Decreased Hepatic 5-HT<Subscript>1A</Subscript> Receptors During Liver Regeneration and Neoplasia in Rats

In the present study we investigated the role of 5-hydroxytryptamine (5-HT) and 5-HT_1A receptor during liver regeneration after partial hepatectomy (PH) and N-nitrosodiethylamine (NDEA) induced hepatocellular carcinoma in male Wistar rats. 5-HT content was significantly increased during liver regeneration after PH and NDEA induced hepatocellular carcinoma. Scatchard analysis using 8-OH-DPAT, a 5-HT_1A specific agonist showed a decreased receptor during liver regeneration after PH and NDEA induced hepatocellular carcinoma. 5-HT when added alone to primary hepatocyte culture did not increase DNA synthesis but was able to increase the EGF mediated DNA synthesis and inhibit the TGFβ1 mediated DNA synthesis suppression in vitro. This confirmed the co-mitogenic activity of 5-HT. 8-OH-DPAT at a concentration of 10⁻⁴ M inhibited the basal and EGF-mediated DNA synthesis in primary hepatocyte cultures. It also suppressed the TGFβ1-mediated DNA synthesis suppression. This clearly showed that activated 5-HT_1A receptor inhibited hepatocyte DNA synthesis. Our results suggest that decreased hepatic 5-HT_1A receptor function during hepatocyte regeneration and neoplasia has clinical significance in the control of cell proliferation.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

Enhanced 5-HT<Subscript>2A</Subscript> Receptors in Brain Stem and ALDH Activity in Brain Stem and Liver: 5-HT<Subscript>2A</Subscript> Regulation on ALDH in Primary Hepatocytes Cultures In Vitro

Brain serotonin (5-HT) modulates the neural effects of ethanol. In the present study, we investigated the changes in 5-HT level, 5-HT_2A receptor binding and aldehyde dehydrogenase (ALDH) activity in brain stem and liver of ethanol treated rats and 5-HT_2A regulation on ALDH in hepatocyte cultures in vitro. The 5-HT content in the brain stem and liver significantly decreased with an increased 5-HIAA/5-HT ratio in the ethanol treated rats compared to control. Scatchard analysis of [³H] (±)2,3-dimethoxyphenyl-1-[2-(-4-piperidine)-methanol] [³H] MDL 100907 against ketanserin in brain stem of ethanol treated rats showed a significant increase in B _max without any change in K _d compared to control. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in both control and ethanol treated rats with unity as Hill slope value. A significant increase in V _max of ALDH activity in liver and a significant decrease in K _m in liver and brain stem of ethanol treated rats compared to control was observed. In 24 h culture studies, an increase in enzyme activity was observed in cells in medium with 10% ethanol. The elevated ALDH activity in ethanol treated cells was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT. Ketanserin, an antagonist of 5-HT_2A, reversed the effect of 5HT on 10% ethanol induced ALDH activity in hepatocytes. Our results showed that there was a decreased 5-HT content with an enhanced 5-HT_2A receptor and aldehyde dehydrogenase activity in the brain stem of alcohol treated rats and in vitro hepatocyte cultures. The enhanced ALDH activity in ethanol supplemented hepatocytes was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT.     

Enhanced 5-HT<Subscript>2A</Subscript> Receptor Status in the Hypothalamus and Corpus Striatum of Ethanol-Treated Rats

