Rat Frontal Cortex Membranes Research Articles

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of alpha 2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [35S]-guanosine 5'-O-(gamma-thiotriphosphate) ([35S]GTP gamma S) binding assay. alpha 2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [35S]GTP gamma S binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat alpha 2 adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the alpha 2A-adrenergic system. Only using compromising concentration of agonist (up to 1 microM UK14304) and antagonist (up to 1 microM RS79948) to inhibit agonist's effect, allowed us to filtrate out alpha 2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.

Decrease of adenylyl cyclase activity and expression by a sigma1 receptor ligand and putative atypical antipsychotic.

We examined whether changes in the adenylyl cyclase system could be induced by the administration of the sigma1 receptor ligand and putative atypical antipsychotic 4-[4-fluorophenyl]-1,2,3,6-tetrahydro-1-[4-[1,-2,4-triazol-1-il]butyl]pyridine citrate) (E-5842). Repeated (21 days) but not acute (2 h) treatment with E-5842 induced a significant decrease in adenylyl cyclase type I immunoreactivity and adenylyl cyclase activity in rat frontal cortex membranes, with less or no effect in other brain regions such as the hippocampus or the striatum. Changes in immunoreactivity were not observed in other adenylyl cyclases (type V/VI). The reported changes, observed only after a chronic treatment, could be related to the mechanism of action of sigma receptor ligands in general or to that of E-5842 in particular and should be taken into account, given the long duration of treatment in psychiatric patients.

Changes in phosphoinositide signalling activity and levels of the alpha subunit of G q/11 protein in rat brain induced by E-5842, a sigma 1 receptor ligand and potential atypical antipsychotic

Changes in the phosphoinositide (PPI) signal transduction system induced by E-5842, a new sigma 1 (σ 1) receptor ligand and potential atypical antipsychotic, were studied in the rat frontal cortex, hippocampus and striatum. Acute treatment with E-5842 increased phospholipase C (PLC) activity in the striatum and the hippocampus. Chronic treatment with E-5842 induced an increase in the activity of PLC in the frontal cortex and the striatum. Similar up-regulation of the activity of the enzyme was also observed in rat frontal cortex membranes in presence of GTPγS. After chronic treatment with E-5842, it was also observed a significant increase of the immunoreactivity levels of G q/11α in the frontal cortex. Our results suggest that part of the antipsychotic effects of E-5842 could be related to the regulation of the PPI signal transduction pathway, especially after a prolonged treatment.

C5-Substituted Derivatives of 5-OMe-BPAT: Synthesis and Interactions with Dopamine D2 and Serotonin 5-HT1A Receptors

Eight new C5-substituted derivatives of the potential atypical antipsychotic agent 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) have been prepared by chemical conversion of the 5-trifluoromethylsulfonyloxy (triflate) analogue 4 via various Stille-type cross-couplings, a Heck reaction, and an amidation in moderate to good yields. The 5-acetyl, 5-cyano, 5-methyl, 5-(2-furyl), 5-phenyl, methyl 5-carboxylate, and the 5-carboxamido analogues 5–11 thus obtained, the previously disclosed 5-methoxy, 5-hydroxy, and 5-unsubstituted analogues 1–3, and the 5-triflate analogue 4 were evaluated for their ability to compete for [3H]-spiperone binding to rat striatal membranes containing dopamine D2 receptors, and their ability to compete for [3H]-8-OH-DPAT binding to rat frontal cortex membranes containing serotonin 5-HT1A receptors in vitro. Compounds 1–11 displayed weak to high affinities for dopamine D2 receptors, with Ki-values ranging from 550 nM for the 5-carboxamido analogue to 4.9 nM for the 5-hydroxy analogue. The relative affinities of the 5-methoxy, 5-hydroxy, and 5-unsubstituted analogues suggested that these compounds may bind to the same site and in a similar way as the 5-oxygenated DPATs, with the 5-methoxy substituent of 1 functioning as a hydrogen bond acceptor. The serotonin 5-HT1A receptor tolerated more structural diversity at the C5-position of 1, as revealed by the higher Ki-values of 1–11, which ranged from 60 nM for the 5-carboxamido analogue to 1.0 nM for the 5-unsubstituted analogue. Partial least-squares (PLS) analysis of a set of 24 molecular descriptors, generated for each analogue, revealed no significant correlation between the dopamine D2 receptor affinities of 1–11 and their molecular properties, supporting the view that they may have different binding modes at this receptor subtype. A PLS model with moderate predictability (Q2=0.49) could be derived for the serotonin 5-HT1A receptor affinities of 1–11. According to the model, a relatively lipophilic, nonpolar C5-substituent should be optimal for a high affinity at this receptor subtype.

Voltage-dependent calcium channel β-subunits in combination with α1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G αo in rat frontal cortex

The dihydropyridine-sensitive calcium channel agonist (−)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by G o in rat frontal cortex membranes. An anti-calcium channel β-subunit antiserum abolished the (−)-BayK 8644-stimulated hydrolysis of GTP by G o and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the β-subunit binding domain of the calcium channel complex, also attenuated (−)-BayK 8644 activation of GTPase. This study suggests that the calcium channel β-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by G o. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal.

