Regional CNS densities of serotonin and dopamine receptors in high alcohol-drinking (HAD) and low alcohol-drinking (LAD) rats.

The densities of subtypes of serotonin (5-HT) and dopamine (DA) receptors were determined in the CNS of male alcohol-naive HAD and LAD lines of rats. Autoradiographic studies were undertaken to measure the densities of (a) 5-HT1A sites labelled with 2 nM [3H]8-OH DPAT, (b) 5-HT2A sites labelled with 2 nM [3H] ketanserin, (c) D1 sites labelled with 1 nM [3H]SCH23390, and (d) D2 sites labelled with 20 nM [3H]sulpiride. Membrane binding, using tissue combined from the olfactory bulb, olfactory tubercle, and nucleus accumbens, was carried out to determine Kd and Bmax values for the binding of 0.25-8.0 nM [3H]7-OH DPAT to D3 sites. Among the 14 regions measured for densities of 5-HT1A sites, no interline differences were found in the cerebral cortical regions or in the septal nuclei; however, within the hippocampus, 15-20% lower binding of [3H]8-OH DPAT was observed in the posterior dorsal CA3 and dentate gyrus of the HAD line. There were no interline differences in any of the 10 regions examined for [3H]ketanserin binding to 5-HT2A sites, or in the densities of D1 and D2 sites in the mesolimbic and nigrostriatal DA systems, except for a 35% higher density of D2 sites in the substantia nigra pars compacta of the HAD line. There were no interline differences in the Kd or Bmax values for [3H]7-OH DPAT binding to D3 sites. Overall, these results indicate that no marked interline differences are evident in the densities of 5-HT1A, 5-HT2A, D1, D2, and D3 receptors within the mesolimbic system that could be associated with the disparate alcohol drinking behaviors of the HAD and LAD rats.

Regional CNS densities of monoamine receptors in alcohol-naive alcohol-preferring P and -nonpreferring NP rats.

The densities of subtypes of serotonin (5-HT) and dopamine (DA) receptors were determined in the CNS of alcohol-naive alcohol-preferring P and -nonpreferring NP lines of rats. Autoradiography studies were undertaken to measure the densities of 5-HT1B sites labelled with 100 pM [125I](-)-iodocyanopindolol, 5-HT3 sites labelled with 2 nM [3H]LY 278584, and D1 sites labelled with 1 nM[3H]SCH 23390. Membrane binding, using tissue combined from the olfactory bulb, olfactory tubercle, and nucleus accumbens, was carried out to determine Kd and B max values for the binding of 0.25-8.0 nM[3H]7-OH DPAT to D3 sites. Among the 48 regions measured for differences in 5-HT1B recognition sites, statistically significant differences (p < 0.05) were found only in the cingulate and retrosplenial cortices, in the lateral and medial septum, and in the lateral nucleus of the amygdala, with lower values being found in the P than the NP line. There were no significant differences in the regional CNS densities of D1 or 5-HT3 sites between the P and NP lines. There were also no differences between the rat lines in the Kd or Bmax values for [3H]7-OH DPAT binding to D3 sites. The lower densities of 5-HT1B sites in the CNS of the P compared to the NP rats may be a result of reduced numbers of 5-HT1B presynaptic autoreceptors as well as postsynaptic receptors in the P line. The observation that there are no differences in the amount of radioligand binding to D1, 5-HT3, and D3 sites between the P and NP lines suggests that the disparate alcohol drinking behaviors of these two lines is not associated with an innate alteration in the densities of these receptor subtypes.

Apomorphine and 7-OH DPAT reduce ethanol intake of P and HAD rats.

Adult male rats of the alcohol-preferring (P) line (N = 10) and high alcohol drinking (HAD) line (N = 12) were used to study the effects of IP administration of 0.125-0.50 mg/kg 7-OH DPAT (a putative D agonist) and 0.25-1.0 mg/kg apomorphine (a dopamine agonist with 50-fold higher affinities for the D1 and D2 receptors than for the D3 receptor) on the concurrent intakes of 10% (v/v) ethanol and 0.0125% (g/v) saccharin during a daily 4-h scheduled access period. Control intakes by the P rats for the 4-h period were 17.9 +/- 0.5 and 7.2 +/- 0.4 ml for the ethanol and saccharin solutions, respectively. For the HAD line, ethanol consumption was 18.7 +/- 0.2 ml and saccharin intake was 8.7 +/- 1.6 ml for the 4-h period. In terms of grams ethanol/kg body wt, the 4-h intakes were 2.2 +/- 0.2 for the P line and 3.0 +/- 0.3 for the HAD rats. Both P and HAD rats consumed approximately 40% of their total ethanol intake in the first 15 min of access while consuming only about 15% of their total saccharin intake during this 15-min period. The putative D3 agonist 7-OH DPAT produced a decrease in ethanol intake in the first h to 45-55% of control levels for the P rat (p < 0.01) and to 25-70% of control values in the HAD line (p < 0.001). Apomorphine caused a dose-dependent decrease in ethanol intake in the first hour to 15-70% of control values in the P rat (p < 0.001) and to 25-60% of control levels in the HAD line (p < 0.001). Saccharin and 4-h food intakes for both lines were not altered by either 7-OH DPAT or apomorphine. Overall, these results suggest that D2 and D3 dopamine receptors may play a role in mediating alcohol drinking behavior of the selectively bred HAD and P lines of rats.

