PubMed Health⌕ Search

Biomedical subjects

R K Gordon

Publications and source records attributed to R K Gordon.

At least 37 records · Page 2Linked to original sources

Distance geometry of alpha-substituted 2,2-diphenylpropionate antimuscarinics.

Quantitative structure-activity relationships between pharmacological activities and physical properties of a series of 2,2-diphenylpropionate compounds were used to define the topography of the antagonist binding site of muscarinic receptors. XICAMM, a computer molecular modeling program, was used to calculate geometrical and topological values of the compounds. The compounds were tested for their antimuscarinic activities by: (a) inhibition of [N-methyl-3H]scopolamine binding to the muscarinic receptors of N4TG1 neuroblastoma cells, (b) inhibition of carbachol-induced alpha-amylase release from rat pancreas acini, and (c) blocking of acetylcholine-induced contraction of guinea pig ileum. To evaluate as clearly as possible only the effect of the bond distance on the potency of the synthesized antimuscarinics, the compounds contained as many constant features as possible. Neither the hydrophobic nor the ester moieties of the compounds were changed, and the rings containing the protonated nitrogen were saturated and restricted. The antimuscarinic activities obtained from the three assays were significantly correlated with each other, with the exception of two compounds, 9 and 13. The latter two compounds demonstrated specificity for the m3 muscarinic receptor subtype expressed in the pancreas. Furthermore, it was demonstrated that the antimuscarinic activities were significantly related to the bond distances between the carbonyl oxygen (constant electronegative locus) and the protonated nitrogen (center of cationic charge) of the 2,2-diphenylpropionate compounds. Parabolic relationships between the pharmacological activities and bond distances were empirically established. The shortest calculated bond distance of these compounds was approximately 4.4 A, whereas the longest was about 5.9 A. The maximum antimuscarinic potency was observed with a calculated bond distance of about 5.2 A in all three assays.

Animals↗

A microtechnique for quantification of detergent-solubilized muscarinic and nicotinic acetylcholine receptors using a semi-automated cell harvestor.

A specific method for the rapid assay of muscarinic acetylcholine receptors (mAChR), either detergent-solubilized or in neuroblastoma cells, is described. This method is also applicable to the assay of nicotinic acetylcholine receptors. The procedure employs a cell harvestor and microtiter plates, and has the advantage of requiring small quantities of radioligand, microgram quantities of detergent-solubilized cholinergic receptor or less cells. The binding parameters such as the equilibrium dissociation constants (Kd) of mAChR and nicotinic acetylcholine receptor (nAChR) and inhibition constants (Ki) for antagonists determined by the present method are in excellent agreement with values determined by other methods. This assay procedure for mAChR and nAChR should facilitate the rapid screening of cholinergic agonists/antagonists and also the further purification and characterization of mAChR.

Animals↗

Comparison of in vitro actions with behavioral effects of antimuscarinic agents.

In vitro potencies of a series of muscarinic antagonists were compared with their effects on operant behavior. Ki values for inhibition of [3H]N-methylscopolamine binding in N4TG1 neuroblastoma cells correlated positively with ED50 values for the inhibition of carbachol-induced alpha-amylase release from pancreatic acini cells and with KB values for inhibition of acetylcholine-induced contractions of guinea pig ileum. The rank order of potency for inhibition of [3H]N-methylscopolamine binding was quinuclidinyl benzilate = quinuclidinyl xanthene-9-carboxylate greater than (methyl atropine = atropine) greater than benactyzine greater than azaprophen greater than (adiphenine = aprophen) greater than pirenzepine greater than ethyl aprophen. The M1 antagonist, pirenzepine, was a weak inhibitor in the guinea pig ileum and alpha-amylase assays relative to its ability to inhibit [3H]N-methylscopolamine binding; azaprophen exhibited the opposite relationship. Lever-press responses of rats were maintained by food delivery under a schedule requiring 10 responses for each food presentation. The high response rates engendered by this schedule were decreased in a dose-dependent manner by all compounds. The order of potency for this behavioral effect (ED50) was atropine = azaprophen greater than aprophen greater than (methyl atropine = benactyzine) greater than pirenzepine greater than adiphenine. Behavioral depressant actions of the antimuscarinics correlated positively with their potencies in inhibiting alpha-amylase secretion. Pirenzepine was unique in being relatively more potent in its behavioral effects than in its actions in vitro. In contrast to the other antimuscarinic agents studied, the benzilates, benactyzine, aprophen and adiphenine, but not azaprophen, increased behavioral response rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Endocytosis of particulate and soluble IgG immune complexes: differential effects of cytoskeletal modulating agents.

