Thomas A. Pugsley

A rapid filtration assay for soluble receptors using polyethylenimine-treated filters

Ligand bound to detergent-solubilized or cytosolic receptors can be separated from free ligand by filtration through glass-fiber filters which have been pretreated with polyethylenimine (PEI). Receptors which can be assayed by this technique include detergent-solubilized muscarinic, adenosine A1, alpha 1-adrenergic, alpha 2-adrenergic, beta-adrenergic, dopamine D2, opiate, bradykinin, and benzodiazepine receptors as well as naturally soluble estradiol receptors. For muscarinic, adenosine, alpha 2, dopamine, and estradiol receptors, specific binding measured by the PEI-filter technique was 84-110% of specific binding measured by gel filtration, demonstrating that the technique gave almost quantitative recovery of bound ligand.

Mice lacking dopamine D4 receptors are supersensitive to ethanol, cocaine, and methamphetamine

The human dopamine D4 receptor (D4R) has received considerable attention because of its high affinity for the atypical antipsychotic clozapine and the unusually polymorphic nature of its gene. To clarify the in vivo role of the D4R, we produced and analyzed mutant mice (D4R-/-) lacking this protein. Although less active in open field tests, D4R-/- mice outperformed wild-type mice on the rotarod and displayed locomotor supersensitivity to ethanol, cocaine, and methamphetamine. Biochemical analyses revealed that dopamine synthesis and its conversion to DOPAC were elevated in the dorsal striatum from D4R-/- mice. Based on these findings, we propose that the D4R modulates normal, coordinated and drug-stimulated motor behaviors as well as the activity of nigrostriatal dopamine neurons.

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907

Abstract The selective D3-dopamine receptor agonist 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-[N-propyl-2,3-3H]-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol ([3H]PD 128907) was used to visualise D3-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [3H]PD 128907 has an 18- to 40-fold selectivity for D3- over D2-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [3H]7-OH-DPAT. [3H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [3H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D2/D3-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D3-dopamine receptors in the human brain. The binding obtained with [3H]PD 128907 was qualitatively similar to that using [3H]7-OH-DPAT in the presence of GTP. However, [3H]7-OH-DPAT labelled, in contrast to [3H]PD 128907, also D3-dopamine receptors in neocortex. The new compound [3H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D3-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype.

Isoindolinone enantiomers having affinity for the dopamine D4 receptor.

PD 108635 (1) was identified as a potent dopamine D4 ligand and we wanted to replace the benzylic alcohol with a metabolically more stable moiety. Investigations led to the discovery of a series of isoindolinones having D4 affinity.

Characterization of the human dopamine D3 receptor expressed in transfected cell lines

A full-length cDNA clone of the human dopamine D3 receptor was obtained by the polymerase chain reaction (PCR) using reverse-transcribed RNA from human brain as the template. The cDNA was inserted into an expression vector which was then stably transfected into either Chinese hamster ovary (CHO), SK-N-MC human epithelioma or mouse CCL1.3 fibroblast cell lines. Post-transfection, the Bmax for D3 receptor expression was 1.9, 1.1 and 0.4 pmol/mg protein in the CHO-K1, SK-N-MC and CCL1.3 cell lines, respectively. The D3 receptor expressed in CHO-K1 and CCL1.3 cells exhibited similar radioligand binding profiles, especially for the D3-selective compound, 7-hydroxy-2-(di-n-propylamino)tetralin (7-OH-DPAT). Radioligand-binding competition curves of presumed D3 agonists were shifted to the right by the addition of guanine nucleotides and Na+ to the assay buffer. Presumed D3-receptor agonists had no effect on cAMP accumulation in any of the D3-transfected cell lines although cAMP accumulation was inhibited by dopamine D2 receptor activation in D2-transfected CHO and CCL1.3 cells and by activation of the exogenously expressed neuropeptide Y receptor in SK-N-MC cells. Also, D3 receptor activation neither potentiated ATP-stimulated arachidonic acid release from CHO cells nor stimulated inositol phosphate production in CCL1.3-cells although both of these responses were elicited by D2 agonists in D2-transfected cells. We conclude that the signalling properties of the D3 receptor differ from those of its closest homolog, the D2 receptor.

Meclofenamate sodium is an inhibitor of both the 5-lipoxygenase and cyclooxygenase pathways of the arachidonic acid cascade in vitro

Meclofenamate sodium was compared to other nonsteroidal antiinflammatory drugs in terms of its potency to inhibit the formation of 5-HETE and LTB4 in human leukocytes and the formation of prostaglandin E2 in bovine seminal vesicles as measures of its ability to inhibit the 5-lipoxygenase and cyclooxygenase pathways of the arachidonic acid cascade. Meclofenamate sodium was about 2-4 times less potent than BW-755C in inhibiting 5-lipoxygenase enzyme activity and three times more potent than benoxaprofen, while naproxen, ibuprofen, and indomethacin showed IC50 greater than 100 microM. Meclofenamate sodium and indomethacin were the most potent inhibitors of the formation of PGE2 in bovine seminal vesicles followed by ibuprofen, naproxen, and benoxaprofen in this order. Meclofenamate sodium, like BW-755C, can be considered a dual inhibitor of 5-lipoxygenase and cyclooxygenase pathways of arachidonic acid cascade. This finding may explain in part the antiinflammatory activity of meclofenamate sodium.