Aim Brain is the major target for the actions of ethanol and it can affect the brain in a variety of ways. In the present study we have investigated the changes in 5-HT level and the 5-HT_2A receptors in the ethanol-treated rats. Methods Wistar adult male rats of 180–200 g body weight were given free access to 15% (v/v) (approx.7.5 g/Kg body wt./day) ethanol for 15 days. Controls were given free access to water for 15 days. Brain 5-HT and its metabolites were assayed by high performance liquid chromatography (HPLC) integrated with an electrochemical detector (ECD) fitted with C-18-CLS-ODS reverse phase column. 5-HT_2A receptor binding assay was done with different concentrations of [³H] MDL 100907. Results The hypothalamic 5-HT content significantly increased (P < 0.001) with a decreased (P < 0.001) 5-HIAA/5-HT turnover in the ethanol-treated rats when compared to control. The corpus striatum 5-HT content significantly decreased (P < 0.01) with increased (P < 0.01) 5-HIAA/5- HT turnovers in the ethanol-treated rats when compared to control. Scatchard analysis of [³H] MDL 100907 against ketanserin in hypothalamus showed a significant increase (P < 0.001) in B_max with a decreased affinity (P < 0.001) in ethanol-treated rats when compared to control. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in all the groups with unity as Hill slope value. An increased K_i and log (EC₅₀) value were also observed in ethanol-treated rats when compared to control. Scatchard analysis of [³H] MDL 100907 against ketanserin in the corpus striatum of ethanol-treated rats showed a significant increase (P < 0.001) in B_max and in affinity (P < 0.01) when compared to control. The change in affinity of the receptor protein in both corpus striatum and hypothalamus shows an altered receptor. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in all the groups with unity as Hill slope value. There was no significant change in K_i and log (EC ₅₀) value in ethanol-treated rats when compared to control. Conclusion The present study demonstrated the enhanced 5-HT_2A receptor status in hypothalamus and corpus striatum. The ethanol-induced enhanced 5-HT_2A receptors in the hypothalamus and corpus striatum has clinical significance in the better management of ethanol addiction. This will have therapeutic application.     

Prefrontal Serotonergic Denervation Induces Increase in the Density of 5-HT<Subscript>2A</Subscript> Receptors in Adult Rat Prefrontal Cortex

The 5-HTergic system and particularly 5-HT_2A receptors have been involved in prefrontal cognitive functions, but the underlying mechanisms by which the serotonin (5-HT) system modulates these processes are still unclear. In this work, the effects of prefrontal 5-HTergic denervation on the density and expression levels of 5-HT_2A receptors were evaluated by immunohistochemical and molecular biology studies in the prefrontal cortex (PFC). The [³H]-Ketanserin binding study revealed an increase in the B_max, along with no change in the binding affinity (K_D) for 5-HT_2A receptors. The increase in PFC of 5-HT_2A receptor density in response to denervation was accompanied by increase in 5-HT_2A receptor mRNA and protein levels. This increase in the number of 5-HT_2A receptors may be interpreted as an adaptive plastic change, i.e., hypersensitivity; resulting from the selective pharmacological lesion of the raphe-proceeding 5-HTergic fibers to the PFC. Based on previous evidence, this could be strongly related to the abnormal expression of short-term memory.     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

Decreased 5-HT2C receptor binding in the cerebral cortex and brain stem during pancreatic regeneration in rats

The purpose of this study was to investigate the role of central 5-HT_2C receptor binding in rat model of pancreatic regeneration using 60–70% pancreatectomy. The 5-HT and 5-HT_2C receptor kinetics were studied in cerebral cortex and brain stem of sham operated, 72 h pancreatectomised and 7 days pancreatectomised rats. Scatchard analysis with [³H] mesulergine in cerebral cortex showed a significant decrease (p < 0.05) in maximal binding (B_max) without any change in K_d in 72 h pancreatectomised rats compared with sham. The decreased B_max reversed to sham level by 7 days after pancreatectomy. In brain stem, Scatchard analysis showed a significant decrease (p < 0.01) in B_max with a significant increase (p < 0.01) in K_d. Competition analysis in brain stem showed a shift in affinity towards a low affinity. These parameters were reversed to sham level by 7 days after pancreatectomy. Thus the results suggest that 5-HT through the 5-HT_2C receptor in the brain has a functional regulatory role in the pancreatic regeneration.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Internalization of Human 5-HT<Subscript>4a</Subscript> and 5-HT<Subscript>4b</Subscript> Receptors is Splice Variant Dependent

The family of 5-HT₄ receptors comprises 16 putative splice variants. We have previously shown that there are differences in signal transduction of the h5-HT_4a and h5-HT_4b receptors. In the present study, the internalization of these two splice variants following receptor stimulation was investigated with confocal microscopy on living cells. Chimeric receptors, h5-HT_4a-GFP and h5-HT_4b-GFP were generated by fusing the coding sequence of the 5-HT₄ receptor with the coding sequence of the GFP. The agonist stimulation of fluorescent receptors resulted in a time-dependent internalization of the h5-HT_4b-GFP receptor, but not of the h5-HT_4a-GFP receptor. The h5-HT_4b receptor displays a dual coupling to Gαi,o and Gαs proteins, in contrast to the h5-HT_4a receptor, which couples to Gαs proteins only. We investigated whether the difference in internalization of the two splice variant receptors was related to their differential coupling. Therefore, we performed agonist-stimulation of the receptor following inhibition of the Gαi,o protein coupling using PTX. The h5-HT_4b receptor internalization is PTX insensitive. We co-transfected the fluorescent chimeric receptors with other wild-type variants, which did not produce an alteration of the receptor trafficking. These findings provide the first evidence of differential internalization between the two splice variants, 5-HT_4a and 5-HT_4b receptors.     