We determined the ability of cytoskeletal modulating agents to affect endocytosis of particulate and soluble immune complexes. Either immunoglobulin G-opsonized 51Cr-labelled erythrocytes (E) or 125I-labelled soluble IgG anti-dinitrophenyl immune complexes (IC) were added to adherent murine peritoneal macrophages and the extent of internalization was measured. Either intracellular 51Cr radioactivity or trichloroacetic acid soluble 125I radioactivity was monitored as an indication of uptake by the cell. In addition to cytochalasin B and colchicine, the phospholipid methylation inhibitors, 3-deazaadenosine and 3-deaza(+/-)aristeromycin, were used. Whereas incubation of macrophages with colchicine alone resulted in 50% inhibition of uptake of E-IgG, there was no effect on the degradation of soluble IC. Both cytochalasin B and colchicine produced a 95% inhibition of E-IgG uptake by macrophages, but these drugs only minimally inhibited (17%) the degradation of soluble immune complexes. Both inhibitors of methylation produced a 50% decrease in phospholipid methylation in treated cells. However, only 3-deazaadenosine inhibited phagocytosis (50% of control for E-IgG and 75% of control for soluble IC). These data suggest that an intact cytoskeleton is necessary for the uptake of a particulate immune complex and much less important for the internalization and degradation of these model soluble immune complexes. In addition, inhibition of phospholipid methylation reactions alone do not impare the uptake and degradation of either a soluble or a particulate immune complex.

Adenosine↗

Enzymatic synthesis of a false cholinergic neurotransmitter: diethylaminoethyl acetate as a muscarinic agonist analog of acetylcholine.

Diethylaminoethyl acetate, an acetylcholine analog, was formed upon the incubation of diethylaminoethanol and acetyl-CoA with bovine brain choline acetyltransferase (acetyl-CoA: choline O-acetyltransferase; EC 2.3.1.6). The new product co-chromatographed with authentic diethylaminoethyl acetate on thin layer plates, and its formation was proportional to the duration of incubation and enzyme concentrations. When tested on guinea-pig ileum, diethylaminoethyl acetate was found to be an agonist with an ED50 of 1.3 X 10(-4) M, compared to an ED50 of 2.0 X 10(-7) M for acetylcholine. The contraction of guinea-pig ileum induced by diethylaminoethyl acetate was blocked by atropine. Moreover, diethylaminoethyl acetate induced a secretion of alpha-amylase from isolated pancreatic acini cells; this effect was also blocked by atropine. It is entirely possible that diethylaminoethyl acetate can be a false cholinergic transmitter generated in vivo when drugs such as aprophen or procaine are administered to animals, since either of these drugs can undergo enzymatic hydrolysis to generate diethylaminoethanol. A method for the synthesis of radioactive diethylamino [1,2-14C]ethyl acetate was also described.

Acetylcholine↗

Tumor cell autocrine motility factor.

A cell motility-stimulating factor has been isolated, purified, and partially characterized from the serum-free conditioned medium of human A2058 melanoma cells. We term this activity "autocrine motility factor" (AMF). AMF has the properties of a protein with an estimated size of 55 kDa. At concentrations of 10 nM or less, AMF stimulated the random or directed motility of the producer cells. However, AMF is not an attractant for neutrophils. Amino acid analysis of the purified AMF protein revealed a high content of serine, glycine, glutamic acid, and aspartic acid residues. The activity of AMF was not replaced or blocked by known growth factors such as epidermal growth factor or type beta transforming growth factor. Mechanistic studies showed that AMF stimulated the incorporation of [3H]methyl into cell membrane phospholipids after incubation with [methyl-3H]methionine with a sustained increase in the methylation of phosphatidyldimethylethanolamine to phosphatidylcholine. In contrast, AMF did not affect the incorporation of [1,2-14C]choline into phosphatidylcholine. AMF was produced in large amounts by three different clones of ras oncogene-transfected metastatic NIH 3T3 cells but not by the nontransformed parental cells. AMF may play a major role in the local invasive behavior of tumor cells and may also facilitate the concerted invasion by groups of tumor cells.

Animals↗

Disposition of aprophen in rats.

