1 The relationships between the density of dopamine D 4.4 receptors and the agonist e cacies of L-745,870 (3-(4-[4-chlorophhenyl]piperazin-1-yl)-methyl-1H-pyrrolo [2,3-b]pyridine) and U-101958 ((1-benzyl-piperidin-4-yl)-(3-isopropoxy-pyridin-2-yl)-methyl-amine) were investigated in Chinese hamster ovary (CHO) cells, after treatment with the gene expression enhancer, sodium butyrate. 2 In CHO cells expressing D 4.4 receptors (CHO/D 4 cells), dopamine inhibited forskolin-stimulated cyclic AMP accumulation (E max 56+1% inhibition, pEC 50 7.4+0.1, n=10). U-101958 behaved as a partial agonist (39+7% the e cacy of dopamine, pEC 50 8.1+0.3, n=4), whereas L-745,870 had no detectable agonist e ect. 3 Receptor density, as estimated by [ 3 H]-spiperone saturation binding was 240+30 fmol mg 71 protein (n=8) in CHO/D 4 cell homogenates. It reached 560+150 (n=6), 1000+190 (n=4) and 840+120 (n=4) fmol mg 71 protein after treatment with sodium butyrate (5 mM) for 6, 18 and 48 h, respectively. 4 The increase in receptor density was associated with a gradual enhancement of the agonist e ects (increased E max and pEC 50 values) of dopamine. The e cacy of U-101958 (relative to dopamine) doubled and L-745,870 was turned into a partial agonist (e cacy 49% relative to dopamine, pEC 50 8.6+0.2, n=6, after 48 h treatment with sodium butyrate). These agonist e ects of U-101958 and L-745,870 could be antagonized by spiperone (0.1 mM) but not by raclopride (10 mM). 5 The results show that U-101958 and L-745,870 are partial agonists at human dopamine D 4.4 receptors expressed in CHO cells. Their e cacy is governed by receptor density. Agonist e ects of these two compounds in vivo cannot be excluded under circumstances of increased receptor levels.
1 Dopamine D 4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-eects. Two such compounds, L-745,870 and U-101958 have been recently introduced. ]-spiperone binding was observed with spiperone (pK i 9.6+0.1, n=3), clozapine (pK i 7.4+0.1, n=4), L-745,870 (pK i 8.5+0.1, n=3) and U-101958 (pK i 8.9+0.1, n=3). By contrast, raclopride was very weak (pK i 55, n=3). 4 Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D 4 cells in a concentration-dependent fashion (E max 71+2% inhibition of forskolin-stimulated levels, pEC 50 8.7+0.1, n=10). This eect was mimicked by the dopamine D 2 -like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT).
IContractile responses of rabbit aortic rings elicited by KCI-depolarization, angiotensin II (All), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) have been investigated in the presence of cromakalim (BRL 34915) and isradipine .2 Above 10-6M, cromakalim inhibited contractile responses to low (,<32mM) but not to higher KCl concentrations. The 5-HT and All concentration-response curves were antagonized noncompetitively by cromakalim (10-7-1O-M) and the maximal responses were inhibited by 40 and 55%, respectively. 3 Isradipine caused less inhibition of All and 5-HT contractile responses than cromakalim, and in the presence of isradipine (10-7M), cromakalim was still able to antagonize further the contractions to All in this vessel. 4 NA-induced contractions were relatively insensitive to inhibition by cromakalim and isradipine, both drugs causing a small rightward shift of the NA concentration-response curve. This result suggests that NA utilizes different Ca2 + pools from those involved in All-and 5-HT-induced contractions of this vessel. 5 The sustained (tonic) part of the NA response was inhibited in a concentration-dependent manner by cromakalim (10--1O-I M), but not by isradipine. 6 In aortic rings partially depolarized with 3.5 x 10-2 M KCI, the ability of cromakalim, but not of sodium nitroprusside, atriopeptin III or hydralazine, to inhibit All-and tonic NA-induced contractions was abolished. 7 Antivasoconstrictor activity of cromakalim on the rabbit aorta appears to involve factors in addition to an indirect inhibition of Ca2 + entry through dihydropyridine-sensitive Ca2+ channels. 8 The ability of cromakalim to open K+ channels and thereby modify the membrane potential would appear to underlie these antivasoconstrictor effects. This mechanism of action of cromakalim clearly differs from that of other vasodilators such as sodium nitroprusside and hydralazine.
A cDNA clone encoding the rat metabotropic glutamate receptor mGluR3 was stably transfected into human embryonic kidney 293 cells. Receptor-expressing cell lines were characterized by centrifugation binding assays using [3H]glutamate as radioligand. The rank order of affinity was L-glutamate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) > L(+)-2-amino-3-phosphonopropionic acid (L-AP3) > quisqualic acid > L(+)-2-amino-4-phosphonobutyric acid (L-AP4) > ibotenic acid. The active enantiomers of several phenylglycines displayed Ki values of 300 to 400 microM. The nonactive enantiomers and the standard ionotropic glutamate receptor ligands N-methyl-D-aspartic acid (NMDA), (R,S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainic acid only weakly displaced [3H]glutamate. In this cell line, L-glutamate and (2S,3S,4S)-alpha-(Carboxycyclopropyl)-glycine (L-CCG-I) reduced cAMP levels in a dose-dependent manner. The sensitivity of this system and its easy applicability make it feasible to envisage ligand binding assays on cell lines expressing cloned receptors as useful screening tools to discover and characterize new and specific agonists and antagonists.
In order to test the ability of aged animals to compensate a cerebrovascular insufficiency, common carotid ligation was performed on young (12 months) and aged (30 months) rats. By using gold microelectrodes, the pO2 distribution in the cerebral cortex was registered for 1 h following the occlusion of the carotid arteries. It could be shown that, while the young group was able to compensate the reduction in arterial blood supply, the cortical pO2 distribution in the group of aged rats shifted towards a hypoxic profile. These results provide further evidence of the limited adaptability to functional demands with increasing age.
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