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Biomedical subjects

L G Abood

Publications and source records attributed to L G Abood.

At least 19 recordsLinked to original sources
PubMed1992

4-Bromoacetamidoprocaine: an affinity ligand for brain muscarinic and nicotinic cholinergic receptors.

This study describes the synthesis, receptor binding characteristics, and some behavioral effects of p-bromoacetamidoprocaine (BAP), a new affinity ligand for brain muscarinic and nicotinic cholinergic receptors. The reversible binding of [3H]QNB to rat brain membranes was inhibited in a concentration dependent and saturable manner by both procaine and BAP, with Ki values of 4 x 10(-6) and 3 x 10(-7) M, respectively, and complete inhibition at 1 x 10(-5) M. Both procaine and BAP, although at much concentrations, inhibited the binding of [3H]methylcarbamylcholine in a concentration dependent manner, with Ki values of 5 x 10(-5) and 1 x 10(-5) M, respectively, and complete inhibition for both at 1 x 10(-3) M. Plots of the % irreversible inhibition of [3H]QNB, [3H]nicotine, and [3H]MCC vs [BAP] yielded Ki values of 7 x 10(-8), 1 x 10(-4), and 6 x 10(-5) M, respectively. In behavioral studies BAP was able to antagonize the QNB-induced hyperactivity in mice; however, BAP did not appear to alter nicotine-induced seizure activity or other behavioral effects in mice. A plot of the time course of inhibition by BAP for [3H]QNB binding revealed that the inhibition was almost complete within 10 min exposure at 37 degrees. The findings indicate that BAP is a useful affinity ligand for examining the biochemical and functional characteristics of brain cholinergic receptors, particularly the muscarinic which has an affinity near the nM concentration range.

Animals↗
PubMed1992

Opioid receptor antagonist affinity ligands: 6 beta-bromoacetamido-6-desoxynaltrexone and 6 beta-thioglycolamido-6-desoxynaltrexone.

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6 beta-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6 beta-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [3H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had Ki values of 1 x 10(-9) and 1 x 10(-10) M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5 x 10(-8) and 1 x 10(-8) M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [3H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1 x 10(-7) M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED50 dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.

Affinity Labels↗
PubMed1990

[3H]mecamylamine binding to rat brain membranes. Studies with mecamylamine and nicotine analogues.

Mecamylamine, an antagonist to nicotine, does not compete at the nicotinic recognition site, but is believed to block the ion channel of the nicotinic receptor. The present study demonstrates specific, saturable [3H]mecamylamine binding in rat brain membranes. [3H]Mecamylamine binding was destroyed by heating at 100 degrees and trypsin. Scatchard analysis revealed the presence of two sites with Kd values of 9.6 x 10(-8) and 1.1 x 10(-6) M and Bmax values of 7 x 10(-12) and 3 x 10(-11) mol/mg protein respectively. A good correlation was observed between the Ki values for [3H]mecamylamine binding of a number of mecamylamine and related analogues and their ability to block nicotine-induced prostration in rats and seizures in mice. Inorganic cations, particularly divalent, and various ion channel blockers, such as phencyclidine and verapamil, exhibited a high affinity for the [3H]mecamylamine site. Although mecamylamine did not block nicotine binding, nicotine and its analogues exhibited a high affinity for the [3H]mecamylamine site, a finding which suggests that nicotine acts directly on ion channels as well as the nicotinic cholinergic recognition sites. The data are consistent with the notion that mecamylamine interacts with the open ion channel of the nicotinic receptor.

Animals↗
PubMed1989

Interaction of putative vasopressin receptor proteins of rat brain and bovine pituitary gland with an antibody against a nanopeptide encoded by the reverse message of the complementary mRNA to vasopressin.

An antibody directed against the reverse message of the complementary mRNA for arginine vasopressin was demonstrated to be immunoreactive with 62 and 55 kdalton proteins, obtained by affinity chromatography of rat brain and bovine posterior pituitary extracts and believed to be associated with the vasopressin binding site. A similar pattern of immunoreactivity was seen with an anti-idiotypic antibody for arginine vasopressin.

Animals↗
PubMed1988

Inhibition of phosphoinositide turnover by selective muscarinic antagonists in the rat striatum. Correlation with receptor occupancy.

