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Biomedical subjects

D E Schmidt

Publications and source records attributed to D E Schmidt.

At least 37 records · Page 2Linked to original sources
PubMed1996

Elevations of hepatic quinone reductase, glutathione, and alpha- and mu-class glutathione S-transferase isoforms in mice with chronic hepatitis: a compensatory response to injury.

Hepatic levels of GSH and Phase II detoxication enzymes were compared to biochemical and histological indices of hepatic damage in 4- to 76-week-old nontransgenic mice and their transgenic littermates that overexpress the hepatitis B virus large envelope protein. The mice were fed a low-sucrose AIN-76A diet ad libitum. Hepatic-specific activities of quinone reductase (QR) and glutathione S-transferase (GST) were increased 2- to 10-fold beginning at 12 weeks of age in transgenic mice and correlated with increases in serum alanine aminotransferase (ALT) (r = 0.84 and 0.59, respectively). Quantitative histological analysis demonstrated that apoptosis was the predominant feature in 4- to 12-week-old transgenic mice, whereas necrosis and inflammation predominated at later time points. Surprisingly, 3-fold elevations in ALT were observed beginning at 52 weeks of age in nontransgenic mice, and hepatic-specific activities of QR and GST were also modestly increased in elderly nontransgenic animals. In contrast to transgenic mice, apoptosis was not a prominent feature. The strongest histological correlates to ALT in 4- to 76-week-old nontransgenic mice were necrosis and inflammation (r > 0.96), which in turn may have been evoked by hepatic fat accumulation. Profiles of specific GST isoforms were quantitated chromatographically and identified by sequencing tryptic digests. The Ya1 subunit of alpha-class GST was markedly increased from undetectable levels in transgenic mice, while more modest increases were observed in nontransgenic mice more than 1 year old. Fivefold elevations of the Yb1 subunit, a constitutively expressed mu-class GST, were found in transgenic mice older than 4 weeks of age, while 2-fold increases were observed in nontransgenic animals that were more than 1 year old. These studies demonstrate that selected increases in Phase II detoxication enzymes are a stereotyped response to chronic hepatitis that is strikingly reminiscent of the treatment of mice with anticarcinogenic enzyme inducers.

Aging↗
PubMed1996

Salt-induced immobilizations of DNA oligonucleotides on an epoxide-activated high-performance liquid chromatographic affinity support.

Synthetic oligonucleotides, possessing a recognition sequence for the transcription factor NF-kappa B, were immobilized onto an epoxide-activated hydroxyethylmethacrylate HPLC affinity support in the presence of high concentrations of potassium phosphate. The extent of immobilization increased with salt concentration in a manner analogous to that which has been reported for salt-induced immobilizations of proteins. High immobilization efficiencies were achieved, and at 2.7 M potassium phosphate, 85-90% of the DNA initially present in the reaction mixture was immobilized. Reactions were examined for double stranded DNA, one strand of which was modified with a 5'-mercaptohexyl spacer arm, and for each of the strands comprising the duplex. For double stranded immobilizations, about 85% of the non-modified strand (the d22 strand) was released from the support under melting conditions, suggesting that d22 exhibited low reactivity when organized as the duplex. For immobilizations of single stranded DNA, mild acid hydrolysis of the products was used to provide information concerning the mode of attachment. For reactions of the d22 strand alone, only about 60% each of guanine and adenine were recovered from the immobilized oligonucleotide following mild acid hydrolysis. This suggests that when d22 is immobilized as the single strand, significant attachment occurs through the purine bases, in contrast with the low reactivity exhibited by d22 in the duplex. Purified p50 protein, the DNA binding element of NF-kappa B, and nuclear extracts from phorbol ester-stimulated HeLa cells were injected onto a column packed with the double stranded product. In both cases p50 was retained on the column and was recovered upon elution with a salt gradient.

Base Sequence↗
PubMed1996

Glutathione-associated enzymes in the human cell lines of the National Cancer Institute Drug Screening Program.

