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Biomedical subjects

E R Korpi

Publications and source records attributed to E R Korpi.

At least 37 records · Page 2Linked to original sources
PubMed2001

Do antiepileptics phenytoin, carbamazepine, and loreclezole show GABA(A) receptor subtype selectivity in rat brain sections?

[35S]t-Butylbicyclophosphorothionate ([35S]TBPS), a convulsant site ligand of GABA(A) receptors, was used in autoradiography with rat brain sections to test suggested receptor subtype-selective actions of antiepileptics phenytoin, carbamazepine and loreclezole on native GABA(A) receptors. At maximal 100 microM concentration, both phenytoin and carbamazepine decreased [35S]TBPS binding only by 20%, indicating that their low potency and efficacy prevents their use as alpha1 subunit-identifying compounds. Ten microM loreclezole did not affect the binding, but a further increase in loreclezole concentration strongly decreased it. The action of loreclezole, assumed to reflect beta2/3 subunit-containing receptors, varied from brain region to region, but the effects were unrelated to the regional expression profiles of beta subunit variants. We conclude that in autoradiographic [35S]TBPS binding assay neither carbamazepine, phenytoin nor loreclezole are useful tools in characterizing brain regional heterogeneity of GABA(A) receptors in rats and that only loreclezole exhibits high, pharmacologically relevant efficacy.

Animals↗
PubMed2001

Uncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptors alter the mRNA expression of proteins associated with the NMDA receptor complex.

N-methyl-D-aspartate (NMDA) receptor function appears to be under complex control during physiological and pharmacological states. We have investigated the effects of acute administration of uncompetitive NMDA receptor antagonists on mRNA levels of NMDA receptor subunits and on molecules known to cluster or phosphorylate the receptor utilizing in situ hybridization on rat brain sections. A high dose (5 mg/kg; 4 hr) of dizocilpine (MK-801) decreased mRNA levels of NMDA receptor subunits NR2C and NR2B in the entorhinal and parietal cortices, respectively. MK-801 increased mRNA levels of synapse-associated protein-90/postsynaptic density-95 (SAP90/PSD-95) and a gamma-isoform of protein kinase C (PKCgamma) in cortical regions. Synapse-associated protein-97 (SAP97) mRNA levels were increased in the entorhinal cortex layer III after MK-801 or after relatively high doses of other uncompetitive NMDA receptor antagonists: phencyclidine (15 mg/kg; 6 hr) and memantine (50 mg/kg; 6 hr). Memantine also increased SAP97 mRNA expression in other cortical regions, but this effect was not observed with MK-801 or phencyclidine. NMDA receptor uncompetitive antagonists alter the expression of multiple receptor components and such events may ultimately play a role in adaptation or toxic responses.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Assembly of functional alpha6beta3gamma2delta GABA(A) receptors in vitro.

Transgenic mice deficient in the alpha6 subunit of the GABA(A) receptor show reduced levels of the delta subunit protein and an altered GABA(A) receptor pharmacology, suggesting selective assembly mechanisms. Delta reduced the binding of [3H]Ro15-4513 or t-butylbicyclophosphoro[35S]thionate and, to a lesser extent, [3H]muscimol to recombinant alpha1beta1gamma2(delta), alpha4beta1gamma2(delta) and alpha6beta1gamma2(delta) receptors, paralleled by diminished GABA-evoked maximal currents in electrophysiological recordings for the latter one. The delta subunit gave rise to a lower EC50 for GABA and a slowed desensitization indicating its assembly in alpha6beta2delta, alpha6beta1gamma2delta and alpha6beta2gamma2delta receptors. The data show that the delta subunits assemble in various functional GABA(A) receptor subtypes in vitro to reduce GABA-evoked maximal currents and ligand binding, but increase the potency for GABA.

Affinity Labels↗
PubMed2000

Coupling between agonist and chloride ionophore sites of the GABA(A) receptor: agonist/antagonist efficacy of 4-PIOL.

Eight gamma-aminobutyric acid (GABA) mimetics were tested on their ability to differentiate native GABA(A) receptor subtypes present in various rat brain regions. In rat brain cryostat sections, little regional variations by the agonistic actions of muscimol, thiomuscimol, 4,5,6,7-tetrahydroisoazolo(5,4-c)pyridin-3-ol, piperidine-4-sulphonic acid, taurine and beta-alanine on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABA(A) receptor channels were found. They were very similar to those found for GABA itself and indicated no direct correlation with single subunit distributions for any of these compounds. Only the low-efficacy GABA mimetic 5-(4-piperidyl)isoxazol-3-ol (4-PIOL) acted like a weak partial agonist or antagonist depending on the brain area. As the cerebellar granule cell layer was relatively insensitive to both modes of action, we tested 4-PIOL in recombinant alpha1beta2gamma2 (widespread major subtype) and alpha6beta2gamma2 (cerebellar granule cell restricted) receptors where it had different effects on GABA-modulated [35S]TBPS binding and on electrophysiological responses. 4-PIOL may thus serve as a potential lead for receptor subtype selective compounds.