In the rat corpus striatum, receptor occupancy and the inhibition of phosphoinositide turnover by muscarinic antagonists have been examined under very similar conditions with respect to tissue preparation and buffer composition. The results suggest a good correlation between receptor occupancy and inhibition by muscarinic antagonists, of the carbachol-stimulated turnover of inositol phospholipids, measured by the accumulation of [3H]inositol phosphates in the presence of 5 mM LiCl. In the presence of 10 mM carbachol (CCh), the accumulation of labeled inositol phosphates was increased 8-fold above basal levels (EC50 = 95 microM). Inclusion of antagonists resulted in a dose-dependent inhibition of the 0.1 mM CCh-stimulated inositol phosphate accumulation, with a rank order of potency of atropine greater than trihexyphenidyl greater than pirenzepine greater than or equal to gallamine. Radioligand binding studies with [3H]-l-quinuclidinyl benzilate [( 3H]QNB) in a cell aggregate preparation revealed a single class of saturable, high affinity [3H]QNB binding sites exhibiting a Kd of 74 pM and a Bmax of 2.85 pmol/mg protein. The antagonists examined were able to inhibit the binding of [3H]QNB with the same rank order of potency as for the inhibition of carbachol-stimulated phosphoinositide turnover (atropine greater than trihexyphenidyl greater than pirenzepine greater than or equal to gallamine). Although the inhibition of phosphoinositide turnover and [3H]QNB binding by the nonselective antagonist atropine was best described by interaction at a single site, inhibition of phosphoinositide turnover and [3H]QNB binding by both pirenzepine, which is selective for M1 receptors, and gallamine, which is selective for M2 receptors, is complex. Pirenzepine was much more potent than gallamine for both binding to receptors and inhibiting phosphoinositide turnover. Nonlinear curve-fitting analysis indicated that slope factors for inhibition of phosphoinositide turnover (analogous to Hill coefficient for binding) by only subtype selective antagonists were significantly less than unity. The above-mentioned antagonist interactions together with the apparently multicomponent stimulation of phosphoinositide turnover by carbachol suggest that phosphoinositide turnover may be coupled to more than one muscarinic receptor subtype in the corpus striatum.

Animals↗
PubMed1988

Structure-activity studies of carbamate and other esters: agonists and antagonists to nicotine.

A number of aromatic, cycloalkyl, and heterocyclic carbamic acid esters, thiocarbamic acid esters, and carboxylic acid esters of di- and trial-kylaminoalkyl and heterocyclic amino alcohols have been synthesized and tested for their pharmacologic and receptor binding characteristics at the nicotine receptor. Receptor binding was measured in rat brain membranes using (-)-3H-nicotine or 3H-methylcarbamylcholine as radioligands. The compounds were tested for their ability to produce seizures and prostration and to antagonize the nicotine-induced prostration and seizures as well as the hypertensive action of nicotine in rats. Among the potent agonists were the N-methylcarbamyl and N-methylthiocarbamyl esters of choline (trimethylaminoethanol), with the tertiary amino derivatives between considerably less potent than the quaternary. Potent antagonists included trimethylaminoethyl benzoate, 3-quinuclidinyl benzoate, and trimethylaminoethyl esters of phenyl and phenylthiocarbamic acids. One of the most potent antagonists to nicotine was alpha-lobeline.

Animals↗
PubMed1988

Synthesis and biological characterization of pyridohomotropanes. Structure-activity relationships of conformationally restricted nicotinoids.

The recently discovered nicotinic agonist pyrido[3,4-b]norhomotropane [corrected] (PHT) as well as its N-methyl and 2'-methyl derivatives (syntheses reported herein) were compared with nicotine, nornicotine, and anatoxin a in a series of in vitro and in vivo assays. The results reveal that PHT possesses activity comparable to that of the highly potent agonist, anatoxin a. The inactivity observed relative to PHT of N-methyl- and 2'-methyl-PHT has helped to further define the structure-activity requirements of conformationally restricted nicotinoids.

Animals↗
PubMed1987

Receptor binding characteristics of a 3H-labeled azetidine analogue of nicotine.

