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J R Hammond

Publications and source records attributed to J R Hammond.

At least 37 records · Page 2Linked to original sources
PubMed1991

Comparative pharmacology of the nitrobenzylthioguanosine-sensitive and -resistant nucleoside transport mechanisms of Ehrlich ascites tumor cells.

A variety of nucleoside transport inhibitors and substrates were compared for their capacities to inhibit the zero-trans influx of [3H]uridine in Ehrlich ascites tumor cells. ATP-depleted cells accumulated [3H]uridine primarily by facilitated diffusion (Vmax = 16 pmol/sec/microliter cell water) via both nitrobenzylthioguanosine (NBTGR)-sensitive (IC50 = 0.53 nM, 100 microM [3H]uridine) and NBTGR-resistant (IC50 = 71 microM, 100 microM [3H]uridine) mechanisms with uridine Km estimates of 99 and 284 microM, respectively. Dilazep also distinguished between the transporter subtypes with IC50 values of 1.4 nM and 1.8 microM, respectively, for inhibiting 100 microM [3H]uridine influx. Incubation of cells with 50 nM NBTGR allowed the selective study of inhibitor effects on NBTGR-resistant [3H]uridine influx. Dipyridamole, cyclopentyladenosine, 2-phenylaminoadenosine, etoposide, teniposide, diazepam, chlordiazepoxide, triazolam and the lidoflazine derivative 2-(aminocarbonyl)-N-(4-amino-2,6-dichlorophenyl)-4-[5,5-bis-(4- fluorophenyl)pentyl]-1-piperazineacetamide (R75231), were significantly less potent as inhibitors of NBTGR-resistant influx, when compared with their capacities to inhibit the total mediated influx of [3H]uridine. In contrast, 2-fluoroadenosine, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine and soluflazine were relatively more effective as inhibitors of the NBTGR-resistant component. Mioflazine, a compound related to both soluflazine and R75231, did not distinguish between transporter subtypes. The NBTGR-resistant transporter also had a distinctive substrate specificity; guanosine, 2'-deoxyguanosine, cytidine and 2'-deoxycytidine were significantly less effective as inhibitors of NBTGR-resistant [3H]uridine influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Kinetic analysis of ligand binding to the Ehrlich cell nucleoside transporter: pharmacological characterization of allosteric interactions with the [3H]nitrobenzylthioinosine binding site.

Kinetic analysis of the binding of [3H]nitrobenzylthioinosine ([3H] NBMPR) to Ehrlich ascites tumor cell plasma membranes was conducted in the presence and absence of a variety of nucleoside transport inhibitors and substrates. The association of [3H] NBMPR with Ehrlich cell membranes occurred in two distinct phases, possibly reflecting functional conformation changes in the [3H]NBMPR binding site/nucleoside transporter complex. Inhibitors of the equilibrium binding of [3H]NBMPR, tested at submaximal inhibitory concentrations, generally decreased the rate of association of [3H]NBMPR, but the magnitude of this effect varied significantly with the agent tested. Adenosine and diazepam had relatively minor effects on the association rate, whereas dipyridamole and mioflazine slowed the rate dramatically. Inhibitors of nucleoside transport also decreased the rate of dissociation of [3H]NBMPR, with an order of potency significantly different from their relative potencies as inhibitors of the equilibrium binding of [3H]NBMPR. Dilazep, dipyridamole, and mioflazine were effective inhibitors of both [3H]NBMPR dissociation and equilibrium binding. The lidoflazine analogue R75231, on the other hand, had no effect on the rate of dissociation of [3H]NBMPR at concentrations below 300 microM, even though it was one of the most potent inhibitors of [3H]NBMPR binding tested (Ki less than 100 nM). In contrast, a series of natural substrates for the nucleoside transport system enhanced the rate of dissociation of [3H]NBMPR with an order of effectiveness that paralleled their relative affinities for the permeant site of the transporter. The most effective enhancers of [3H]NBMPR dissociation, however, were the benzodiazepines diazepam, chlordiazepoxide, and triazolam. Comparable effects of adenosine and dipyridamole on [3H]NBMPR dissociation rate were obtained upon solubilization of the membranes with octylglucoside, suggesting that this phenomenon was not due to changes in membrane fluidity. These results are compatible with the existence of specific ligand recognition sites on the nucleoside transport complex of Ehrlich cells that are pharmacologically distinct from, but allosterically linked to, the high affinity binding sites for [3H]NBMPR. The marked effects on [3H]NBMPR binding kinetics that result from ligand interactions with these sites must be considered in the design and analysis of all studies involving the use of [3H]NBMPR as a high affinity probe for the nucleoside transport system.

