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PubMed2000

Anthelmintic actions on homomer-forming nicotinic acetylcholine receptor subunits: chicken alpha7 and ACR-16 from the nematode Caenorhabditis elegans.

Two homomer-forming nicotinic acetylcholine receptor subunits with 47% identity in their amino acid sequences were employed to compare the actions of cholinergic anthelmintics and ivermectin on expressed vertebrate and nematode nicotinic receptors of known molecular composition. Voltage-clamp electrophysiology was used to study recombinant nicotinic receptors expressed in Xenopus laevis oocytes following nuclear injection of cDNA encoding either chicken alpha7 or Caenorhabditis elegans ACR-16 (Ce21) subunits. Butamisole, morantel and metyridine were without agonist actions on either alpha7 or ACR-16 nicotinic receptors in the range 10nM-1mM. However, butamisole (pIC(50)=4.9 for both alpha7 and ACR-16) and morantel (pIC(50)=5.6 for alpha7 and 5.7 for ACR-16) antagonized responses of both alpha7 and ACR-16 receptors to acetylcholine. Metyridine (1mM) did not affect responses to acetylcholine of either receptor. Oxantel was without agonist actions on ACR-16, but was an acetylcholine antagonist (pIC(50)=5.4). In contrast, it was found to have low efficacy agonist action (pEC(50)=4.4) on alpha7 at concentrations in the range 10-300microM. In agreement with a previous study, ivermectin (30microM), an agonist of L-glutamate-gated chloride channels, enhanced the amplitude of responses to acetylcholine of alpha7 nicotinic receptors. However, this same concentration of ivermectin (30microM) did not potentiate the acetylcholine-induced responses of ACR-16, but rather resulted in a slight attenuation. We conclude that oxantel and ivermectin have identified new pharmacological differences between the chicken alpha7 nicotinic receptor and its C. elegans homologue ACR-16.

Acetylcholine↗

PubMed2000

Flumazenil reversal of carisoprodol (Soma) intoxication.

A 52-year-old woman presented with central nervous system depression and a Glasgow Coma Score of 9 secondary to ingestion of carisoprodol, a centrally acting muscle relaxant analgesic. After administration of i.v. flumazenil, the patient's neurologic status normalized and she required no further therapy. Carisoprodol and its active sedative-hypnotic metabolite, meprobamate, are gamma aminobutyric acid receptor indirect agonists with central nervous system chloride ion channel conduction effects similar to the benzodiazepines, thus making flumazenil a potentially useful antidote in toxic presentations.

Antidotes↗

PubMed1996

Inhibition of regulated neuropeptide secretion from mouse pituitary cells by propofol.

Neuropeptides modulate neuronal responses to stimuli. Secretion of neuropeptides is a potential site for anesthetic action. This paper examines the hypothesis that propofol alters the secretion of beta-endorphin. Cultures of a mouse pituitary cell line (AtT-20) were exposed to propofol in vitro, then induced to secrete beta-endorphin. Secretion was measured by immunoassay. Propofol caused statistically significant inhibition of secretion. Secretion stimulated by phorbol ester was inhibited by propofol with a calculated 50% inhibitory concentration (IC50) value of 48 microM. The propofol IC50 values for secretion stimulated by other secretagogs were 47 microM (barium), 42 microM (Bay K 8644, a calcium channel agonist), and 28 microM (a cyclic adenosine monophosphate [cAMP] analog). AtT-20 cells recovered their ability to secrete beta-endorphin upon removal of the propofol, which demonstrated that they were not damaged permanently by propofol. The effect was relatively specific to neuropeptide secretion, as AtT-20 cells grew normally for 5 days in the presence of 10 or 80 microM propofol. The finding suggests that propofol inhibited a site in neuropeptide exocytosis common to the three studied pathways of secretion.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1997

Effects of protein phosphatase and kinase inhibitors on Ca2+ and Cl- currents in guinea pig ventricular myocytes.