Consumption of sweet, salty, sour, and bitter solutions by selectively bred alcohol-preferring and alcohol-nonpreferring lines of rats.

To determine whether selective breeding for high or low oral ethanol consumption is associated with different preferences for nonpharmacological solutions with various flavors, the oral intake of a range of concentrations of sucrose (0.5-64.0 g/100 ml), NaCl (0.025-3.2 g/100 ml), citric acid (0.008-2.048 g/liter), and sucrose octaacetate (0.002-0.512 g/liter) was studied in alcohol-preferring (P) and alcohol-nonpreferring (NP) rats. Separate groups of 7-8 rats from each line were tested for consumption of each of the four flavors. The flavored solutions were presented continuously with water and food always available, and the concentrations were doubled every 48 hr. Although rats from both lines showed a strong preference for the sucrose solutions, P rats consumed greater amounts than NP rats [F(7,98) = 5.57, p < 0.001]. Rats of the P line drank less of the NaCl solutions than NP rats [F(7,98) = 3.88, p < 0.001], but the effect was not as robust as the line differences seen with sucrose. The P and NP rats did not differ in citric acid or sucrose octaacetate intake at any of the concentrations tested. Selective breeding for high oral ethanol preference appears to be positively associated with consumption of sweet solutions and negatively associated with intake of salty solutions.

Mechanistic studies of the biosynthesis of 3,6-dideoxyhexoses in Yersinia pseudotuberculosis. Purification and stereochemical analysis of CDP-D-glucose oxidoreductase.

An NAD(+)-dependent CDP-D-glucose oxidoreductase which catalyzes the first step of the biosynthesis of CDP-ascarylose (CDP-3,6-dideoxy-L-arabino-hexose), converting CDP-D-glucose to CDP-4-keto-6-deoxy-D-glucose, was isolated from Yersinia pseudotuberculosis. A protocol consisting of DEAE-cellulose, Matrex Blue-A, hydroxylapatite, DEAE-Sephadex, Sephadex G-100, and NAD(+)-agarose column chromatography was used to purify this enzyme 6000-fold to homogeneity. This enzyme consists of two identical subunits, each with a molecular weight of 42,500. Using CDP-D-glucose as the substrate, the Km and Vmax of this catalysis were determined to be 222 microM and 8.3 mumols mg-1 min-1, respectively. Unlike most other oxidoreductases of its class which have a tightly bound NAD+, this highly purified CDP-D-glucose oxidoreductase showed an absolute requirement of NAD+ for its activity. Using chemically synthesized (6S)- and (6R)-CDP-D-[4-2H,6-3H]glucose as substrates, a stereochemical analysis showed this enzymatic reaction involves an intramolecular hydrogen migration from C-4 to C-6, and the displacement of C-6 hydroxyl group by the C-4 hydrogen occurs with inversion. Thus, despite the low cofactor affinity, this enzyme undergoes a mechanism consistent with that followed by other members of its type. Such a mechanistic and stereochemical convergency found for all sugar oxidoreductases so far characterized suggests the presence of a common progenitor of this class of enzyme.

Synthesis of stereospecifically labeled 3,6-dideoxyhexoses.

Preparations of ascarylose (3,6-dideoxy-L-arabino-hexose), abequose (3,6-dideoxy-D-xylo-hexose), and paratose (3,6-dideoxy-D-ribo-hexose) with stereospecific deuterium labeling at C-3 are discussed. The methods used to synthesize these sugars, such as the hydrogenation of olefins, the displacement of halides, the reduction of epoxides, and the substitution of tosyl esters, illustrate a variety of strategies leading to stereospecific deuterium incorporation. Many of the techniques described here should be of general utility for the synthesis of other deuterium-labeled sugars.