Dopamine separation

Application of the CCM to small sets (n < 6) of enzyme inhibitors revealed correlations between the inhibitory activity and the chirality measure of the inhibitors, calculated by Eq. (26) for the entire structure or for the substructure that interacts with the enzyme (pharmacophore) [41], This was done for arylammonium inhibitors of trypsin, Di-dopamine receptor inhibitors, and organophosphate inhibitors of trypsin, acetylcholine esterase, and butyrylcholine esterase. Because the CCM values are equal for opposite enantiomers, the method had to be applied separately to the two families of enantiomers (R- and S-enantiomers). 

The task of the study presented here was to separate a data set of 172 molecules into benzodiazepine agonists 60 compounds) and dopamine agonists (112 compounds) [84]. 

Even in this fiiirly diverse data set of structures, the dopamine and benzodiazepine agonists could be separated quite well only two neurons had collisions between these two types ol compounds. Even more importantly, however, we now know in which chemical space one would have to search For new lead structures for dopamine or for benzodiazepine agonists. 

FIGURE 3-24 Electrophoretic separation of catechols with end-column detection. Detection potential, +0.8 V separation capillary, 20 kV The peaks correspond to 4.6 fmol dopamine (1), 4.1 fmol isoproterenol (2), and 2.7 fmol catechol (3). (Reproduced with permission from reference 60.)... 

Biochemical analyses of 6-OHDA-injected animals revealed a 93 percent depletion of dopamine. The tissue was assayed using electrochemical detection following separation by high-pressure liquid chromatography (Felice et al. 1978). recorded as ng/mg protein in the nucleus accumbens and compared to control rats with sham lesions (sham=65.5 4.4, lesion=4.9 1.5 t(39)=23.4). A lesion was defined as complete if 75 percent or more of the dopamine was determined to be depleted from the nucleus accumbens compared to mean sham group values. 

The majority of DDC-expressing cells in the brain lobes are dopamine cells. Most of these dopamine cells have axons that project into a common axonal fiber extending anteriomedially within the brain lobe and then separating into finer fibers that cross between the lobes. The dopamine cells occur in small clusters of two to six cells, which suggests that these cells might share common lineages. The serotonin cells within the lobes are also found in pairs, and each pair projects axons into closely associated tracts. The pathways of the serotonin tracts often parallel those of the dopamine cells but are distinct (Lundell and Hirsh, 1994). 

Figure 1-1 shows an example of the detection limits that can be reached with EC detection. The chromatogram shows the separation and detection of the catecholamines noradrenaline (nor), adrenaline (adr), dihydroxybenzylamine (dhba), and dopamine (dopa), using a glassy carbon electrode at a working potential of +0.6 V. The minimum detectable quantity is less than 3 Pg-... 

Rose JE, Levin ED (1991) Inter-relationships between conditioned and primary reinforcement in the maintenance of cigarette smoking. Br J Addict 86(5) 605-609 Rosecrans JA (1971) Elfects of nicotine on brain area 5-hydroxytryptamine function in male and female rats separated for differences of activity. Eur J Pharmacol 16(1) 123-127 Rosecrans JA (1972) Brain area nicotine levels in male and female rats with different levels of spontaneous activity. Neuropharmacology ll(6) 863-870 Rosecrans JA, Schechter MD (1972) Brain area nicotine levels in male and female rats of two strains. Arch Int Pharmacodyn Ther 196(l) 46-54 Saigusa T, Takada K, et al (1997) Dopamine efflux in the rat nucleus accumbens evoked by dopamine receptor stimulation in the entorhinal cortex is modulated by oestradiol and progesterone. Synapse 25(1) 37 3... 

Ng, G. Y., Trogadis, J., Stevens, J., Bonvier, M., O Dowd, B. F., and George, S. R. (1995) Agonist-induced desensitization of dopamine D receptor-stimnlated adenylyl cyclase activity is temporally and biochemically separated from D receptor internalization. Proc. Natl. Acad. Sci. U. S. A. 92, 10157-10161. 

Fig. 7.5 Schematic flowchart of the competitive MS-binding assay quantifying the nonbound marker employed for dopamine Di receptors. After incubation of the target (Di receptor) in presence of the marker (SCH 23390) and a test compound, the binding samples are centrifuged to separate bound from nonbound marker. The nonbound marker in the resulting supernatant is quantified by LC-ESI-MS/MS without further sample preparation.

Fig. 7.6 Nonbound SCH 23390 in a competitive MS binding assay for dopamine Di receptors monitored at a transition from 288.1 91.2 m/z from binding samples without or with (+)-butaclamol. Intensity (/) is shown (a) without (+)-butaclamol, (b) with 30 nM (+)-butaclamol, (c) with 10 pM (+)-butaclamol. (a-c) Representative chromatograms after HPLC separation (RP8 column solvent CH3CN/0.1% HCOOH in H2O 1 1 300 pL min ).

Fio. 51. Dependence of retention of catecholamines on volume percent acetonitrile in hetaeric chromatography. The ehient is water-acetonitrile at the volume percent indicated containing 0.2% (v/v) sulfuric acid and 0.1% (w/v) sodium dodecyl sulfote. The catecholamines separated are noradrenaline (NA), adrenaline (A). L-3,4-dihydroxyphenylalanine (LD), normetanephrine (NMA), dopamine (DA), metadrenaline (MA), and 3-methoxytyramine (MDA). Column 5- tm octadecyl silica treated with triroethylchlorosilane, 125 X 5 mm i.d. Reprinted with permission from Knox and Jurand (223). ... 

MAO is a much less discriminating enzyme in that it will catalyze the removal of an amine group from a variety of substrates. The action of MAO on norepinephrine and epinephrine also is indicated in Figure 9.5. The list of its substrates is very large, including endogenous substances (norepinephrine, epinephrine, dopamine, tyramine, 5-hydroxy-tryptamine) and many drugs that are amines. At least in the brain, two separate forms of MAO have been described MAO type A and MAO type B. The two types are differentiated on the basis of substrate and inhibitor specificity. 

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