Synthesis and biological evaluation of tricarbonyl Re(I) and Tc(I) complexes anchored by poly(azolyl)borates: application on the design of radiopharmaceuticals for the targeting of 5-HT<Subscript>1A</Subscript> receptors

The building blocks fac-[^99mTc{κ³-HB(tim^Me)₃}(CO)₃] and fac-[^99mTc{κ³-R(μ-H)B(tim^Me)₂}(CO)₃] [R is H (4a), Ph (5a); tim^Me is 2-mercapto-1-methylimidazolyl] were obtained almost quantitatively by reacting fac-[^99mTc(CO)₃(H₂O)₃]⁺ with the corresponding scorpionate. These compounds cross the intact blood–brain barrier in mice, with significant retention in the case of 4a and 5a. Using 4a as the lead structure, we have synthesized the functionalized complexes fac-[M{κ³-H(μ-H)B(tim^Bu-pip)₂}(CO)₃] [M is Re (8), ^99mTc (8a); tim^Bu-pip is methyl[4-((2-methoxyphenyl)-1-piperazinyl)butyl](2-mercapto-1-methylimidazol-5-yl)methanamide] and fac-[M{κ ³-H(μ-H)B(tim^Me)(tim^Bu-pip)}(CO)₃] [M is Re (9), ^99mTc (9a)] and evaluated their potential as radioactive probes for the targeting of brain 5-HT_1A serotonergic receptors. The Re complexes exhibit excellent affinity [IC₅₀=0.172 ± 0.003 nM (8); IC₅₀=0.65 ± 0.01 nM (9)] for the 5-HT_1A receptor. The radioactive congeners (^99mTc) have shown an initial brain uptake of 1.38 ± 0.46%ID g⁻¹ (8a) and 0.43 ± 0.12%ID g⁻¹ (9a), but suffer from a relatively fast washout.     

Membrane Organization and Function of the Serotonin<Subscript>1A</Subscript> Receptor

(1) The serotonin_1A receptor is a G-protein coupled receptor involved in several cognitive, behavioral, and developmental functions. It binds the neurotransmitter serotonin and signals across the membrane through its interactions with heterotrimeric G-proteins. (2) Lipid–protein interactions in membranes play an important role in the assembly, stability, and function of membrane proteins. The role of membrane environment in serotonin_1A receptor function is beginning to be addressed by exploring the consequences of lipid manipulations on the ligand binding and G-protein coupling of serotonin_1A receptors, the ability to functionally solubilize the serotonin_1A receptor, and the factors influencing the membrane organization of the serotonin_1A receptor. (3) Recent developments involving the application of detergent-based and detergent-free approaches to understand the membrane organization of the serotonin_1A receptor under conditions of ligand activation and modulation of membrane lipid content, with an emphasis on membrane cholesterol, are described.     

Hyperthermia-Induced Seizures Modify the GABA<Subscript>A</Subscript> and Benzodiazepine Receptor Binding in Immature Rat Brain

Effects of hyperthermia-induced seizures (HS) on GABA_A and benzodiazepine (BDZ) receptor binding in immature rat brain were evaluated using in vitro autoradiography. HS were induced in 10-days-old rats by a regulated stream of moderately heated air directed 50 cm above the animals. Rats were killed 30 min, 24 h or 20 days after HS and their brains were used for in vitro autoradiography experiments to determine GABA_A and BDZ receptor binding. GABA_A binding was significantly enhanced in all brain areas evaluated 30 min after HS, an effect that endures 24 h and 20 days after seizures. Concerning BDZ receptor binding, a significant increase was detected in entorhinal and perirhinal cortices and decreased in basolateral amygdala 30 min following HS. One day after HS, animals demonstrated enhanced BDZ binding in the cingulate, frontal, posterior parietal, entorhinal, temporal and perirhinal cortices; striatum, accumbens, substantia nigra pars compacta and amygdala nuclei. Twenty days after HS enhanced BDZ binding was restricted in the cingulated, frontal, anterior and posterior parietal cortices, as well as in substantia nigra pars reticulata, whereas decreased values were found in accumbens nucleus and substantia nigra pars compacta. Our data indicate differential effects of HS in GABA_A and BDZ binding in immature brain. HS-induced GABA_A and BDZ changes are different from those previously described in experimental models of temporal lobe epilepsy in adult animals.     