Affinity Labels↗
PubMed1989

Solubilization and reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Uptake of [3H]uridine by Ehrlich cells was mediated by both nitrobenzylthioinosine (NBMPR)-sensitive (75%) and NBMPR-insensitive (25%) mechanisms. Each cell contained approx. 26,000 high-affinity (KD = 0.19 nM) recognition sites for [3H]NBMPR, and binding was inhibited by dipyridamole and adenosine at concentrations similar to those required for inhibition of [3H]uridine uptake. Calculations show that each cell contains a total of about 35,000 nucleoside transporters. Photoaffinity labelling of a partially purified preparation of plasma membranes with [3H]NBMPR resulted in a single broad 3H-labelled band on SDS/polyacrylamide gels, with an apparent molecular-mass peak of 42 kDa. This is in contrast with human erythrocyte membranes, where [3H]NBMPR photolabelled two broad bands with peaks at 55 and 80 kDa. Treatment of photoaffinity-labelled membranes with endoglycosidase F decreased the apparent molecular masses of both the Ehrlich-cell and erythrocyte [3H]NBMPR-labelled proteins to approx. 40 kDa. These results suggest that the human erythrocyte [3H]NBMPR-binding polypeptides are more extensively glycosylated than the corresponding Ehrlich-cell polypeptides. Octyl beta-D-glucopyranoside [1.0% (w/v) + asolectin] solubilized over 90% of the [3H]NBMPR-binding sites, with near-complete retention of [3H]NBMPR-binding characteristics. The only major change was a 65-fold decrease in affinity for dipyridamole, which was partly reversed upon incorporation of the solubilized proteins into asolectin membranes. Proteoliposomes, prepared by using asolectin and the octyl glucoside-solubilized plasma membranes, were capable of accumulating [3H]uridine via a protein-dependent dipyridamole/nitrobenzylthioguanosine/dilazep-sensitive mechanism. We have thus demonstrated the efficient solubilization and functional reconstitution of a nucleoside-transport system from Ehrlich ascites-tumour cells.

Adenosine↗
PubMed1989

Enhanced efflux of [3H]vinblastine from Chinese hamster ovary cells transfected with a full-length complementary DNA clone for the mdr1 gene.

Multidrug-resistant Chinese hamster ovary cell clones stably transfected with, and overexpressing, the mouse mdr1 complementary DNA clone along with drug-sensitive Chinese hamster ovary control cells were characterized for their capacities to accumulate and retain [3H]vinblastine. Multidrug-resistant mdr1 transfectants show a 3-4-fold decrease in [3H]vinblastine accumulation, compared to their drug-sensitive counterparts. After ATP depletion, this difference in [3H]vinblastine accumulation between mdr1 transfectants and control cells effectively disappears. This ATP-dependent decreased drug accumulation is paralleled in mdr1 transfectants by an enhanced capacity of these cells to extrude the drug in an ATP-dependent manner. In medium containing glucose and glutamine, the mdr1 transfectants release preloaded drug at a rate five times that of control, drug-sensitive cells. In ATP-depleted control and mdr1-transfected cells, there is little difference in the rate or extent of [3H]vinblastine release. The observation that the mdr1 transfectants show a decreased [3H]vinblastine accumulation and an increased vinblastine release, both of which are abolished when cellular ATP levels are reduced, provides a direct demonstration that the product of the transfected mdr1 gene is responsible for a mechanism controlling cellular drug levels in an ATP-dependent manner. However, attempts to establish competition for [3H]vinblastine transport by vincristine, daunomycin, and actinomycin D were only partly successful in mdr1 transfectants.

Adenosine Triphosphate↗
PubMed1988

Evoked secretion of [3H]noradrenaline and ATP from nerve varicosities isolated from the myenteric plexus of the guinea pig ileum.