Coupling of receptors in brain membranes by fusion to the adenylate cyclase system of a foreign effector cell

Receptor-effector coupling in the adenylate cyclase (AC) system was studied using fusion transfer of rat or monkey frontal cortex membranes to Friend erythroleukemia (Fc) cells. The indigenous AC activity of cortex membranes had previously been inactivated with N-ethylmaleimide. In the fusates, the AC activity could be stimulated through β-adrenoceptors using noradrenaline (NA), or through specific receptors fpor vasoactive intestinal polypeptide (VIP), or by fluoride which activates the effector components of the AC-system directly, bypassing receptors. There was a critical stoichiometric relationship between the receptor-stimulated cAMP output and the number of recipient cells of the fusion system, i.e. the total AC capacity as measured by fluoride stimulation. In fusates with rat brain membranes, the β-adrenoceptor coupling increased as the availability of recipient cells increased; on the other hand, the excess of recipient cells did not change the VIP receptor coupling capacity. In fusates with monkey brain membranes, the situation was the opposite: VIP receptor coupling increased as larger amounts of recipient cells were made available, but the β-adrenoceptor coupling capacity remained unchanged. The differences in coupling capacities were related to differences in receptor binding with higher β-adrenoceptor density in rat than in monkey frontal cortex membranes, as opposed to higher VIP receptor density in monkey than in rat frontal cortex membranes. Neuropeptide tyrosine (NPY) attenuated both NA-and VIP-induced activation of AC in the fusates; it was equally potent against both agents. In rat brain membrane fusates, the NA-induced AC activity was attenuated in a dose-dependent and apparently non-competitive manner. Pretreatment with pertussis toxin abolished the effect indicating that the NPY receptor couples to a mechanism involving G i and G o protein. In conclusion, the fusion procedure may allow the determination of the coupling capacity of receptors, activating or inhibiting adenylate cyclase.

Identification of the serotonin-S 2 receptor ligand binding site by photoaffinity labelling with 7-azido-8-[ 125I]ketanserin ([ 125I]AZIK)

7-Azido-8-[ 125I]ketanserin ([ 125I]AZIK) was characterized as a potent photoaffinity probe for serotonin-S 2 receptors. In reversible binding experiments, [ 125I]AZIK bound with high affinity (K d = 0.69 nM) to rat frontal cortex membranes. When incubation with [ 125I]AZIK was followed by UV irradiation, the binding was found to be irreversible. Protection experiments with various drugs demonstrated the serotonin-S 2 nature of the photoaffinity labelling. SDS-polyacrylamide gel electrophoresis of the photolabelled membranes allowed one to identify the serotonin-S 2 receptor ligand binding site as a single polypeptide with a molecular mass of approx. 67500 Da. [ 125I]AZIK will be a valuable tool for the elucidation of the serotonin-S 2 receptor structure.

Antagonism of a peripheral vascular but not an apparently central serotonergic response by xylamidine and BW 501C67

Xylamidine and BW501C67 (α-anilino-N-2-m-chlorophenoxypropylacetamidine), serotonin antagonists that have been reported not to cross the blood-brain barrier, were compared to other serotonin antagonists: mianserin, ketanserin, metergoline and LY 53857. All six compounds were potent inhibitors of the binding of tritiated spiperone to 5HT 2 receptors in rat frontal cortex membranes in vitro and were less potent inhibitors of the binding of tritiated serotonin to 5HT 1 receptors in rat brain membranes. All were potent antagonists of the 5HT 2 receptor-mediated contractile response to serotonin in the rat jugular vein in vitro. At doses of 0.1 and 0.3 mg/kg i.p., xylamidine and BW501C67 antagonized the pressor response to intravenously injected serotonin in pithed rats. In contrast, neither xylamidine nor BW501C67 at doses of 1 or 3 mg/kg i.p. antagonized the elevation of serum corticosterone concentration by quipazine in rats, although the other compounds antagonized this effect with ED 50 values between 0.03 and 0.9 mg/kg. These data corroborate the previously reported antiserotonin activity of xylamidine and BW501C67. Since xylamidine and BW501C67 were potent antagonists of a peripheral serotonergic response in vivo, their inability to antagonize the elevation of serum corticosterone concentration by quipazine suggests that this effect results from activation of central serotonin receptors.

Characterization of 125I-lysergic acid diethylamide binding to serotonin receptors in rat frontal cortex.

125I-Lysergic acid diethylamide (125I-LSD) is the first 125I-labeled ligand for serotonin receptor studies. Its binding to rat frontal cortex membranes is saturable, reversible, and stereospecific. Specific binding is linearly dependent on tissue concentration and represents 70-80% of the total binding. Scatchard plots of the binding data are linear with a KD of 1.5 nM, a Bmax of 12.4 fmol/mg wet weight tissue, and a Hill slope of 1.02. The binding kinetics are highly temperature-dependent. At 37 degrees C the bimolecular association rate constant is 1.28 X 10(8) min-1 M-1 and the dissociation rate constant is 0.087 min-1 (t 1/2 = 8.0 min). At 0 degrees C less than 4% dissociation occurs over 40 min and the association rate is similarly depressed. Inhibition of 125I-LSD binding by a variety of serotonergic, dopaminergic, and adrenergic ligands reveals a 5-hydroxytryptamine2 (5-HT2) serotonergic profile for this binding site. Regional distribution studies of 125I-LSD binding in rat brain show that areas with the highest levels of binding include the cortex and striatum. Iodinated radioligands can be synthesized with specific activities exceeding 2,000 Ci/mmol, which makes them approximately 75-fold more sensitive than tritiated radioligands. This high specific activity, coupled with the selectivity of 125I-LSD for 5-HT2 sites, makes this ligand a sensitive new probe for 5-HT2 serotonin receptors.