The steady state expression of glutathione S-transferases (GSTs) at both the protein and mRNA level is reported for the 60 tumor cell lines that are used for the National Cancer Institute Drug Screening Program. Individual GST isozymes were separated, identified, and quantified (with reverse-phase calibration curves) through a novel high performance liquid chromatographic procedure. GSTP1 was the predominant isozyme and was found at quantifiable levels in all but two of the cell lines. This isozyme ranged from 0.03% to 2.7% of the total cytosolic protein. For the mu family, 90% of the lines had GSTM2, 68% had GSTM3, but only 28% were positive for the M1 phenotype. The M1 proportion is lower than would be expected from the standard M1 null phenotype for human populations. Isozymes of the alpha family were detected only at very low levels in 35% of the lines. Significant quantitative correlations among enzyme activity, total enzyme protein, and mRNA were shown for GSTP1. However, such relationships were not apparent for the mu or alpha families. Levels of glutathione (GSH), and the transcript levels of other enzymes involved in GSH homeostasis were determined. gamma-Glutamyl cysteine synthetase (gamma-GCS) was present in all cell lines, but did not correlate with levels of intracellular GSH. Glyoxalase-I and gamma-glutamyl transpeptidase, both involved in GSH salvage, were found in 100% and 70% of the cell lines, respectively. Using a pattern-matching computer program, COMPARE, we compared and correlated the arrays of mRNA and protein levels with the pattern of chemosensitivity or chemoresistance of the 60 cell lines with 175 agents constituting a standard agent database. This database is composed of compounds to which a putative mechanism of action has been assigned. Although Pearson correlation coefficients relating the target and drug patterns were generally modest, when the patterns for the enzyme protein and mRNA levels for GST pi were correlated to drug sensitivity patterns, the list of 30 agents most closely matching (for which P < 0.05) was enriched with alkylating agents. gamma-GCS also showed an enrichment of alkylating agents in the COMPARE correlations, indicating that high levels of gamma-GCS may be an important determinant of resistance. In contrast, none of the other enzymes or GSH had patterns of expression that resulted in an obvious correlation to the sensitivity or resistance of alkylating agents.

Antineoplastic Agents↗
PubMed1995

Norepinephrine-independent regulation of GRII mRNA in vivo by a tricyclic antidepressant.

Desipramine (DMI), a tricyclic antidepressant drug used in the treatment of depression, has been shown to increase steady-state levels of glucocorticoid receptor type II (GRII) mRNA in vitro and in vivo. To determine whether this effect is secondary to norepinephrine (NE) reuptake inhibition i.e., increases in synaptic NE induced by DMI, GRII mRNA levels were assayed in rat hippocampus following neurotoxic lesioning of NE neurons with DSP4. Chronic DMI treatment significantly increased GRII mRNA levels to the same degree in lesioned and non-lesioned animals. In contrast to DMI, the non-tricyclic antidepressant fluoxetine had no effect on GRII mRNA. These results provide evidence which demonstrates that a tricyclic antidepressant can regulate steady-state mRNA levels in vivo by a mechanism which is independent of its effects on synaptic monoamine levels.

Animals↗
PubMed1994

Variability of glutathione S-transferase isoenzyme patterns in matched normal and cancer human breast tissue.

The determination of GST levels in blood has been proposed to a marker of tumour burden in general, whereas level of the P1 isoenzyme has been identified as a prognostic factor for breast-cancer patients receiving no adjuvant chemotherapy. Particular glutathione S-transferase (GST) isoenzymes differ in their substrate specificity, however, and their presence or absence might therefore account for the resistance of tumours to particular chemotherapeutic drugs, as already established for cultured cell lines. Determination of the GST isoenzyme profile of a cancer tissue could have prognostic value in the selection of treatment if the levels of expression/activity show a degree of variation comparable with that exhibited by actual patient responses. Using reversed-phase h.p.l.c. to quantify affinity-isolated GSTs, we have analysed full isoenzyme profiles in the first large sample of matched normal and cancer human tissues (18 breast-cancer patients). In no patients did the tumour tissues express any isoenzymes that were not found in normal breast tissue. In addition to the GSTs, another enzyme, identified as enoyl-CoA isomerase, was regularly found in breast tissue cytosol following elution from a hexyl-glutathione affinity column. In most cases, the average level of GST was substantially elevated in the cancer tissues above the levels in normal breast tissue from the same patient. Furthermore, the relative levels of the isoenzymes were substantially more variable in the cancer samples than in the normal breast tissue, providing a plausible mechanism for the well established variable response to treatment.