A new radioligand, (+/-)-[3H]-1-methyl-2-(3-pyridyl)-azetidine, which is an analogue of nicotine, has been used to investigate the binding characteristics of the nicotine receptor in rat brain membranes. By Scatchard analysis, the azetidine analogue yielded a curvilinear plot with Kd values of 7 X 10(-11) and 1.7 X 10(-9) M and Bmax values of 0.3 X 10(-14) and 2.5 X 10(-14) mol/mg protein respectively. Thermodynamic analyses yielded negative free enthalpy values for both sites, a decrease in the Bmax of only the lower affinity site, and no effect on either Kd. The psychotropic potency (prostration in rats following intraventricular injection) of the azetidine analogue was about 5-fold greater than (-)-nicotine, being among the greatest of any known nicotine analogues tested to date. Since only the higher affinity Kd differed from that of (-)-nicotine, 3-fold greater, the psychotropic potency appears to be correlated with the higher affinity site. Insofar as [3H]methylcarbamylcholine, a nicotinic ligand resembling acetylcholine, exhibits a linear Scatchard with a Kd of 1 X 10(-9) M, the higher affinity site appears to be characteristic of nicotine analogues.

Animals↗
PubMed1987

Solubilization and purification of the Ni-stimulated arginine-vasopressin binding site of rat brain membranes.

Arginine vasopressin binding sites on rat brain membranes were solubilized and purified by affinity chromatography. Membrane protein solubilized with CHAPS bound arginine vasopressin (AVP) only in the presence of divalent cations. Specific binding to the solubilized tissue was maximally stimulated by Ni2+, and markedly stimulated by Co2+ (30% of maximal binding with Ni2+), Zn2+ (18%), and Fe2+ (11%), parallel to the effects of these ions on the binding of AVP to neural membranes. Binding to solubilized tissue was not stimulated by Mg2+, Cu2+, Mn2+, or Ca2+. In the presence of Ni2+, binding of AVP to solubilized tissue was reversible, and the dissociation constant (10.5 nM), pH optimum, and time course were virtually identical to those of the membrane-bound AVP binding site. Purification of solubilized AVP-binding proteins by affinity chromatography on AVP-sepharose followed by gel electrophoresis yielded a major band of 55 kdalton molecular weight when purified in the presence of 5 mM Mg2+, or a major band of 62 kdaltons when purified in the presence of 1-5 mM Ni2+ or 10 microM Zn2+. By means of a new binding assay involving conjugation of the 62 kdalton fraction to brain membranes, the extent of purification of AVP binding activity was 150-fold in the presence of Ni2+. We suggest that the 62 kdalton protein is a component of the Ni-stimulated AVP binding site.

Animals↗
PubMed1987

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies.

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [3H]nicotine binding to the purified material. An enantiomeric analogue of nicotine, (-)-6-hydroxymethyl-nicotine, was used to prepare the affinity column. In addition, with the use of anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [3H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of stereospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[3H]nicotine-binding characteristics.

Animals↗
PubMed1986

[3H]methylcarbamylcholine, a new radioligand for studying brain nicotinic receptors.

A new radioligand, [3H]methylcarbamylcholine, has been developed for the study of the nicotinic cholinergic and nicotine-like binding sites in rat brain membranes. A Scatchard analysis with the radioligand yielded a Kd of 1.1 X 10(-9) M and a Bmax of 4.0 X 10(-14) moles/mg protein which compares with a lower affinity site for (-)-[3H]nicotine having a Kd of 3 X 10(-9) M and a Bmax of 2 X 10(-14) moles/mg. Comparable values for the Kd were obtained from a Hill plot and from calculations based on rate constants for association and dissociation. A comparison of the binding affinities of various nicotine analogues, nicotinic cholinergic agents, and other neurotropic agents revealed a close similarity between the two radioligands, with the exception that quaternization of nicotine or carbamate esters increased affinity by at least an order of magnitude with [3H]methylcarbamylcholine and resulted in a comparable decrease in affinity with [3H]nicotine as the ligand. The binding of [3H]methylcarbamylcholine, like [3H]nicotine, was not displaceable by muscarinic cholinergic antagonists. It was concluded that, although [3H]methylcarbamylcholine and [3H]nicotine bind to a common receptor in brain, the functional and chemical characteristics of the receptor(s) differ in some respects from peripheral nicotinic cholinergic receptors.

Animals↗
PubMed1986

Effects of the affinity ligands 14-beta-chloroacetylnaltrexone and 14-beta-bromoacetamidomorphine on [3H]-dihydromorphine binding sites in rat brain.