It is well-established that in heart, both the L-type Ca2+ channel and the cystic fibrosis transmembrane conductance regulator Cl- channel are regulated by cAMP-dependent phosphorylation. However, it is not clear whether both of these channels are regulated in concert by protein kinase A (PKA) or whether there are mechanisms that independently control the phosphorylation of these two PKA targets. The purpose of this study was to compare the effects of various protein phosphatase and protein kinase inhibitors on these two ionic currents (ICa and ICl) in guinea pig ventricular myocytes to gain insight into these questions. We found that both the stimulation and washout of the effects of isoproterenol on ICl are about twice as fast as the effects on ICa, probably because the dephosphorylation reaction for ICl is faster than that for ICa. In contrast, inhibition of protein phosphatases with 10 microM microcystin stimulated both ICa and ICl, but the stimulation of ICl was much slower and smaller than the stimulation of ICa. The effect of microcystin was inhibited by staurosporine (Ki = 171.5 and 161 nM for ICa and ICl, respectively), suggesting that the stimulation was due to a kinase. The kinase was not protein kinase C (PKC) because it was not inhibited by the specific pseudosubstrate inhibitor of PKC, PKC(19-31), and it was not PKA because it was not inhibited by adenosine 3',5'-cyclic phosphorothioate. These results suggest that although both the Ca2+ and Cl- channels are regulated by cAMP-dependent phosphorylation, another protein kinase may also regulate these channels, and the kinetics of the response of the channels to phosphorylation can be modulated independently by protein phosphatases.

Adrenergic beta-Agonists↗

PubMed1997

Role of beta1- and beta2-adrenergic receptors in regulation of Cl- and Ca2+ channels in guinea pig ventricular myocytes.

The role of beta1- and beta2-adrenergic receptor stimulation in modulating adenosine 3',5'-cyclic monophosphate (cAMP)-regulated Cl- and Ca2+ currents was investigated with use of guinea pig ventricular myocytes. Activation of the Cl- current by the nonselective beta-receptor agonist isoproterenol (Iso) was not affected by the beta2-receptor antagonist ICI-118,551 (ICI), but it was blocked by the beta1-receptor antagonist atenolol. The inability of beta2-receptor stimulation to activate the Cl- current was confirmed by the lack of response to the selective beta2-receptor agonists salbutamol and zinterol. Responses to beta2-adrenergic receptor stimulation were also looked for in pertussis toxin (PTX)-treated myocytes because PTX increases the sensitivity of responses to Iso, and PTX has been reported to increase the responsiveness to beta2- but not beta1-receptor stimulation. PTX treatment increased the sensitivity of the Cl- current to activation by Iso in the presence of ICI, indicating that PTX increases beta1-receptor responsiveness. PTX treatment also resulted in the ability of salbutamol to activate the Cl- current. However, the response to salbutamol was blocked by atenolol but not by appropriate concentrations of ICI, suggesting that salbutamol was activating beta1-receptors. These results indicate that PTX treatment increases the sensitivity to beta1-receptor stimulation, without affecting beta2-responsiveness. To verify that the lack of response to beta2-receptor stimulation was not unique to the Cl- current, the effects of beta2-receptor agonists on the L-type Ca2+ current were also examined. The Ca2+ current was only affected by high concentrations of zinterol or salbutamol, and such responses were blocked by atenolol, but not by ICI, suggesting that activation of beta1-receptors was involved. These results indicate that beta1- but not beta2-adrenergic receptor stimulation plays an important role in modulating the cAMP-regulated Cl- and Ca2+ currents in guinea pig ventricular myocytes.

Adrenergic beta-Agonists↗

PubMed1989

Sources of calcium for the contraction induced by various agonists in trachealis muscle from normal and sensitized guinea pigs.

Active sensitization of guinea pigs resulted in an increase in the responsiveness and sensitivity of tracheal strips to CaCl2 in K+-depolarized tissue (Emax 0.81 +/- 0.22 g/mm2 and pD2 2.35 +/- 0.10 in normal vs. 0.98 +/- 0.01 g/mm2 and 3.10 +/- 0.08 in sensitized tissue; p less than 0.05), KCl (Emax 0.62 +/- 0.08 g/mm2 and pD2 1.71 +/- 0.03 in normal vs. 0.91 +/- 0.04 g/mm2 and 2.00 +/- 0.01 in sensitized tissue; p less than 0.05) and histamine (Emax 0.70 +/- 0.06 g/mm2 and pD2 5.08 +/- 0.06 in normal vs. 0.94 +/- 0.09 g/mm2 and 5.80 +/- 0.16 in sensitized tissue; p less than 0.05) but not to caffeine 10 mM (20 degrees C, indomethacin 2.8 microM). Generation of responses to these agonists in nonsensitized tissues bathed in a Ca2+-free medium resulted in the abolition of KCl-induced contraction and partial inhibition of the responses to histamine (60% inhibition) and caffeine (40% inhibition). The contraction of sensitized tracheal strips in response to histamine in 0 calcium was greater than that obtained in nonsensitized tissues (0.48 +/- 0.02 g/mm2 vs. 0.28 +/- 0.04 g/mm2, respectively; p less than 0.05). The caffeine-induced contraction of sensitized tracheae was independent of extracellular calcium. These results suggest that a greater calcium entry and/or intracellular calcium release may be an alteration underlying hyperreactivity of sensitized tissues to spasmogens.