Adult↗
PubMed1994

Coupled affinity-reversed-phase high-performance liquid chromatography systems for the measurement of glutathione S-transferases in human tissues.

HPLC affinity and reversed-phase modes were coupled for the direct measurement of glutathione S-transferases (GSTs) in cytosol extracts. Two coupling designs were examined. In the sequential configuration the affinity column served to extract the isoenzymes which were then eluted directly onto the reversed-phase column as a single fraction. Subsequent separation in the reversed-phase mode provided a GST profile based on the subunit composition of the isoenzymes as a whole. In the second configuration (rapid sampling configuration), gradient elution was performed in the affinity mode resulting in resolution of the intact isoenzymes. The eluate from the affinity separation was sampled in continuous, repetitive intervals and automatically subjected to ongoing reversed-phase analysis. This multidimensional approach provided information on the GST subunit content and also gave information about the distribution of the subunits among individual isoenzymes, thereby forming a basis for the determination of the actual isoenzymatic composition of the GSTs. In both configurations, events were automated and co-ordinated through the use of computer and multiport switching valves. Examples of GST separations from these procedures are shown for human lung and liver tissues. A comparison of the GST subunit analyses from normal and cancer lung tissue excised from the same patient showed substantial elevations of GSTs in the cancer sample. Two-dimensional affinity-reversed-phase analysis of a human liver sample illustrates the utility of the technique for determining the isoenzymatic organization of GST subunits. The criteria for extending two-dimensional analysis to more complex GST mixtures are discussed.

Chromatography, Affinity↗
PubMed1994

Examination of glutathione S-transferase isoenzyme profiles in human liver using high-performance affinity chromatography.

A method for the examination of the glutathione S-transferase isoenzyme profiles in human liver using a new HPLC affinity support is described. Liver cytosol was injected directly onto an HPLC column (5 x 0.46 cm) containing a support with a covalently bound affinity ligand (S-octylglutathione) specific for the isoenzymes. Contaminating cytosolic proteins were removed in a washing step. The isoenzymes were eluted with a linear gradient of a different affinity ligand in the mobile phase. Coinciding with the affinity ligand gradient, a salt gradient (0-200 mM sodium chloride) was applied. In this manner the isoenzymes were fractionated into the enzymatically active homodimers and heterodimers. The classes of the affinity fractionated isoenzymes were determined by SDS-PAGE and ELISA while the subunit content was determined by reversed-phase chromatography. For one liver three Alpha class isoenzyme subunits, forming three heterodimers and two homodimers, were detected. Five livers were examined, and the homodimer A1-1 was found to be the predominant glutathione S-transferase isoenzyme. Minor amounts of Pi and Mu class isoenzymes were also detected. This non-denaturing high-performance affinity chromatography method reduced analysis time by a factor of ten when compared to other affinity analysis methods for the glutathione S-transferases.

Chromatography, Affinity↗
PubMed1994

Aromatic and amine substituent effects on the apparent lipophilicities of N-[(2-pyrrolidinyl)methyl]-substituted benzamides.

