera toxin induced net water secretion was inhibited by 1 mg/kg igmesine (median -120 versus -31 microl/min/g, p<0.001). LT and STa induced secretion were also inhibited by 1 mg/kg igmesine (-90 versus -56, p<0.03; and -76 versus -29, p<0.01, respectively). Igmesine reduced established cholera toxin induced secretion. CONCLUSION: The sigma ligand, igmesine, inhibits neurally mediated enterotoxigenic secretion. Its ability to inhibit established secretion makes it an agent with therapeutic potential. (+info)
Kinetics of opiate receptor inactivation by sulfhydryl reagents: evidence for conformational change in presence of sodium ions.
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The role of SH groups in opiate-receptor interactions has been further examined. In activation by N-ethylmaleimide of sterospecific opiate binding by rat brain membrane fractions follows pseudo-first order kinetics and exhibits strong temperature dependence. The kinetics indicate that alkylation of a single SH group suffices to block opiate binding. Considerable protection from SH group inactivation is observed when treatment with N-ethylmaleimide is carried out in the presence of an opiate or an antagonist, suggesting close proximity of the SH group to the opiate binding site. The rate of inactivation of receptor binding by N-ethylmaleimide is markedly slower in buffers containing 100 mM NaCl (t1/2 equals 30 plus or minus 1.4 min) than in sodium-free buffers (t1/2 equals 10 plus or minus 1.0 min). Since the rate of alkylation of model SH compounds is unaffected by sodium ions, this protection seems best explained by a conformational change in the receptors that renders the SH groups less accessible to alkylation. The rate of inactivation is not affected by K+, Rb+, or Cs+ and only slightly by Li+. This cation specificity as well as the concentration-response to Na+ are remarkably similar to those previously shown to lead to increased antagonist and decreased agonist binding. We suggest that the same conformational change is involved in the two phenomena. (+info)
Production of 6-deoxy-13-cyclopropyl-erythromycin B by Saccharopolyspora erythraea NRRL 18643.
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Cyclopropane carboxylic acid was fed to Saccharopolyspora erythraea NRRL 18643 (6-deoxyerythromycin producer), resulting in the production of 6-deoxy-13-cyclopropyl-erythromycin B. These studies provide further evidence that deoxyerythronolide B synthase has a relaxed specificity for the starter unit. (+info)
Adenosine A1 and class II metabotropic glutamate receptors mediate shared presynaptic inhibition of retinotectal transmission.
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Presynaptic inhibition is one of the major control mechanisms in the CNS. Previously we reported that adenosine A1 receptors mediate presynaptic inhibition at the retinotectal synapse of goldfish. Here we extend these findings to metabotropic glutamate receptors (mGluRs) and report that presynaptic inhibition produced by both A1 adenosine receptors and group II mGluRs is due to G(i) protein coupling to inhibition of N-type calcium channels in the retinal ganglion cells. Adenosine (100 microM) and an A1 (but not A2) receptor agonist reduced calcium current (I(Ca2+)) by 16-19% in cultured retinal ganglion cells, consistent with their inhibition of retinotectal synaptic transmission (-30% amplitude of field potentials). The general metabotropic glutamate receptor (mGluR) agonist 1S,3R-1-amino-cyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, 50 microM) and the selective group II mGluR receptor agonist (2S, 2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 300 nM) inhibited both synaptic transmission and I(Ca2+), whereas the group III mGluR agonist L-2-amino-4-phosphono-butyrate (L-AP4) inhibited neither synaptic transmission nor I(Ca2+). When the N-type calcium channels were blocked with omega-conotoxin GVIA, both adenosine and DCG-IV had much smaller percentage effects on the residual 20% of I(Ca2+), suggesting effects mainly on the N-type calcium channels. The inhibitory effects of A1 adenosine receptors and mGluRs were both blocked by pertussis toxin, indicating that they are mediated by either G(i) or G(o). They were also inhibited by activation of protein kinase C (PKC), which is known to phosphorylate and inhibit G(i). Finally, when applied sequentially, inhibition by adenosine and DCG-IV were not additive but occluded each other. Together these results suggest that adenosine A1 receptors and group II mGluRs mediate presynaptic inhibition of retinotectal synaptic transmission by sharing a pertussis toxin (PTX)-sensitive, PKC-regulated G(i) protein coupled to N-type calcium channels. (+info)