Animals↗

PubMed1991

Kinetic evaluation of pharmacological effects based on allosteric coupling of the benzodiazepine/gamma-aminobutyric acidA receptor in the brain.

A mathematical allosteric coupling model has been proposed to describe the process by which binding to the benzodiazepine/gamma-aminobutyric acid (GABA) receptor complex initiates a biological response. The model states that the first receptors (benzodiazepine receptors) can diffuse independently in the plane of the membrane and reversibly associate with the second receptors (GABA receptors) to regulate their activity (induction of increased chloride ion flux due to the opening of the chloride ion channel). The ratio of agonist-bound to total GABA receptor density was defined to be directly proportional to the biological response in the model. The analysis makes the following assumptions: i) the binding affinity of agonists (muscimol or benzodiazepine) to the benzodiazepine receptor/GABA receptor complex is much greater than that to each receptor alone; ii) the double receptor-single agonist (benzodiazepine, muscimol or GABA) ternary complex binds to the other agonist with a high binding affinity as compared with that of each agonist to an agonist-free receptor complex; iii) benzodiazepine receptor-GABA receptor interaction is enhanced in the presence of each agonist; iv) the GABA receptor is desensitized after the binding of GABA agonist (GABA or muscimol) to the receptor. The modeling exercise shows that the benzodiazepine concentration required for half-maximal biological response is lower than that required for half-maximal receptor binding. In the case of the GABA agonist, a linear relationship between receptor occupancy and biological response was observed. The degree of discrepancy between the two profiles (receptor occupancy and biological response) concerning benzodiazepine concentration dependency and time dependency increased with a decrease in the dissociation constants based on the benzodiazepine receptor-GABA receptor interaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain↗

PubMed1992

Activation of T84 cell chloride channels by carbachol involves a phosphoinositide-coupled muscarinic M3 receptor.

Muscarinic agonists stimulate Cl- secretion across monolayers of the colon tumor epithelial cell line, T84. The muscarinic receptor has been characterized in T84 cell homogenates by radioligand binding using [3H]N-methylscopolamine ([3H]NMS). [3H]NMS bound to a single population of sites at 25 degrees C in 100 mM NaCl, 20 mM HEPES, 10 mM MgCl2, pH 7.4 buffer, with calculated Kd = 278 (+/- 44) pM and Bmax = 40 (+/- 6) fmol/mg protein (n = 4). Binding was reversible (diss. t1/2 = 18 +/- 3 min) and stereoselective (dexetimide Ki = 0.3 nM) much greater than levetimide (Ki = 8300 nM). Antagonists exhibited the following rank order of potencies and Ki values (nM): atropine (0.54) greater than 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) (0.84) greater than dicyclomine (14) = hexahydrosiladifenidol (18) greater than pirenzepine (136) greater than AF-DX 116 (3610). The same sequence was observed for inhibition of carbachol-induced 125I efflux from T84 monolayers. This is indicative of an M3 'glandular' muscarinic receptor. Coupling to second messenger systems was examined by labelling monolayers with [14C]arachidonic acid (AA) or [3H]inositol. Carbachol (0.3 mM) did not release [14C]AA from labelled lipids, but ionomycin produced a dose-dependent increase in media [14C]AA. Carbachol (0.3 mM) elevated inositol monophosphate 14-fold. The results suggest that muscarinic agonists stimulate Cl- secretion by interacting with an M3 receptor coupled to inositide lipid hydrolysis.