Lipophilic properties of 92 dopamine D-2 receptor antagonists belonging to the substituted benzamide class of compounds (orthopramides and methoxysalicylamides) were determined by octadecylsilane reversed-phase HPLC. The apparent lipophilicity at pH 7.5 (log kw) was obtained from the chromatographic capacity factors in 0.02 M3-(morpholino)propanesulfonic acid (MOPS) buffer at various concentrations of methanol. The experimental log kw values were validated by comparison with the apparent octanol-water partitioning (log Papp) of 15 compounds of low to medium lipophilicity. The global lipophilicity of the neutral molecule (log kwo) was obtained by correcting for ionization of the amine and the phenol, using known relationships for the effects on the pKa (where Ka is the dissociation constant) of aromatic and aliphatic substituents. Multiple regression analysis showed that log kwo can be expressed as the sum of pi contributions and a cross correlation term (sigma rho sigma) for interactions between the aromatic substituents. Comparison between the methoxysalicylamide (raclopride) series and the orthopramide (sulpiride) series demonstrated that an aromatic 6-hydroxy group increased log kw by 0.4 in the 5-halogen series and by 0.8 in the 5-alkyl series, and that a 6-methoxy group decreased log kw by 0.5. These paradoxical effects can be explained by the masking of the polarity of the amide caused by the 6-hydroxy group forming an intramolecular hydrogen bond with the amide carbonyl group. Introduction of an additional ortho-methoxy substituent had the opposite effect because the resulting steric hindrance prevents the amide moiety from adopting a coplanar conformation with the benzene ring. The presence of a substituent in the aromatic 3-position lowered log kw by 0.3 via a combination of steric and electronic influences on the adjacent 2-methoxy group, causing a weakening of the hydrogen bond between the amide and the oxygen atom of the 2-methoxy group. As a result, halogen and alkyl substituents in the 3-position increase the apparent lipophilicity only half that of similar substituents in the 5-position. Substitution with omega-fluoroalkoxyl groups in the aromatic 2- and 3-positions and with omega-fluoroalkyl groups in the 5-position reduced lipophilicities by 0.5 as compared with the corresponding desfluoro derivatives, thereby making them equivalent to an alkyl derivative with one less carbon atom in the chain. In contrast, substitution on the pyrrolidine nitrogen atom with a 2-fluoroethyl or a 3-fluoropropyl group produced compounds with apparent lipophilicities approximately 1.5 and approximately 0.5 higher, respectively, than those of the corresponding N-ethyl derivatives.(ABSTRACT TRUNCATED AT 400 WORDS)

Amines↗
PubMed1993

Identification of extrastriatal dopamine D2 receptors in post mortem human brain with [125I]epidepride.

The regional distribution of striatal and extrastriatal dopamine D2 receptors in human brain was studied in vitro with (S)-N-[(1-ethyl-2- pyrrolidinyl)methyl]-5-[125I]iodo-2,3-dimethoxybenzamide, [125I]epidepride, using post mortem brain specimens from six subjects. Scatchard analysis of the saturation equilibrium binding in twenty-three regions of post mortem brain revealed highest levels of binding in the caudate (16.5 pmol/g tissue) and putamen (16.6 pmol/g tissue) with lower levels seen in the globus pallidus (7.0 pmol/g tissue), nucleus accumbens (7.2 pmol/g tissue), hypothalamus (1.8 pmol/g tissue), pituitary (1.3 pmol/g tissue), substantia innominata (1.0 pmol/g tissue), and amygdala (0.87 pmol/g tissue). Of note was the presence of dopamine D2 receptors in the four thalamic nuclei studied, i.e. anterior nucleus (1.0 pmol/g tissue), dorsomedial nucleus (0.96 pmol/g tissue), ventral nuclei (0.72 pmol/g tissue), and pulvinar (0.86 pmol/g tissue), at levels comparable to the amygdala (0.87 pmol/g tissue) and considerably higher than levels seen in anterior cingulate (0.26 pmol/g tissue) or anterior hippocampus (0.36 pmol/g tissue). The frontal cortex had very low levels of dopamine D2 receptors (0.17-0.20 pmol/g tissue) while the inferior and medial temporal cortex had relatively higher levels (0.31-0.46 pmol/g tissue). Inhibition of [125I]epidepride binding by a variety of neurotransmitter ligands to striatal, ventral thalamic and inferior temporal cortical homogenates demonstrated that [125I]epidepride binding was potently inhibited only by dopamine D2 ligands. The present study demonstrates that dopamine D2 receptors are present in basal ganglia, many limbic regions, cortex and in the thalamus. The density of thalamic D2 receptors is comparable to many limbic regions and is considerably higher than in cortex.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Antagonists↗
PubMed1993