Genetic polymorphism c.1438A>G of the 5-HT<Subscript>2A</Subscript> receptor is associated with abdominal obesity in Chinese Northern Han population

Objective We examined the potential impact of the 5-hydroxytryptamine 2A receptor (5-HT_2AR) c.1438A>G promoter polymorphism on obesity and estimates of insulin, glucose as well as lipid metabolism. Methods The genotypes and allelic frequencies of the 5-HT_2AR c.1438A>G were examined with polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 210 patients with overweight/obesity and 216 unrelated healthy subjects. Results The genotype (AA, AG, and GG) distribution of c.1438A>G polymorphism of the 5-HT_2AR gene promoter was 35%, 46%, and 19% in patients, and 32%, 56%, 12% in controls, respectively, no significant difference was found between two groups. Association of genetic diversity of 5-HT_2AR c.1438A>G with the total body fat, fat distribution and clinical characteristics revealed that overweight/obese men carrying G allele were associated with increased body mass index (P = 0.043), waist circumference (P = 0.038), waist-to-hip ratio (P = 0.045), in comparison with patients who carrying A allele, but there were no significant difference between the c.1438A>G genotype groups in overweight/obese women. Conclusion No significant associations were detected. However, the present study suggests the possibility that an abnormal production rate of the 5-HT_2AR c.1438A>G gene product might lead to the development of abdominal obesity in men but not in women. Su Ying and Yan-Ming Sun equally contributed to this study.     

5-HT<Subscript>6/7</Subscript> Receptor Antagonists Facilitate Dopamine Release in the Cochlea via a GABAergic Disinhibitory Mechanism

In humans, serotonin (5-HT) has been implicated in numerous physiological and pathological processes in the peripheral auditory system. Dopamine (DA), another transmitter of the lateral olivocochlear (LOC) efferents making synapses on cochlear nerve dendrites, controls auditory nerve activation and protects the sensory nerve against overactivation. Using in vitro microvolume superfusion techniques we tested 5-HT₆ and 5-HT₇ receptor antagonists whether they can influence dopamine (DA) release from the guinea-pig cochlea in control and in ischemic conditions using currently available and new 5-HT₆ and 5-HT₇ antagonists and mixed antagonists, which were synthesized and characterized for the current study. While the 5-HT₇ antagonist SB-258719 was ineffective, SB-271046, which blocks the 5-HT₆ receptor, caused a significant increase in cochlear DA release what is contradictory with the excitatory nature of this type of receptor. Moreover, the mixed 5-HT_6/7 antagonist EGIS-12233 induced an even more pronounced increase in the resting DA release. To understand why the block of an excitatory receptor results in an increase instead of a decrease in function, we investigated the possible involvement of an indirect neural mechanism through an inhibitory system. In the presence of the GABA_A receptor blocker bicuculline, EGIS-12233 failed to increase the release of DA, suggesting that the serotonin receptor modulation of DA release from the lateral olivocochlear efferents in the cochlea was produced indirectly by decreasing the GABAergic inhibitory tone on dopaminergic nerve endings. The mixed 5-HT₇/D₄ receptor antagonist EGIS-11983 significantly increased both the stimulation-evoked and the resting DA release, while the selective D4 blocker L-741,741 alone had no significant effect. Ischemia, simulated by oxygen and glucose deprivation from the perfusion solution had no action on the effect of the drugs. Drugs that can increase the release of DA from LOC terminals in the cochlea may have a role in the treatment of sensorineural hearing loss.     