Neuronal varicosities, isolated from the myenteric plexus of guinea pig ileum longitudinal muscle, were incubated with [3H]noradrenaline to label the contents of the noradrenergic secretory vesicles. Exposure of these varicosities to KCl, nicotine, or acetylcholine resulted in the Ca2+ -dependent release of [3H]noradrenaline. Veratridine also evoked a large efflux of [3H] from this preparation, but this release was only partially Ca2+ dependent. The alpha 2-adrenoceptor agonist, clonidine, inhibited the K+-, nicotine-, and acetylcholine-induced release of [3H]noradrenaline. Similarly, exogenously administered (-)noradrenaline was an effective inhibitor of the K+ -evoked release of [3H]noradrenaline. The alpha 2-adrenoceptor antagonist, yohimbine, antagonized the inhibitory actions of both clonidine and (-)noradrenaline on the K+ -evoked release of [3H]noradrenaline from myenteric varicosities. Nicotine, acetylcholine, KCl, and veratridine also released ATP from these guinea pig ileal myenteric varicosities. However, the evoked release of ATP was unaffected by clonidine. These results indicate that myenteric varicosities can take up and release [3H]noradrenaline and that they possess presynaptic alpha 2-adrenoceptors which, when activated, inhibit the release of [3H]noradrenaline. These receptors may play a role in modulating the release of noradrenaline in the myenteric plexus in vivo. In addition, the present results suggest that ATP and [3H]noradrenaline may not be released from the same population of secretory vesicles in neuronal varicosities isolated from guinea pig ileum longitudinal muscle.

Acetylcholine↗
PubMed1987

Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes).

Vesicles are released during the in vitro culture of sheep reticulocytes which can be harvested by centrifugation at 100,000 X g for 90 min. These vesicles contain a number of activities, characteristic of the reticulocyte plasma membrane, which are known to diminish or disappear upon reticulocyte maturation. The activities include acetylcholinesterase, cytochalasin B binding (glucose transporter) nucleoside binding (i.e. nucleoside transporter), Na+-independent amino acid transport, and the transferrin receptor. Enzymes of cytosolic origin are not detectable or are present at low activity in the vesicles. Cultures of whole blood, mature red cells, or white cells do not yield comparable levels of these activities, supporting the conclusion that the activities arise from the reticulocytes. In addition, the lipid composition of the vesicles shows the high sphingomyelin content characteristic of sheep red cell plasma membranes, but not white cell or platelet membranes, also consistent with the conclusion that the vesicles are of reticulocyte origin. It is suggested that vesicle externalization may be a mechanism for shedding of specific membrane functions which are known to diminish during maturation of reticulocytes to erythrocytes.

Acetylcholinesterase↗
PubMed1987

Modulation of [3H]flunitrazepam binding to rat cerebellar benzodiazepine receptors by phosphatidylserine.

The influence of phosphatidylserine on ligand binding to the benzodiazepine/GABA receptor complex was assessed in rat cerebellar synaptic membranes and in a detergent-solubilized membrane preparation. Intact synaptic membranes or membranes solubilized with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate) were incubated with a range of concentrations of phosphatidylserine for 2 h at 4 degrees C, prior to use in radioligand binding assays. Phosphatidylserine, an endogenous membrane phospholipid, facilitated the site-specific binding of [3H]flunitrazepam to synaptic membranes and CHAPS-solubilized preparations. In addition, phosphatidylserine inhibited the facilitation of [3H]flunitrazepam binding induced by either cartazolate or gamma-aminobutyric acid (GABA). Although the maximum effect (38% facilitation of [3H]flunitrazepam binding; greater than 90% inhibition of the cartazolate action) was produced using 130 microM phosphatidylserine, a significant enhancement of [3H]flunitrazepam binding could be observed upon preincubation of synaptic membranes with concentrations of phosphatidylserine as low as 5 microM. These results suggest that endogenous phosphatidylserine may play a role in the regulation of benzodiazepine/GABA receptor function, possibly through modulation of the mechanisms which functionally link the various components of this complex receptor system.

Animals↗
PubMed1986

Solubilization of the benzodiazepine/gamma-aminobutyric acid receptor complex: comparison of the detergents octylglucopyranoside and 3-[(3-cholamidopropyl)-dimethylammonio]1-propanesulfonate (CHAPS).

