Immediate-early gene response to methamphetamine, haloperidol, and quinolinic acid is not impaired in Huntington's disease transgenic mice.

Striatal neurons in symptomatic Huntington's disease (HD) transgenic mice are resistant to a variety of toxic insults, including quinolinic acid (QA), kainic acid and 3-nitropropionic acid. The basis for this resistance is currently unknown. To investigate the possibility that the immediate-early gene (IEG) response is defective in symptomatic HD mice leading to a lack of response to these compounds, we examined the expression of c-Fos and Krox 24 after administration of the indirect dopamine agonist methamphetamine, the dopamine D(2) receptor antagonist haloperidol and the neurotoxin QA in 5- and 10-week-old R6/2 transgenic HD and wild-type mice. Unlike wild-type and pre-symptomatic R6/2 transgenic HD mice, 10-week-old symptomatic HD mice were resistant to methamphetamine-induced gliosis and QA lesion. There was, however, no difference in the number or distribution of c-Fos-immunoreactive nuclei 2 hr after single injections of methamphetamine or haloperidol among 5- and 10-week-old wild-type mice and 5- and 10-week-old R6/2 HD mice. Similarly, despite their resistance to QA-induced lesioning and lower basal levels of krox-24 mRNA, the symptomatic R6/2 mice had equivalent increases in the amount of c-fos and krox-24 mRNA compared to wild-type and pre-symptomatic R6/2 HD mice as determined by in situ hybridization and densitometry 2 hr after QA administration. These data demonstrate that the c-Fos and Krox 24 IEG response to dopamine agonists, dopamine antagonists and neurotoxic insult is functional in symptomatic R6/2 HD mice. Resistance to toxic insult in R6/2 mice may be conferred by interactions of mutant huntingtin with proteins or transcriptional processes further along the toxic cascade.

L-prolyl-l-leucyl-glycinamide and its peptidomimetic analog 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) attenuate haloperidol-induced c-fos expression in the striatum.

Acute treatment of rats with haloperidol results in a rapid and transient increase in striatal c-fos mRNA and Fos immunoreactivity. The induction of immediate early genes by haloperidol may be involved in the development of extrapyramidal side effects. L-Prolyl-L-leucyl-glycinamide (PLG, or MIF-1) has been observed to antagonize the development of haloperidol-induced D(2) receptor supersensitivity in rats. We investigated the modulatory effects of PLG on haloperidol-induced c-fos and Fos protein expression in the rat striatum. We report that coadministration of either PLG or the potent analog of PLG, 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetam ide (PAOPA), attenuated haloperidol-induced c-fos and Fos expression. Haloperidol induced [2 mg/kg, intraperitoneally (i.p.)] c-fos and Fos expression by 500% and 100%, respectively. These responses were attenuated by 170% and 75%, respectively, when coadministered with PLG (20 mg/kg, i.p.) or by 79% by PAOPA (10 microg/kg, i.p.).

Modulatory effects of PLG and its peptidomimetics on haloperidol-induced catalepsy in rats.

A behavioral model of dopaminergic function in the rat was used to examine the anticataleptic effects of L-prolyl-L-leucyl-glycinamide (PLG) and peptidomimetic analogs of PLG. Administration of 1 mg/kg PLG intraperitoneally significantly attenuated haloperidol (1 mg/kg)-induced catalepsy (as measured by the standard horizontal bar test), whereas doses of 0.1 and 10 mg/kg PLG did not. Eight synthetic PLG peptidomimetics (Calpha, alpha-dialkylated glycyl residues with lactam bridge constraint [1-4] and without [5-8]) were tested in the same manner (at a dose of 1 microg/kg) and categorized according to their activity, i.e. very active (5), moderately active (2, 3, 4, and 6), and inactive (1, 7, and 8). The catalepsy-reversal action of the diethylglycine-substituted peptidomimetic 5 was examined further and found to exhibit a U-shaped dose-response effect with an optimal dose of 1 microg/kg. The similarity between the effects of PLG and the synthetic peptidomimetics suggests a common mechanism of action. Finally, the synthetic peptidomimetics examined here, particularly peptidomimetic 5, were more effective than PLG in attenuating haloperidol-induced catalepsy.

Modulation of agonist stimulated adenylyl cyclase and GTPase activity by L-pro-L-leu-glycinamide and its peptidomimetic analogue in rat striatal membranes.

L-prolyl-L-leucyl-glycinamide (PLG), also known as melanocyte-stimulating hormone release inhibiting factor (MIF-1), is an endogenous brain tripeptide. Previous studies have shown that PLG, and its peptidomimetic analogues, render dopamine D2 receptors more responsive to agonists by maintaining the high-affinity binding state of the receptors. In the present study, we examined the effect PLG and its analogue 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetam ide (PAOPA) on dopamine-stimulated adenylyl cyclase and NPA-stimulated GTPase activity in rat striatal membranes. Dopamine-stimulated adenylyl cyclase activity was inhibited by both PLG and PAOPA in a dose-dependent manner, whereas R(-)-propylnorapomorphine (NPA)-stimulated low Km GTPase activity was significantly increased by 1 microM PLG or 1 nM PAOPA. These results suggest that PLG and PAOPA maintain the high affinity state of the D2 receptor by increasing GTP hydrolysis through stimulation of agonist-induced GTPase activity.