Evaluation of 5-[18F]fluoropropylepidepride as a potential PET radioligand for imaging dopamine D2 receptors.

This study evaluated the utility of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-(3-[18F]fluoropropyl)-2,3 - dimethoxybenzamide ([18F]fluorpropylepidepride), [18F]5-FPrEpid, as a ligand for PET studies of cerebral dopamine D2 receptors. The in vitro affinity for the rat striatal dopamine D2 receptor, KD 138 pM, was determined by Scatchard analysis of in vitro binding to rat striatal homogenate. The apparent lipophilicity, log kw 1.6, was measured with reverse phase HPLC at pH 7.5. The receptor specificity was determined by competitive displacement of [18F]5-FPrEpid by a variety of neurotransmitter ligands. Only dopamine D2 ligands displaced [18F]5-FPrEpid with high affinity. Positron tomographic imaging studies in primates of [18F]5-FPrEpid demonstrated a stable striatal uptake of 0.02% injected dose/ml for up to 5 h after injection. The striatal: cerebellar ratio increased from 2 at 15 min, to 7 at 200 min, and to 10 at 300 min. Striatal uptake was displaceable by haloperidol (1 mg/kg) or raclopride (2.5 mg/kg) to cerebellar levels with a t1/2 of washout of 9 or 15 min. Striatal uptake was mildly susceptible to displacement by d-amphetamine (1-2 mg/kg) released endogenous dopamine; d-amphetamine administration produced a 10% h increase in the rate of striatal washout. Although uptake in the striatum is reversible, an equilibrium between receptor bound [18F]5-FPrEpid in striatum and [18F]5-FPrEpid in plasma is not reached within 5 h postinjection.

Animals↗
PubMed1993

High affinity dopamine D2 receptor radioligands. 3. [123I] and [125I]epidepride: in vivo studies in rhesus monkey brain and comparison with in vitro pharmacokinetics in rat brain.

Studies of [123I]epidepride uptake in rhesus monkey brain were performed using single photon tomography. Striatal uptake peaked at 0.85% of administered dose/g at 107 min post-injection, then declined slowly to 0.70% of administered dose/g at 6 h. Striatal:posterior brain ratios rose from 2 at 25 min to 6.8 at 105 min, to 15 at 4 h and to 58 at 6.4 h. [123I]Epidepride was displaced by haloperidol (0.1 and 1 mg/kg) with a half-life of washout of 55 min. Little displacement of [123I]epidepride was observed following administration of 1 or 2 mg/kg d-amphetamine, respectively, indicating [123I]epidepride is not easily displaced by endogenous dopamine. In vitro equilibrium binding studies using rat striatum revealed a KD of 46 pM and Bmax of 33 pmol/g tissue at 37 degrees C, while at 25 degrees C the KD was 25 pM and the Bmax 32 pmol/g tissue. In vitro kinetic analysis of association and dissociation curves revealed a half-life for receptor dissociation at 37 degrees C of 15 min and 79-90 min at 25 degrees C. Allowing for the temperature difference, there is good correspondence between in vivo and in vitro dissociation kinetics at 25 degrees C. Increasing in vitro incubation temperature from 25 to 37 degrees C caused a 6-fold increase in the dissociation rate, suggesting that there is a change in binding kinetics at the dopamine D2 receptor at 37 degrees C compared to in vivo binding. The results of this study indicate that [123I]epidepride is an excellent radioligand for SPECT studies of the dopamine D2 receptor in man.