Octylglucopyranoside (OCTG) was three times more efficient than 3-[(3-cholamidopropyl)-dimethylammonio]1-propanesulfonate (CHAPS) in solubilizing the benzodiazepine (BDZ)/gamma-aminobutyric acid (GABA) receptor complex from rat cerebellar synaptic membranes. OCTG-solubilized receptor preparations had ligand binding characteristics that were significantly different from those of the CHAPS-solubilized receptors. The inclusion of phospholipids in the solubilization media improved the binding characteristics of both soluble receptor preparations and appeared absolutely necessary for the maintenance of chloride facilitation of flunitrazepam (FNZ) binding to OCTG-solubilized receptors. FNZ and ethyl-beta-carboline-3-carboxylate bound to OCTG-solubilized preparations with equilibrium dissociation constants of 2.2 nM and 1.6 nM, respectively, and chloride (150 mM) and GABA (100 microM) + chloride facilitated the binding of FNZ by 15% and 55%, respectively; these ligand binding characteristics are similar to those of membrane-located BDZ receptors. Cartazolate, a pyrazolopyridine that facilitated the binding of FNZ to membrane-located and CHAPS-solubilized receptors, did not facilitate FNZ binding to OCTG-solubilized receptors. These results are discussed in terms of an interaction between the membrane lipid phosphatidylserine (PS) and cartazolate; PS appears to have the capacity to inhibit the effects of cartazolate on FNZ binding. Storage of the soluble receptor preparations for 24 h at 4 degrees C resulted in the loss of several characteristic BDZ receptor binding properties. Incorporation of the OCTG-solubilized receptor complex into liposomes prevented these losses but this procedure did not protect the CHAPS-solubilized receptors. We conclude that OCTG may have some advantages over CHAPS as the detergent of choice for the solubilization and reconstitution in liposomes of a functional BDZ/GABA receptor-chloride ionophore complex.

Animals↗
PubMed1985

Species differences in the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system in mammalian central nervous system membranes: evidence for interconvertible conformations of the binding site/transporter complex.

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific sites in CNS membranes was investigated using cortical tissue from a variety of mammalian species. Mass law analysis of the site-specific binding of NBMPR data revealed that rat, mouse, guinea pig, and dog cortical membranes each contained an apparent single class of high-affinity (KD 0.11-4.9 nM) binding sites for NBMPR; rabbit cortical membranes, however, exhibited two distinct classes of NBMPR binding sites with KD values of 0.4 nM and 13.8 nM. Dipyridamole, a potent inhibitor of nucleoside transport, produced a biphasic profile of inhibition of the binding of NBMPR to guinea pig, rabbit, and dog membranes (IC50 less than 20 nM and IC50 greater than 6 microM for NBMPR binding sites displaying high and low affinity for dipyridamole, respectively). These results are indicative of heterogeneity of NBMPR binding sites in mammalian cortical membranes. Rat and mouse cortical membranes appear to possess only one type of NBMPR binding site, which has low affinity for dipyridamole. Detailed analysis of inhibitor-induced dissociation of NBMPR from its sites in each species led to the conclusion that these multiple forms of NBMPR binding sites are different conformations of a single site associated with the CNS nucleoside transport system, rather than two distinct sites. It is also suggested that the affinity of dipyridamole for each conformation of NBMPR site indicates the susceptibility of that conformation of the nucleoside transport system to inhibition by dipyridamole.

Adenosine↗
PubMed1985

Photoaffinity labelling of benzodiazepine receptors: lack of effect on ligand binding to the nucleoside transport system.

This study was undertaken to investigate the possibility of an allosteric interaction between benzodiazepine receptors and the CNS nucleoside transport system. Irreversible (photoaffinity) labelling of the benzodiazepine receptors in guinea pig cortical membranes resulted in a marked reduction in the binding (Bmax) of both [3H]flunitrazepam (71%) and [3H]ethyl-beta-carboline-3-carboxylate (22%) to the benzodiazepine receptors but had no effect on the binding of [3H]nitrobenzylthioinosine to the nucleoside transport system. Furthermore, although photoaffinity labelling resulted in a significant decrease in the affinities of flunitrazepam (approximately equal to 16-fold) and dipyridamole (approximately equal to sevenfold) for the [3H]Ro 15-1788 binding site of the benzodiazepine receptor complex, the affinities of these compounds for the nucleoside transport system were unaltered. These results suggest that the CNS nucleoside transport system and the benzodiazepine receptor complex are distinct, noninteractive ligand recognition sites.

Affinity Labels↗
PubMed1985

Affinity of calcium channel inhibitors, benzodiazepines, and other vasoactive compounds for the nucleoside transport system.