Animals↗
PubMed2000

Stereospecific modulation of GABA(A) receptor function by urocanic acid isomers.

A deamination product of histidine, urocanic acid, accumulates in the skin of mammals as trans-urocanic acid. Ultraviolet (UV) irradition converts it to the cis-isomer that is an important mediator in UV-induced immunosuppression. We have recently shown that urocanic acid interferes with the agonist binding to GABA(A) receptors. We now report that the effects of urocanic acid on binding of a convulsant ligand (t-butylbicyclo[35S]phosphorothionate) to GABA(A) receptors in brain membrane homogenates are dependent on pH of the incubation medium, the agonistic actions being enhanced at the normal pH of the skin (5.5). Using Xenopus laevis oocytes expressing recombinant rat alpha1beta1gamma2S GABA(A) receptors, the low pH potentiated the direct agonistic action of trans-urocanic acid under two-electrode voltage-clamp, whereas cis-urocanic acid retained its low efficacy both at pH 5.5 and 7.4. The results thus indicate clear differences between urocanic acid isomers in functional activity at one putative receptor site of immunosuppression, the GABA(A) receptor, the presence of which in the skin remains to be demonstrated.

Animals↗
PubMed2000

GABA(A) receptor epsilon and theta subunits display unusual structural variation between species and are enriched in the rat locus ceruleus.

Previously, GABA(A) receptor epsilon and theta subunits have been identified only in human. Here, we describe properties of the epsilon and theta subunit genes from mouse and rat that reveal an unusually high level of divergence from their human homologs. In addition to a low level of amino acid sequence conservation ( approximately 70%), the rodent epsilon subunit cDNAs encode a unique Pro/Glx motif of approximately 400 residues within the N-terminal extracellular domain of the subunits. Transcripts of the rat epsilon subunit were detected in brain and heart, whereas the mouse theta subunit mRNA was detectable in brain, lung, and spleen by Northern blot analysis. In situ hybridization revealed a particularly strong signal for both subunit mRNAs in rat locus ceruleus in which expression was detectable from the first postnatal day. Lower levels of coexpression were also detected in other brainstem nuclei and in the hypothalamus. However, the expression pattern of theta subunit mRNA was more widespread than that of epsilon subunit, being found also in the cerebral cortex of rat pups. In contrast to primate brain, neither subunit was expressed in the hippocampus or substantia nigra. The results indicate that GABA(A) receptor epsilon and theta subunits are evolving at a much faster rate than other known GABA(A) receptor subunits and that their expression patterns and functional properties may differ significantly between species.

Animals↗
PubMed2000

Quantification of GABA(A) receptor subunit mRNAs by non-radioisotopic competitive RT-PCR utilizing plate-based EIA methodology.

We developed a non-radioisotopic quantitative competitive RT-PCR method for the measurement of gamma-aminobutyric acid (GABA) type A receptor subunit mRNA levels. The specificity of the method was optimized by the use of four subunit-specific oligonucleotides in the sequential steps: reverse transcription, polymerase chain reaction (PCR), and detection. The biotinylated PCR products were bound on streptavidin-coated microtiter plates allowing detection of the products using dinitrophenyl (DNP)-labeled probes and anti-DNP alkaline phosphatase conjugate. The method was set up for the six major cerebellar GABA(A) receptor subunits: alpha1; alpha6; beta2; beta3; gamma2 and delta. The method is quantitative and rapid. With a large dynamic range from 10 fg to 1 ng of subunit mRNA, the accuracy was 12 and 19% (intra- and interassay coefficients of variation, respectively), which might be improved by using a smaller range of standards. The use of a double logarithmic standard curve [log (standard to competitor signal) vs. log (standard mRNA originally present)] requires only one reaction from each sample, allowing the analysis of a large number of samples in one experiment.

Animals↗
PubMed2000

Long-range interactions in neuronal gene expression: evidence from gene targeting in the GABA(A) receptor beta2-alpha6-alpha1-gamma2 subunit gene cluster.

Clustering of GABA(A) receptor alpha1, alpha6, beta2, and gamma2 subunit genes on mouse chromosome 11/human chromosome 5 may have functional significance for coordinating expression patterns, but until now there has been no evidence for cross-talk between the genes. However, altering the structure of the alpha6 gene, specifically expressed in the cerebellum, with neomycin gene insertions in two different experiments unexpectedly reduced the expression of the widespread alpha1 and beta2 genes in the forebrain. There were corresponding reductions in the levels of alpha1 and beta2 subunit proteins and in autoradiographic ligand binding densities to GABA(A) receptors in the forebrain of alpha6-/- mice. The gamma2 mRNA level was not changed, nor were beta3 and delta mRNAs. The data suggest that elements in the neo gene may have an influence over long distances in the GABA(A) subunit gene complex on as yet undefined structures coordinating the expression of the alpha1 and beta2 genes.