The pharmacokinetics of [14C]aprophen and its distribution were determined after intravenous administration to rats. The drug was distributed rapidly with a t1/2 (alpha) of 4 min to highly perfused organs like the brain, kidney and adrenals. An elimination phase was apparent 10 min after injection with a t1/2 (beta) of 23.5 min. The high plasma clearance of the drug was due both to a large volume of distribution and to a high metabolic rate. Aprophen could be hydrolysed to diphenylpropionic acid and diethylaminoethanol in-vivo and in-vitro. Diethylaminoethanol competed with [3H]QNB binding to muscarinic receptors of N4TG1 cells, whereas diphenylpropionic acid did not. The lower plasma concentrations and lower binding activity of diethylaminoethanol compared with aprophen indicate that unchanged aprophen is largely responsible for the in-vivo actions.

Animals↗

Induction of HL-60 cell differentiation by 3-deaza-(+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase.

The metabolically stable inhibitor of S-adenosylhomocysteine hydrolase (AdoHcyase), 3-deaza-(+/-)-aristeromycin (dzAri) has recently been shown to induce differentiation in HL-60 cells. The present study was undertaken to characterize the cytostatic, cytotoxic, and differentiation inducing properties of dzAri in HL-60 cells and to investigate biochemical consequences of AdoHcyase inhibition. A dye exclusion test and a clonogenic assay were used to test cytotoxic and cytostatic properties. dzAri had reversible cytostatic effects on HL-60 cells at concentrations lower than 10 microM and partially reversible cytotoxic effects above 10 microM. The induction of differentiation was dependent upon concentration and time of exposure, with maximal effect after 6 days incubation with 5-10 microM dzAri. Washout experiments demonstrated that the cells were not committed to differentiation after 48 h of incubation with dzAri. The AdoHcyase of HL-60 cells was inhibited with a Ki of 20 nM. The concentration of S-adenosylhomocysteine increased dose dependently 48 h after incubation with 0.1-100 microM dzAri. The incorporation of [3H]methyl from [methyl-3H]methionine into 5-methylcytosine of DNA was reduced by 26% at 5 microM dzAri. The findings indicate that continuous presence of dzAri is necessary to induce differentiation and inhibit proliferation in HL-60 cells. The inhibition of AdoHcyase perturbs levels of transmethylation metabolites and the incorporation of [3H]methyl into 5-methyl-cytosine of DNA.

Adenosine↗

Antimuscarinic activities of hycanthone analogs: possible relationship with animal toxicity.

The antimuscarinic activity of hycanthone and five antischistosomal analogs was determined in three biological assays of cholinergic systems. A linear relationship was established between the LD50 values of hycanthone analogs in mice and 1) the Ki values obtained from the inhibition of [3H]quinuclidinyl benzilate binding to the muscarinic receptors of N4TG1 neuroblastoma cells; 2) the I50 values obtained from the inhibition of alpha-amylase secretion induced by carbachol in pancreatic acini cells; and 3) the KB values obtained from the inhibition of guinea-pig ileum contraction induced by acetylcholine. The linear relationship established between antimuscarinic potency and toxicity in mice suggests that a possible relationship exists between the toxicity of the hycanthone analogs and their antimuscarinic activities. On the other hand, no correlation was established between antischistosomal efficacy and antimuscarinic potency. The Ki and I50 values ranged from 10(-7) to 10(-5) M for the inhibition of the binding of [3H]quinuclidinyl benzilate to the muscarinic receptors and for the inhibition of alpha-amylase secretion. The KB values determined by the guinea-pig ileum assays were approximately 10(-5) to 10(-6) M. The ranking of antimuscarinic potency of the compounds in the three different assays were in good agreement.

Animals↗

Antimalarial activity of neplanocin A with perturbations in the metabolism of purines, polyamines and S-adenosylmethionine.

Neplanocin A, a novel carbocyclic analog of adenosine, was found to be a mixed type inhibitor of S-adenosylhomocysteine hydrolase of human red blood cells with a Ki of 2 nM and an inactivating constant, Ki, of 3 nM. When tested against Plasmodium falciparum cultured in human red blood cells, neoplanocin A inhibited malarial growth with an ED50 of 3 microM. Above 2.5 microM, some red blood cells showed morphological aberrations and became echinocytes (spiculated red blood cells). In infected red blood cells, neplanocin A caused an elevation of the concentrations of S-adenosylmethionine and S-adenosylhomocysteine in a dose-dependent manner. Concurrently, a new analog of S-adenosylmethionine, S-neplanocinylmethionine, was formed. Analysis of polyamines showed that only putrescine was decreased significantly; the others were not changed. Purine analyses showed two putative neplanocinyl nucleotides, possibly the di-and the triphosphates. Neplanocin A most likely exerted its in vitro antimalarial effect via the inhibition of S-adenosylhomocysteine hydrolase and the attendant perturbation of methylation reactions.