The aim of the present study was to examine the inhibitory effects in vitro of the affinity ligands 14-beta-chloroacetylnaltrexone (CAN) and 14-beta-bromoacetamidomorphine (BAM) to characterize the pharmacological specificity of the ligands for high and low affinity opioid binding sites. Rat brain membranes were incubated with 2.0 microM BAM or CAN, or their parent compounds (morphine and naltrexone, respectively) at 37 degrees for 45 min, and the membranes were washed extensively to remove the unbound ligand. The specific binding of 0.3 nM [3H]dihydromorphine ([3H]DHM) was reduced 32 +/- 7% in membranes treated with CAN and BAM, whereas specific binding in preparations treated with morphine and naltrexone was not significantly different from controls. An increased affinity of BAM and CAN relative to morphine and naltrexone could not account for the observed irreversible inhibition, since the relative affinity of CAN was similar to that of naltrexone and that of BAM was 10-fold less active than morphine. Saturation binding assays revealed that the affinity ligands selectively abolished a high affinity binding site (Kd = 0.3 nM, Bmax 95 fmoles/mg protein), which comprised approximately one-third of the total number of sites. The affinity of the remaining site (Kd = 4.0 nM) was not altered significantly. The results indicate that the inhibition caused by the affinity ligands is irreversible and represents inactivation of high affinity opioid binding sites in a relatively selective manner.

Animals↗
PubMed1986

Mechanism of phospholipase A2-induced conduction block in bullfrog sciatic nerve. I. Electrophysiology and morphology.

The effects of exogenously added phospholipase A2 (PLA2) and its hydrolytic products in isolated bullfrog sciatic nerve were investigated. Nerves were pretreated for 3 h with a dose of trypsin which did not affect conduction in order to enhance penetration of the added agents. Treatment of nerves with beta-glucosidase, neuraminidase or chymotrypsin had no effect on conduction. Whereas incubation of the nerves with normal Ringers for 2 h had no significant effect on conduction, incubation with PLA2 in Ringers caused decrements in the height of the compound action potential in a dose-related manner. In addition, incubation of the nerves with 10 mg/ml lysolecithin, arachidonic acid, or docosahexaenoic acid caused marked decrements in the height of the compound action potential. Electron microscopic analysis of nerves after each treatment which caused conduction block revealed varying levels of myelin damage. Although myelin was damaged at the paranodal and/or internodal region, depending on the agents used, the axonal membrane appeared to be intact at the ultrastructural level. It was concluded that the block in conduction resulting from PLA2 was due to the formation of lysolecithin and long chain polyunsaturated fatty acids.

Animals↗
PubMed1986

Mechanism of phospholipase A2-induced conduction block in bullfrog sciatic nerve. II. Biochemistry.

The biochemical changes associated with conduction block following exogenous application of purified phospholipase A2 to bullfrog sciatic nerves were investigated. Nerves were treated with concentrations of phospholipase A2 needed to produce at least a 50% decrease in the compound action potential after a 2-2 1/2 h incubation. This phospholipase A2-induced conduction block was associated with lipid hydrolysis, depletion of high energy phosphates and decreases in [3H]saxitoxin binding. Forty-two percent of the nerve phosphatidylcholine and 45% of the nerve phosphatidylethanolamine were hydrolyzed. [3H]Saxitoxin binding was decreased by 85% in association with conduction block attained with phospholipase A2 incubation. There were significant decrements in adenosine triphosphate levels (-58%) and in phosphocreatine levels (-78%), but no difference in the specific activities of these phosphate compounds. It is concluded that a number of mechanisms can account for the conduction failure resulting from phospholipase A2, including disruption of sodium channels needed for propagation of regenerative nerve impulses and the depletion of high energy phosphates needed to maintain ionic gradients.

Adenine Nucleotides↗
PubMed1986

Barrel rotation and prostration by vasopressin and nicotine in the vestibular cerebellum.

The aim of this study was to determine whether the primary sites for the action of vasopressin and nicotine in producing barrel rotation and prostration in rats were located in the modular cerebellum, i.e., lobule X. When arginine vasopressin was administered into either the fourth ventricles or directly into the nodular cerebellum via chronically implanted cannulae, the rats displayed intermittent barrel rotation and clonic convulsions. The administration of nicotine into the same areas resulted in prostration, atonia and, occasionally, clonic convulsions. A few days after the nodular cerebellum was lesioned with kainic acid, the motor disturbances resulting from either agent were virtually abolished. Histologic studies revealed that kainic acid had destroyed Purkinje and other large neurons, but had left the granular neurons relatively intact. The administration of procaine into either the fourth ventricles or nodular cerebellum blocked the behavioral responses of either vasopressin or nicotine given into the fourth ventricles. It was concluded that the nodular cerebellum is a primary site for the motor disturbances produced by vasopressin and nicotine.

Animals↗