There is evidence to suggest that several different groups of drugs including the so-called coronary vasodilators, benzodiazepines, and calcium channel inhibitors may owe their vasoactivity, in part, to the potentiation of the vasorelaxant effects of endogenous adenosine. To measure the affinity of some of these agents for the membrane-located nucleoside transport system, competition binding assays have been performed using the high-affinity radioligand [3H]nitrobenzylthioinosine (NBMPR). Experiments were performed on human erythrocytes and cardiac membranes from guinea pigs and rats. Recognized nucleoside transport inhibitors had high affinity (less than 50 nM) for NBMPR recognition sites associated with the nucleoside transporter complex in human erythrocytes, whereas calcium channel inhibitors and benzodiazepines had predominantly low affinity (greater than 1 microM). Although some recognized transport inhibitors, such as dipyridamole, show marked differences in affinity for NBMPR sites in guinea pig and rat tissues, benzodiazepines and calcium channel blockers displayed no such species selectivity and had low affinity (greater than 1 microM) for NBMPR sites in both guinea pig and rat cardiac membranes. Consequently, it is unlikely that agents such as benzodiazepines and calcium channel inhibitors cause significant inhibition of adenosine transport, and hence potentiate adenosine actions, at the concentrations required to induce effects through occupation of their respective, specific high-affinity sites.

Animals↗
PubMed1984

[3H]nitrobenzylthioinosine binding to the guinea pig CNS nucleoside transport system: a pharmacological characterization.

The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes.

Adenosine↗
PubMed1984

Heterogeneity of high affinity nitrobenzylthioinosine binding sites in mammalian cortical membranes: multiple forms of central nervous system nucleoside transporters?

Specific binding of [3H]nitrobenzylthioinosine to cortical membranes from several mammalian species was investigated. Rat, mouse, guinea pig, and dog membranes contained an apparent single class of binding sites; there was, however, a marked species-dependent variation in their affinity for [3H]nitrobenzylthioinosine. Rabbit cortical membranes contained two classes of binding sites and the high and low affinity components were similar to those found in guinea pig and dog cortical membranes, respectively. The [3H]nitrobenzylthioinosine binding sites in rat and the low affinity sites in rabbit were atypical in that they exhibited a low affinity for dipyridamole. It is proposed that these latter sites may represent a form of the central nervous system nucleoside transport system which is less susceptible to inhibition by dipyridamole.

Animals↗
PubMed1983

Benzodiazepine inhibition of nucleoside transport in human erythrocytes.

The interaction of several benzodiazepines (BDZs) with the nucleoside transport system of fresh erythrocytes from humans was investigated. The affinities of BDZs for the nucleoside transport system were estimated by measuring BDZ inhibition of (a) the site-specific binding of nitrobenzylthioinosine, a potent and specific inhibitor of nucleoside transport, and (b) the uridine transport processes, zero-trans influx, zero-trans efflux, and equilibrium exchange influx. The BDZs inhibited both the inward and outward transport processes, and, for individual agents, inhibition constants (Ki) were similar for the inhibition of each transport process and for the inhibition of the site-specific binding of nitrobenzylthioinosine. The order of potencies of the BDZs in their interactions with the nucleoside transport mechanism (Ro 5-4864 greater than diazepam greater than clonazepam greater than lorazepam greater than flurazepam) is distinct from the potencies of these compounds at BDZ recognition sites. The affinities of the BDZs for the nucleoside transport system, which are about 1000-fold lower than for BDZ recognition sites, suggest that significant inhibition is unlikely to occur with the plasma concentrations (less than 1 microM) that result from usual anxiolytic doses of these agents.

Benzodiazepines↗
PubMed1983

Nucleoside transport in human erythrocytes. A simple carrier with directional symmetry in fresh cells, but with directional asymmetry in cells from outdated blood.

Kinetic characteristics of the transport of uridine, a non-metabolized permeant in human erythrocytes, have been compared in erythrocytes from fresh and outdated stored blood. Uridine transport kinetics in fresh cells conformed to the predictions of a simple carrier model operating with directional symmetry, but in erythrocytes from outdated blood the kinetic characteristics of uridine transport were those of an asymmetric system. The latter result agrees with earlier reports by others. The mobility of the loaded and empty carriers differed by about 6- and 12-fold in fresh and outdated blood, respectively.

Binding Sites↗
PubMed1982

Species differences in nucleoside transport. A study of uridine transport and nitrobenzylthioinosine binding by mammalian erythrocytes.

A kinetic study of the inward transport of uridine in erythrocytes of rabbit, human, mouse, rat and guinea-pig demonstrated that the apparent Km of this process was similar (about 0.2mM) in these cell types, but Vmax. values differed markedly. In this array of cell types, Vmax. values were proportional to the number of transport-inhibitory, high-affinity binding sites present per cell of each type. Transport of uridine or adenosine was not detected in dog erythrocytes, nor was saturable, high-affinity binding of nitrobenzylthioinosine demonstrable. These findings demonstrate that species differences in nucleoside transport capacity are attributable to differences in the cell-surface content of functional nucleoside transport sites, rather than to differences in the kinetic properties of these sites.

Animals↗