Characterization of binding of [3H]PD 128907, a selective dopamine D3 receptor agonist ligand, to CHO-K1 cells.

PD 128907 [4a R, 10 b R-(+)-trans-3, 4, 4a, 10 b - tetrahydro - 4- n -propyl2 H,5H-[1]benzop-yrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (Ki, 1 nM) versus human D2 receptors (Ki, 1183 nM) and a 10000-fold selectivity versus human D4 receptors (Ki, 7000 nM) using [3H]spiperone as the radioligand in CHO-K1-cells. Studies with [3H]PD 128907, showed saturable, high affinity binding to human D3 receptors expressed in CHO-K1 cells (CHO-K1-D3) with an equilibrium dissociation constant (Kd) of 0.99 nM and a binding density (Bmax) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D2 receptors (CHO-K1-D2). The rank order of potency for inhibition of [3H]PD 128907 binding with reference DA agents was consistent with reported values for D3 receptors. These results indicate that [3H]PD 128907 is a new, highly selective D3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D3 receptors.

Differential effects of the nonpeptide neurotensin antagonist, SR 48692, on the pharmacological effects of neurotensin agonists

In in vitro studies, SR 48692, a nonpeptide neurotensin receptor antagonist, inhibited the binding of [3H] or [125I]neurotensin to membrane preparations from 10-day-old mouse brains and from HT-29 cells with Ki values of 3.9 and 8.6 nM, respectively. SR 48692 also antagonized the neurotensin-induced mobilization of intracellular calcium in HT-29 cells, in agreement with previous findings. In rat cerebellar slices SR 48692 blocked the increase in cyclic GMP levels evoked by neurotensin in a dose-dependent manner. In vivo, SR 48692 antagonized the increase in rat brain mesolimbic dopamine turnover induced by the systemically active neurotensin peptide, EI [(N-Me)Arg-Lys-Pro-Trp-tert-Leu-Leu]. No effects on dopamine turnover of either EI or SR 48692 were observed in the striatum. SR 48692 did not antagonize the EI-induced decreases in mouse body temperature and spontaneous locomotor activity (LMA) or the decreases in LMA induced by ICV-administered neurotensin. Although other explanations are possible, these findings support the hypothesis that a subtype of the NT receptor may mediate the locomotor and hypothermic actions of this peptide and that it is different from the NT receptor that is involved in dopamine turnover.

The Pharmacology of the Novel and Selective Sigma Ligand, PD 144418

The pharmacology of PD 144418 (1-propyl-5-(3-p-tolyl-isoxazol-5-yl)-1,2,3,6-tetrahydropyridine) was characterized using neurochemical, biochemical and behavioral techniques. For sigma (sigma 1 and sigma 2 respectively) sites, PD 144418 affinities were determined using whole guinea pig brain membranes with [3H](+)-pentazocine and neuroblastoma x glioma cell membranes using [3H]1,3,di-O-tolylguanidine (DTG) in the presence of 200 nM (+)-pentazocine. PD 144418 exhibited an affinity for sigma 1 of 0.08 nM (Ki) versus a K1 of 1377 nM for sigma 2 site. Additional receptor binding studies indicated that PD 144418 lacked affinity for dopaminergic, adrenergic, muscarinic and a variety of other receptors. In vitro studies indicated that PD 144418 reversed the N-methyl-D-aspartate (NMDA)-induced increase in cyclic GMP (cGMP) in rat cerebellar slices without affecting the basal levels, suggesting that sigma 1 sites may be important in the regulation of glutamine-induced actions. PD 144418 potentiated the decrease in 5-hydroxytryptophan caused by haloperidol in the mesolimbic region, but by itself had no effect in 5-hydroxytrypamine (5-HT) and dopamine (DA) synthesis. Behaviorally, similar to other sigma ligands, PD 144418 antagonized mescaline-induced scratching at doses that did not alter spontaneous motor activity. This action is suggestive of potential antipsychotic property. It exhibited no anxiolytic and antidepressant properties in the models used. These results show that PD 144418 is a very selective sigma 1 agent, devoid of any significant affinity for other receptors and that sigma 1 site may modulate actions in the CNS.

Loss of muscarinic M1 receptors with aging in the cerebral cortex of fisher 344 rats

Age-related changes in central cholinergic muscarinic receptors were measured in young (3-6 month), middle-aged (15-17 month), and aged (22-26 month) male Fisher 344 rats by receptor binding techniques. Using [3H]-quinuclidinyl benzilate as the ligand, a significant decrease (14-19%) in the number of muscarinic cortical receptors was measured in aged rats compared to both young and middle-aged rats. With the selective M1 antagonist, [3H]-pirenzepine, a 17% decrease in receptor density was observed in the cortex of aged animals compared to young rats. For both ligands no differences were observed in the striatum or hippocampus between any age group and there was no change in affinity (Kd) in any of the three brain regions for the three age groups. Additionally, there was no difference in choline acetyltransferase activity measured in cortex, hippocampus, or striatum of young and aged rats. Thus, there is a loss of M1 muscarinic receptors in the cerebral cortex of aged male Fisher 344 rats.