Amphetamine↗
PubMed1993

An advanced solid support for immunoassays and other affinity applications.

A 6-mm diameter glass bead that has a hydrophilic coating containing epoxy groups has been developed for immunoassays and other affinity applications. These QuantAffinity beads covalently bind protein in the range of 200-400 ng per bead. Derivatization with protein in the presence of high concentrations of potassium phosphate (pH 7.0-8.0) generally gives complete coupling in 16 hours. No preactivation of the support or the protein is necessary. Low nonspecific binding and high retention of bound protein activity are inherent in the system due to the special surface chemistry. Several enzymatic and radioactive immunoassays as well as a biotin-avidin-based assay are described. A use of QuantAffinity beads in small-scale purification is also presented.

Animals↗
PubMed1992

Visualization of extrastriatal dopamine D2 receptors in the human brain.

[123I]Epidepride, a potent and selective dopamine D2 radioligand, was administered to a 27 year old normal male volunteer. Single photon tomography revealed that peak striatal uptake occurred at 4 h after injection with a striatal:cerebellar ratio of 7.8 rising to over 100 at 18 h post injection. Uptake above the levels seen in cerebellum was also noted in the thalamus, pituitary, hypothalamus and temporal lobe, particularly medially. Single photon tomography with [123I]epidepride allows visualization of extrastriatal dopamine D2 receptors in man.

Adult↗
PubMed1991

High affinity dopamine D2 receptor radioligands. 2. [125I]epidepride, a potent and specific radioligand for the characterization of striatal and extrastriatal dopamine D2 receptors.

Epidepride, (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-5-iodo-2,3-dimethoxybenzamide+ ++, the iodine analogue of isoremoxipride (FLB 457), was found to be a very potent dopamine D2 receptor antagonist. Optimal in vitro binding required incubation at 25 degrees C for 4 h at pH 7.4 in a buffer containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2 and 1 mM MgCl2. Scatchard analysis of in vitro binding to striatal, medial frontal cortical, hippocampal and cerebellar membranes revealed a KD of 24 pM in all regions, with Bmax's of 36.7, 1.04, 0.85, and 0.37 pmol/g tissue, respectively. The Hill coefficients ranged from 0.91-1.00 in all four regions. The IC50's for inhibition of [125I]epidepride binding to striatal, medial frontal cortical, and hippocampal membranes for SCH 23390, SKF 83566, serotonin, ketanserin, mianserin, naloxone, QNB, prasozin, clonidine, alprenolol, and norepinephrine ranged from 1 microM to greater than 10 microM. Partial displacement of [125I]epidepride by nanomolar concentrations of clonidine was noted in the frontal cortex and hippocampus, but not in the striatum. Scatchard analysis of epidepride binding to alpha 2 noradrenergic receptors in the frontal cortex and hippocampus revealed an apparent KD of 9 nM. At an epidepride concentration equal to the KD for the D2 receptor, i.e. 25 pM, no striatal alpha 2 binding was seen and only 7% of the specific epidepride binding in the cortex or hippocampus was due to binding at the alpha 2 site. Correlation of inhibition of [3H]spiperone and [125I]epidepride binding to striatal membranes by a variety of D2 ligands revealed a correlation coefficient of 0.99, indicating that epidepride labels a D2 site. In vitro autoradiography revealed high densities of receptor binding in layers V and VI of prefrontal and cingulate cortices as well as in striatum. In vivo rat brain uptake revealed a hippocampal:cerebellar and frontal cortical:cerebellar ratio of 2.2:1 which fell to 1.1:1 following haloperidol pretreatment. These properties suggest that [125I]epidepride is a superior radioligand for the in vitro and in vivo study of striatal and extrastriatal dopamine D2 receptors.

Animals↗
PubMed1991

Strangers in a strange land: a psychobiological study of infant monkeys before and after separation from real or inanimate mothers.