Arachidonic Acid↗

PubMed2001

EBIO, an agent causing maintained epithelial chloride secretion by co-ordinate actions at both apical and basolateral membranes.

The effect of 1-ethyl-2-benzimidazolone (EBIO) on electrogenic chloride secretion in murine colonic and nasal epithelium was investigated by the short-circuit technique. In the colon, EBIO produces a sustained current increase in the presence of amiloride, which is sensitive to furosemide. In nasal epithelium EBIO causes only a small, transient current increase. Sustained increases in current were obtained in response to forskolin in both epithelia. To examine the mechanisms by which EBIO increases chloride secretion, the effects on intracellular mediators were measured in colonic crypts. There was no effect on [Ca(2+)]i but cAMP content was increased, more so in the presence of IBMX, indicating a direct effect on adenylate cyclase. In colonic epithelia in which the apical surface was permeabilized by nystatin, and the tissue subjected to an apical to basolateral K(+) gradient, EBIO caused a current increase that was entirely sensitive to charybdotoxin (ChTX). In similarly permeabilized colons Br-cAMP caused a current increase that was entirely sensitive to 293B. Thus EBIO increases chloride secretion in the colon by coordinated actions at both the apical and basolateral faces of the cells. These include direct and indirect actions on Ca(2+)-sensitive and cAMP-sensitive K(+) channels respectively, and indirect actions on the basolateral cotransporter and apical CFTR chloride channels via cAMP. In CF colonic epithelia EBIO did not evoke chloride secretion. It is not clear why the nasal epithelium responds poorly to EBIO whereas it gives a sustained response to the related compound chlorzoxazone.

Animals↗

PubMed2004

Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.

To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR.

Animals↗

PubMed1997

Flunitrazepam has an inverse agonistic effect on recombinant alpha6beta2gamma2-GABAA receptors via a flunitrazepam-binding site.

gamma-Aminobutyric acid type A (GABAA) receptor subtypes containing the alpha6-subunit are generally thought to be insensitive to the action of benzodiazepine agonists. We describe the specific binding of the benzodiazepine agonist flunitrazepam to alpha6beta2gamma2-containing GABAA receptors, which has not been observed before and differs from previous reports. With the whole-cell voltage-clamp technique, we observed a functional discrimination between alpha1beta2gamma2- and alpha6beta2gamma2-receptors. Different benzodiazepines had different effects on GABA-evoked chloride currents. The agonist flunitrazepam had an inverse agonistic effect, whereas the antagonist flumazenil increased GABA-induced chloride currents. The action of flunitrazepam on the channel activity of alpha6beta2gamma2-receptors was opposite to its action on alpha1beta2gamma2-receptors. We conclude that flunitrazepam can act as either an agonist or an inverse agonist, depending on the GABAA receptor configuration.

Affinity Labels↗

PubMed2001

Nipecotic acid directly activates GABA(A)-like ion channels.

The GABA-related compound nipecotic acid is commonly used to inhibit GABA uptake. This report shows that nipecotic acid can also directly activate GABA(A)-like chloride channels. When applied to outside-out patches of paraventricular neurones, nipecotic acid (1 mM) activated inward unitary currents (approximately 3 pA at a holding potential of -60 mV, E(Cl)+44 mV). The EC(50) for ion channel activation was approximately 300 microM, 3 fold greater than that found for GABA itself in this preparation. The nipecotic acid activated channels had similar conductance and kinetic properties to those of GABA activated channels in the same patches, reversed near E(Cl) and were inhibited by bicuculline (3 microM). This study indicates that for experiments in which relatively high concentrations of nipecotic acid are used, possible direct GABA(A) receptor agonist properties should be considered.

Animals↗

PubMed1988

Partial agonists of the benzodiazepine receptor: from animal data to results in patients.

The BZR is an allosteric modulatory site of the GABAA receptor, which is unique insofar as it mediates both a positive (facilitatory) and a negative (inhibitory) modulation of the GABA-mediated gating of the chloride channel. Ligands acting as positive modulators are called BZR agonists; ligands producing negative modulation, inverse BZR agonists. The effects of both classes of ligands are blocked by antagonists. A large number of compounds belonging to these three groups of ligands are known. Concepts are presented here that could explain the modulatory function of the BZR, and a number of factors are proposed to determine the pharmacological and therapeutic profile of BZR ligands: intrinsic efficacy at the BZR, density of GABAA-BZR on neurons, functional reserve in GABAA-BZRs, the intensity of the GABAergic input to a neuron, and the excitatory neuronal state. The effect of these factors on the pharmacology of full agonists, partial agonists, antagonists, partial inverse agonists, and inverse agonists is illustrated.