Design, synthesis, and dopamine receptor modulating activity of diketopiperazine peptidomimetics of L-prolyl-L-leucylglycinamide.

The diketopiperazine "C5" conformational mimic has been incorporated into the L-prolyl-L-leucylglycinamide (PLG, 1) structure and into the bicyclic lactam PLG peptidomimetic structure 3 to give compounds 5 and 6, respectively. These analogues were designed to explore the idea that the N-terminal "C5" conformation, which was found in the crystal structure of 2 and which was mimicked in 4 by the diketopiperazine function, was a factor in the high potency of these two agents. Through the use of the [3H]spiroperidol/N-propylnorapomorphine (NPA) D2 receptor competitive binding assay, both 5 and 6 were found to increase the affinity of the dopamine receptor for agonists and both were found to increase the percentage of D2 receptors which existed in the high-affinity state. These effects were observed when Gpp(NH)p was either absent or present, and they were analogous to the effects observed previously for PLG and the PLG peptidomimetics 2 and 4. However, the potency seen with 5 and 6 was less than that seen for 2 and 4, suggesting that while the N-terminal "C5" conformation may play a role in the potency of the gamma-lactam peptidomimetics of PLG, it does not appear to be the primary factor. In the 6-hydroxydopamine-lesioned animal model of Parkinson's disease, 5 altered apomorphine-induced rotational behavior in a dose-dependent manner. The maximum effect occurred at a dose of 0.01 mg/kg i.p. and resulted in a 52.27 +/- 13.96% (p < 0.001, n = 7) increase in rotations compared to apomorphine administered alone.

Alterations in inotropy, nitric oxide and cyclic GMP synthesis, protein phosphorylation and ADP-ribosylation in the endotoxin-treated rat myocardium and cardiomyocytes.

To evaluate the effects of the in vivo endotoxin treatment of the rat on (1) the contractile responses in the subsequently isolated papillary muscle to adrenergic and cholinergic agonists and (2) the biochemical parameters (cyclic GMP, nitric oxide synthesis, protein phosphorylation and ADP-ribosyslation) in the subsequently isolated cardiomyocytes. Following the in vivo endotoxin treatment (4 mg/kg i.p., 18 h), contractile responses to increasing amounts of isoprenaline or to increasing amounts of oxotremorine in the presence of a fixed amount of isoprenaline were determined in isolated papillary strips. Activities of nitric oxide synthase, guanylyl cyclase, as well as phosphorylation of phospholamban and troponin-inhibitory subunit, and pertussis toxin-catalyzed and endogenous ADP-ribosylations were determined in the intact cardiomyocytes and subcellular fractions. The increase in the force of contraction by isoprenaline was reduced, while its inhibition by oxotremorine was greater in the endotoxin-treated papillary strips. The activities of both nitric oxide synthase, primarily of the inducible form of the enzyme, and cytosolic guanylyl cyclase were higher while the phosphorylations of both phospholamban and troponin-inhibitory subunit were of lesser magnitude in the cardiomyocytes following the in vivo endotoxin treatment. Pertussis toxin-catalyzed ADP-ribosylation of the 41 kDa polypeptide, which is the alpha subunit of Gi, was also decreased. The results of the present study support the postulate that alterations in both the cyclic AMP and cyclic GMP signalling cascade contribute to the myocardial dysfunction caused by endotoxin and cytokines.

Expressed alpha 1-adrenoceptors in adult rat brown adipocytes are primarily of alpha 1A subtype.

The main objective of this study was to characterize the alpha 1-adrenoceptors expressed in adult rat brown adipocytes. For this purpose, membrane fractions were prepared from brown adipose tissue as well as from isolated brown adipocytes. The following are major findings: (i) BAT membranes were considerably enriched in alpha 1-adrenoceptors (specific [3H]prazosin binding, Bmax, 79.49 +/- 16.77 fmol/mg protein; KD, 0.24 +/- 0.04 nM); (ii) among the cells that comprise brown adipose tissue, brown adipocytes were enriched in alpha 1-adrenoceptors; (iii) > 95% of total alpha 1-adrenoceptors were resistant to inactivation by 20 microM chloroethylclonidine, which readily and essentially completely inactivated alpha 1B-adrenoceptors in rat liver membranes; (iv) brown adipose tissue membrane alpha 1-adrenoceptors showed high affinity towards 5-methyl urapidil (KD 7.23 +/- 2.49 nM) and WB 4101 (KD 0.66 +/- 0.30 nM) and low affinity towards BMY 7378 (KD 0.34 +/- 0.03 microM); essentially similar affinities for these drugs were seen for membranes prepared from brown adipocytes; and (v) EBDA/LIGAND analysis of 5-methyl urapidil, WB 4101, and BMY 7378 competition curves revealed the presence of a single binding site for these drugs. Recent work has documented that 5-methyl urapidil and WB 4101 interact with high affinity with alpha 1A-adrenoceptors, while BMY 7378 interacts with high affinity with alpha 1D-adrenoceptors. Taken together, these findings are consistent with the view that alpha 1-adrenoceptors expressed in adult rat BAT are mainly of the alpha 1A subtype.