Some rhesus monkey (Macaca mulatta) infants have a "despair" or depression-like response to mother-infant separation, while others do not. The presumed interrelation between early rearing conditions and the neurobiological status of the infant that might lead to increased risk for despair is not understood. In this study, the characteristics of the "mother" were controlled by rearing infant rhesus monkeys with their biological mothers, or with inanimate mothers. Behavioral data were collected before and after separation at 6-7 months of age. The neurobiological status of the infants was evaluated by measuring the concentration of norepinephrine, its major metabolite, and the metabolites of dopamine and serotonin in cerebrospinal fluid. The results suggest that despair is not simply a behavioral response to separation. Instead, despair may reflect the inability to cope with the separation environment. Coping with the separation environment appears to depend on neurobiological and behavioral characteristics of the infant that are related to, if not determined by, characteristics of the mother.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1991

High affinity dopamine D2 receptor radioligands. 1. Regional rat brain distribution of iodinated benzamides.

Five 125I-labeled substituted benzamides, which are close structural analogues of (S)-sulpiride, eticlopride, and isoremoxipride, were evaluated for their selective in vivo uptake into dopamine D2 receptor rich tissue of the rat brain. "Iodopride" (KD 0.88 nM), an iodine substituted benzamide structurally related to sulpiride, displayed a maximal striatum: cerebellar uptake ratio of 7.6. Demonstration of saturation of the receptor with [125I]iodopride in striatum required uptake in frontal cortex to be used, rather than cerebellar uptake, to define nonspecific binding. Two other ligands structurally related to eticlopride, "iclopride" (KD 0.23 nM) and "itopride" (KD 0.16 nM), displayed maximal striatal: cerebellar uptake ratios of 9.8 and 3.3, respectively. The most potent ligands, "epidepride" (KD 0.057 nM) and "ioxipride" (KD 0.070 nM) showed striatal:cerebellar uptake ratios of 234 and 65, respectively. The observed uptake ratios correlated poorly with the affinity constants for the dopamine D2 receptor alone, but were highly correlated (r = 0.92) with the product of the receptor dissociation constant (KD) and the apparent lipophilicity (kw), as determined by reverse-phase HPLC at pH 7.5. Total striatal uptake also appeared dependent on lipophilicity, with maximal uptake occurring for ligands having log kw 2.4-2.8.

Animals↗
PubMed1990

Sodium-dependent isomerization of dopamine D-2 receptors characterized using [125I]epidepride, a high-affinity substituted benzamide ligand.

We have characterized the in vitro binding of a new ligand, [125I]epidepride, and used this substituted benzamide to assess the sensitivity of dopamine D-2 receptors to sodium. Both direct and indirect binding studies with [125I]epidepride and unlabeled epidepride, respectively, demonstrated that the affinity of D-2 receptors for the ligand was decreased from 20 to 30 pM in the presence of sodium to 350 to 500 pM in the absence of sodium. The density of binding sites for [125I]epidepride was identical in the presence and absence of NaCl. The time courses for association of [125I]epidepride to and dissociation from D-2 receptors in the presence of sodium were not consistent with simple bimolecular reactions, suggesting the possibility of a sodium-dependent ligand-induced receptor isomerization. Thus, dissociation of [125I]epidepride was biphasic in the presence of sodium, but monophasic in the absence of sodium. The rank order of potency for inhibition of [125I]epidepride binding by drugs was identical in rat striatum and cells expressing a D-2 receptor cDNA, and similar to the previously described pharmacological profile of D-2 receptors labeled by [3H]spiperone. [125I]Epidepride bound to two classes of binding sites in rat medial prefrontal cortex. One class, present at a density of 10 fmol/mg of protein and with a Kd value of approximately 40 pM, was pharmacologically indistinguishable from D-2 receptors in striatum and transfected cells. The pharmacological profile of the second class of sites was similar to that of alpha-2 adrenergic receptors. [125I]Epidepride had 50- to 100-fold lower affinity (approximately 2 nM) for alpha-2 receptors than for D-2 receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