Affinity Labels↗

PubMed1999

Membrane potential and conductance of frog skin gland acinar cells in resting conditions and during stimulation with agonists of macroscopic secretion.

Frog skin glands were stripped of connective tissue and investigated using the nystatin-permeabilized whole-cell patch-clamp configuration. The membrane potential in unstimulated acinar cells was -69.5+/-0.7 mV, and the conductance was dominated by K+, based on ion substitution experiments. The cells were electrically coupled through heptanol- and halothane-sensitive gap junctions. During application of gap junction blockers, the whole-cell current/voltage relationship displayed strong outward rectification. Outward currents were blocked by barium. Stimulation by agonists known to cause increases in either cytosolic cAMP ([cAMP]c) (isoproterenol, prostaglandin E2, both at 2 microM) or free cellular Ca2+ concentration ([Ca2+]c) (noradrenaline, 10 microM, added with propranolol, 5 microM; carbachol, 100 microM) in the frog skin glands caused reversible depolarization: by 34+/-3 mV, 36+/-3 mV, 25+/-3 mV (plateau-phase), and 20+/-3 mV, respectively. Ion substitution experiments showed that stimulation through either pathway (cAMP or Ca2+) resulted in the activation of a Cl- conductance. Application of noradrenaline or adrenaline resulted in a faster depolarization (rates 22 mV/s, 26 mV/s) than stimulation by isoproterenol or prostaglandin E2 (5.6-5.7 mV/s). Cells that were depolarized by exposure to isoproterenol or prostaglandin E2 partially repolarized when stimulated by noradrenaline. The repolarization was blocked by Ba2+ (5 mM) or prazosine (1 microM), consistent with the activation of Ca(2+)-dependent K+ channels via alpha1-adrenergic receptors. We conclude that in the frog skin gland both Ca(2+)-dependent and cAMP-dependent Cl- channels are present in the apical membrane. Increases in free [Ca2+]c in the cAMP-stimulated gland results in the activation of K+ channels, thereby increasing the driving force for Cl- exit.

Adrenergic alpha-Agonists↗

PubMed1997

Genistein directly induces cardiac CFTR chloride current by a tyrosine kinase-independent and protein kinase A-independent pathway in guinea pig ventricular myocytes.

With one-suction electrode voltage-clamp technique, we demonstrated that genistein, a tyrosine kinase (TK) inhibitor, could directly activate cystic fibrosis transmembrane regulator (CFTR) chloride current in guinea pig ventricular myocytes. The activation showed concentration-dependent effect with the estimated IC50 of 39.7 microM. Tyrphostin 51, another TK inhibitor, had no effect, suggesting that genistein's effect might be unrelated to TK inhibition. After the chloride current had been activated by the maximally elevated intracellular cAMP content by saturating concentration of isoproterenol, forskolin and IBMX, genistein could further enhance the current. Pre-treatment with saturating concentration of a specific protein kinase A (PKA) inhibitor, H-89, or other protein kinase inhibitors H-8 and H-9 in the perfusate or intracellularly could not prevent the activation of the current by genistein, suggesting a PKA-independent activity. Furthermore, saturating concentration of calyculin A, a specific inhibitor of phosphotase 1 and 2A, in the perfusate or intracellularly could not block genistein's action. It is possible that genistein opens the channels directly or inhibits the dephosphorylation process of CFTR, which is not sensitive calyculin A.