Structure-based 3D-QSAR studies on thiazoles as 5-HT<Subscript>3</Subscript> receptor antagonists

Structure-based 3D-QSAR studies were performed on 20 thiazoles against their binding affinities to the 5-HT₃ receptor with comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). The thiazoles were initially docked into the binding pocket of a human 5-HT_3A receptor homology model, constructed on the basis of the crystal structure of the snail acetylcholine binding protein (AChBP), using the GOLD program. The docked conformations were then extracted and used to build the 3D-QSAR models, with cross-validated values 0.785 and 0.744 for CoMFA and CoMSIA, respectively. An additional five molecules were used to validate the models further, giving satisfactory predictive values of 0.582 and 0.804 for CoMFA and CoMSIA, respectively. The results would be helpful for the discovery of new potent and selective 5-HT₃ receptor antagonists.      

Membrane Organization of the Serotonin<Subscript><Emphasis Type="Bold">1A</Emphasis></Subscript> Receptor Monitored by a Detergent-Free Approach

1. Insolubility of membrane constituents in nonionic detergents such as Triton X-100 has been a widely used biochemical criterion to indicate their localization in membrane domains. However, concerns on the possibility of membrane perturbation in the presence of detergents have led to the development of detergent-free approaches. 2. We have explored the organization of the serotonin_1A receptor, an important G-protein coupled receptor, from bovine hippocampus and CHO cells using a detergent-free approach in order to address the points of agreement with our previous results using Triton X-100. 3. A significant fraction of the serotonin_1A receptor has been found to be localized in a heavy density fraction obtained using a detergent-free approach to isolate membrane domains. In addition, we have characterized the membrane fractions isolated in terms of their lipid composition and membrane physical properties. 4. The results obtained on the membrane localization of the serotonin_1A receptor from the present experiments using a detergent-free approach correlate well with our earlier findings obtained using a detergent-based method (Kalipatnapu, S., and Chattopadhyay, A., FEBS Lett. 576:455–460, 2004). These results provide important information on the membrane organization of the hippocampal serotonin_1A receptor and are relevant in view of the concerns on the use of detergent in determination of membrane organization of constituent proteins and lipids.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

Hyperthermia-Induced Seizures Modify the GABA<Subscript><Emphasis Type="Bold">A</Emphasis></Subscript> and Benzodiazepine Receptor Binding in Immature Rat Brain

Summary Effects of hyperthermia-induced seizures (HS) on GABA_A and benzodiazepine (BDZ) receptor binding in immature rat brain were evaluated using in vitro autoradiography. HS were induced in 10-day-old rats by a regulated stream of moderately heated air directed 50 cm above the animals. Rats were killed 30 min, 24 h, or 20 days after HS and their brains were used for in vitro autoradiography experiments to determine GABA_A and BDZ receptor binding. GABA_A binding was significantly enhanced in all brain areas evaluated 30 min after HS, an effect that endures 24 h and 20 days after seizures. Concerning BDZ receptor binding, a significant increase was detected in entorhinal and perirhinal cortices and decreased in basolateral amygdala 30 min following HS. One day after HS, animals demonstrated enhanced BDZ binding in the cingulate, frontal, posterior parietal, entorhinal, temporal, and perirhinal cortices; striatum, accumbens, substantia nigra pars compacta, and amygdala nuclei. Twenty days after HS enhanced BDZ binding was restricted in the cingulated, frontal, anterior and posterior parietal cortices, as well as in substantia nigra pars reticulata, whereas decreased values were found in accumbens nucleus and substantia nigra pars compacta. Our data indicate differential effects of HS in GABA_A and BDZ binding in immature brain. HS-induced GABA_A and BDZ changes are different from those previously described in experimental models of temporal lobe epilepsy in adult animals.     

Investigating the Putative Binding-mode of GABA and Diazepam within GABA<Subscript>A</Subscript> Receptor Using Molecular Modeling

The three-dimensional structure of the GABA A receptor that included the ligand/agonist binding site was constructed and validated by using molecular modeling technology. Moreover, the putative binding-mode of GABA and diazepam with GABAA receptor were investigated by means of docking studies. Based on an rmsd-tolerance of 1.0 angstroms, the docking of GABA to alpha1/beta2 interface resulted in three multi-member conformational clusters and model 2 was supported by homologous sequence alignment data and experimental evidence. On the other hand, the docking of diazepam to alpha1/gamma2 interface revealed five multi-member conformational clusters in the binding site and model 1 seemed to represent the correct orientation of diazepam in the binding site.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.