Adenosine↗

Inhibition of muscarinic receptor binding and acetylcholine-induced contraction of guinea pig ileum by analogues of 5'-(isobutylthio)adenosine.

(Isobutylthio)adenosine (SIBA, 1) and its derivatives have been shown to produce a variety of biological effects on the basis of the hypothesis that such agents act directly as inhibitors of transmethylation reactions, as inhibitors of S-adenosylhomocysteine hydrolase, or as inhibitors of polyamine biosynthesis. We report here the ability of selected analogues of SIBA to inhibit the binding of the muscarinic antagonist quinuclidinyl benzilate (QNB) to cultured N4TG1 neuroblastoma cells and to antagonize the acetylcholine-induced contraction of guinea pig ileum. The most potent inhibitors were 5'-deoxy-5'-(isobutylthio)-1-deazaadenosine (1-deaza-SIBA, 5) and 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine (3-deaza SIBA, 3), while the parent nucleoside SIBA and the carbocyclic derivative 5'-(isobutylthio)-3-deazaaristeromycin were less active. The same agents had no effect on the nicotinic receptors of NG108-15 neuroblastoma X glioma hybrid cells. The acyclic derivative 9-[[2-(isobutylthio)ethoxy]methyl]adenine, 3-deazaadenosine, 5'-(isobutylthio)tubercidin, and 5'-(isobutylamino)adenosine were inactive at the 1-mM level. These results suggest that SIBA and 3-deaza-SIBA may have profound effect on membrane-mediated phenomenon, including inhibition of muscarinic receptor binding.

Acetylcholine↗

24-hour ambulatory manometry in diagnosis of esophageal motor disorders causing chest pain.

The clinical diagnosis of motor disorders of the esophagus leading to chest pain usually lacks documentation by manometry despite the use of sophisticated equipment as well as pharmacologic manipulation. We have developed an ambulatory manometry system that allows us to monitor motility in the esophagus on an outpatient basis for 24 hours. This method seems well tolerated, and tracings obtained are of excellent quality, allowing careful inspection and evaluation of details. Simultaneous 24-hour esophageal pH and surface ECG recordings were also obtained. We believe the technique, when perfected, will significantly enhance the understanding and documentation of the cause of chest pain in patients with motor dysfunction of the esophagus.

Ambulatory Care↗

Analysis of S-adenosylmethionine and related sulfur metabolites in animal tissues.

A high-performance liquid chromatographic method was devised that separates S-adenosylmethionine and related sulfur metabolites on a Radial-PAK SCX cation-exchange column using a four-step NH4COOH/(NH4)2SO4 elution gradient. This new procedure permits, in a single run of 60 min, the quantitative analysis of S-adenosylmethionine, S-adenosylhomocysteine (AdoHcy), 5'-deoxy-5'-methylthioadenosine, decarboxylated S-adenosylmethionine, decarboxylated AdoHcy, inosylhomocysteine, and other related metabolites. Furthermore, this method allows the detection in rat tissues of novel sulfur metabolites, S-inosylhomocysteine and decarboxylated AdoHcy. Perturbation of the levels of some of these metabolites could be detected in rat livers and spleens after the administration of 3-deazaadenosine, an inhibitor of AdoHcy hydrolase, but could not be detected in rat adrenal glands. It is notable that decarboxylated AdoHcy disappeared in the livers of rats treated with 3-deazaadenosine. HeLa cells incubated with [35S]methionine displayed the incorporation of the labeled sulfur into S-adenosylmethionine, AdoHcy, decarboxylated S-adenosylmethionine, S-inosylhomocysteine, and 5'-deoxy-5'-methylthioadenosine.

Adrenal Glands↗

Inhibition of adenosylmethionine decarboxylase and perturbation of polyamine metabolism by 3-deaza-(+/-)aristeromycin.

3-Deaza-(+/-)aristeromycin, previously known mainly as a potent inhibitor of adenosylhomocysteine hydrolase, can also inhibit the activity of adenosylmethionine decarboxylase. The release of [14C]CO2 from HeLa cells labeled with [carboxyl-14C]methionine was inhibited by more than 70% after 4 hours in the presence of 4 microM 3-deaza-(+/-)aristeromycin. Concomitant with this inhibition, there was a significant increase in the amount of putrescine in the HeLa cells. Adenosylmethionine decarboxylase isolated from HeLa cells could also be inhibited by 3-deaza-(+/-)aristeromycin and 3-deazaadenosine, 3-deazaadenosylhomocysteine, and 3-deaza-(+/-)aristeromycinylhomocysteine.

Adenosine↗