Adrenergic beta-Agonists↗

PubMed1996

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

Many heterologously expressed mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit residual chloride channel activity that can be stimulated by agonists of the adenylate cyclase/protein kinase A pathway. Because of clinical implications for cystic fibrosis of activating mutants in vivo, we are investigating whether deltaF508, the most common disease-associated CFTR mutation, can be activated in airway epithelial cells. We have found that, 36Cl- efflux can be stimulated 19-61% above baseline by beta-adrenoreceptor agonists and cGI-phosphodiesterase inhibitors in transformed nasal polyp (CF-T43) cells homozygous for the deltaF508 mutation. The increase in 36Cl- permeability is diminished by protein kinase A inhibitors and is not mediated by an increase in intracellular calcium concentrations. Preincubation of CF-T43 cells with CFTR anti-sense oligonucleotides prevented an increase in 36Cl- efflux in response to beta-agonist and phosphodiesterase inhibitor. Primary cells isolated from CF nasal polyps gave similar results. These data indicate that endogenous levels of deltaF508 protein can be stimulated to increase 36Cl- permeability in airway epithelial cells.

1-Methyl-3-isobutylxanthine↗

PubMed1981

Snake alpha-toxin effects on cholinergic and noncholinergic responses of Aplysia californica neurons.

The effects of alpha-toxins from Bungarus multicinctus (alpha BuTX) and Naja naja siamensis (alpha NTX) were studied on synaptic responses and on extrasynaptic responses to focally applied acetylcholine (ACh), histamine (Hm), gamma-aminobutyric acid (GABA), and glutamate (glu) in neurons of the marine mollusc, Aplysia californica. Binding of 125I-alpha BuTX to Aplysia ganglia homogenates was pharmacologically characterized and compared with the pharmacological sensitivity of 125I-alpha BuTX binding to rat brain membrane preparations. Both alpha BuTX and alpha NTX at 10(-5) M reversibly inhibited the ACh-induced increase in Cl- conductance (GCl) without affecting ACh responses mediated by an increase in Na+ conductance (GNa) or an increase in K+ conductance (GK). In addition, both alpha-toxin reversibly inhibited the GCl responses induced by Hm and, in some cases, glu. GABA-induced GCl responses were not affected by either toxin even at concentrations as high as 10(-3) M. Both toxins also inhibited synaptic cholinergic GCl responses as well as the GCl component of postsynaptic potentials mediated by noncholinergic presynaptic neurons. Studies of 125I-alpha BuTX binding to Aplysia ganglia homogenates demonstrated the presence of a single saturable, high affinity site with a dissociation constant (KD) of 3.6 X 10(-9) M. The pharmacological profile of agents which inhibit 125I-alpha BuTx binding to Aplysia ganglia was similar to the profile of vertebrate ganglionic and brain preparations; d-tubocurarine, nicotine, and carbamylcholine effectively and competitively inhibit binding, whereas hexamethonium and decamethonium are less effective and noncompetitive. Strychnine and bicuculline, agents known to block responses to a variety of agonists in Aplysia, were effective competitive inhibitors of toxin binding in Aplysia and in rat brain. Strychnine is not as potent in inhibiting toxin binding to electric organ preparations. Chloride channel blockers, such as penicillin and picrotoxin, and noncholinergic agonists, such as Hm and glu, had no effect on toxin binding. The alpha-toxins appear to inhibit noncholinergic GCl responses through a cholinergic receptor associated with the Cl- conductance mechanism for the noncholinergic agonist.

Acetylcholine↗

PubMed1992

Functionally relevant gamma-aminobutyric acidA receptors: equivalence between receptor affinity (Kd) and potency (EC50)?

Many neurotransmitter receptors bind agonists with high affinity (Kd in the nanomolar range), whereas micromolar concentrations of the same agonists are required to elicit a functional effect. We have identified low affinity agonist binding sites for the gamma-amino-butyric acidA (GABAA) receptor-chloride channel under conditions normally used in 36Cl- uptake assays (a measure of receptor function). The GABAA agonist [3H]muscimol bound to a population of receptors with a Kd (2 microM) similar to its EC50 value for 36Cl- uptake. Binding was inhibited by the GABA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol and by the GABA antagonist bicuculline methiodide. A reduction in the number of [3H]muscimol binding sites (Bmax) by a thiol-modifying reagent produced a corresponding decrease in the Emax for muscimol. The benzodiazepine diazepam enhanced the potency of muscimol in ion flux experiments but did not alter the affinity of [3H]muscimol binding sites. We propose that benzodiazepines enhance GABAergic function by increasing receptor-ion channel coupling, rather than by increasing GABAA receptor affinity. These studies suggest that the study of physiologically relevant (low affinity) binding sites is necessary when examining regulation of receptors by cellular processes, drugs, and disease.

Animals↗