Animals↗
PubMed2000

Enhanced locomotor stimulation by NMDA receptor antagonists in alcohol-sensitive ANT rats.

The ability of the antagonists for the N-methyl-D-aspartate (NMDA) type of glutamate receptor to modulate locomotor activity were compared in alcohol-sensitive (or alcohol-nontolerant, ANT) and alcohol-insensitive (or alcohol-tolerant, AT) rat lines. Both rat lines showed altered locomotor activity after acute injections of a competitive antagonist (LY235959), a glycine-site antagonist (L-701,324), or noncompetitive antagonists [MK-801, phencyclidine (PCP), and ketamine] of the NMDA receptor. MK-801 at 0.5 mg/kg caused a strong increase in horizontal activity in both rat lines, the effect being significantly greater in the ANT rats. There was a subpopulation among AT rats that was almost completely unresponsive to MK-801. This insensitivity to MK-801 correlated with the lack of c-fos induction in the retrosplenial and cingulate cortices. Fos immunoreactive cells in these brain regions after MK-801 treatment were more numerous in ANT than AT rats, although c-fos induction in the inferior olivary nucleus was similar in all animals after MK-801. The ANT rats showed greater locomotor stimulation also after ketamine and LY235959, while stimulation induced by PCP and depression induced by L-701,324 did not differ between the rat lines. The data suggest that altered NMDA receptor-mediated processes may correlate with differences in innate alcohol sensitivity in the ANT/AT rat model.

Alcohol Drinking↗
PubMed2000

Neonatal 6-hydroxydopamine treatment affects GABA(A) receptor subunit expression during postnatal development of the rat cerebellum.

Neurotoxic elimination of noradrenergic terminals by 6-hydroxydopamine (6-OHDA) leads to alteration of the granule cell layer formation. We have studied the developmental expression of GABA(A) receptor subunits in rat cerebellum after neonatal administration of 6-OHDA during the first postnatal month of life. 6-OHDA was injected subcutaneously. The expression of GABA(A) receptor subunits was studied by in situ hybridization and immunohistochemistry. The alterations were observed in the neocerebellum - the part of the cerebellum which starts development postnatally. The migration of granule cells was delayed, and the total area of the granule cell layer in the neocerebellum from 6-OHDA-treated rats was reduced to 22.6+/-5% of the corresponding area from control rats. In situ hybridization with subunit-specific antisense oligonucleotide probes was performed for alpha1, alpha2, alpha3, alpha5, alpha6, beta1, beta2, gamma1 and gamma2 subunits of the GABA(A) receptor. In neocerebellum, 6-OHDA treatment caused a significant reduction in the alpha1, alpha6 and gamma2 subunit mRNA levels. The expression of the other subunits was not changed. It has been shown that in the postnatal cerebellum alpha1 and alpha6 subunits can be detected in granule cells only when the cells had migrated to their final destination. Our findings indicate that a noradrenergic influence may be necessary for the normal maturation and migration of cerebellar granule cells.

Adrenergic Agents↗
PubMed2000

Halothane and desflurane requirements in alcohol-tolerant and -nontolerant rats.

On the basis of data implicating GABAA receptors in the effects of volatile general anaesthetics, we hypothesized that alcohol-, barbiturate-, and benzodiazepine-sensitive alcohol-nontolerant (ANT) rats would also be more sensitive than alcohol-tolerant (AT) rats to two clinical general anaesthetics with differing potencies, halothane and desflurane. The obtunding effect of halothane and desflurane on mature ANT (n = 17) and AT (n = 16) rats was assessed by the loss-of-righting reflex endpoint. ANT rats were significantly (P < 0.0001) more sensitive to the obtunding effects of both halothane and desflurane (ED50 = 0.45 +/- 0.03% atm for ANT vs 0.95 +/- 0.04% atm for AT and 2.16 +/- 0.17 vs 3.69 +/- 0.13% atm, respectively). The immobilization effect of halothane and desflurane was assessed with the tail clamp/withdrawal endpoint. ANT rats were more sensitive to the effects of halothane (ED50 = 1.10 +/- 0.08% atm for ANT vs 1.72 +/- 0.09% atm for AT; P < 0.0001) but not desflurane (ED50 = 6.25 +/- 0.25% atm for ANT vs 5.85 +/- 0.21% atm for AT). The data presented support the hypothesis that volatile anaesthetics interact with specific neuronal proteins (possibly GABAA receptors) and agree with recent hypotheses that different elements of the anaesthetic state are produced by separate sites or mechanisms.

Anesthetics, Inhalation↗
PubMed2000

Neonatal 6-hydroxydopamine treatment affects GABA(A) receptor subunit expression in the frontal cortex but not the hippocampus of rats during postnatal development.

The influence of neonatal administration of 6-hydroxydopamine (6-OHDA) on the maturation of GABA(A) receptors in the frontal cortex and hippocampus was studied using 5- to 40-day-old rats. In situ hybridization with antisense oligonucleotide probes was performed for alpha(1), alpha(2), alpha(5), beta(2), beta(3) and gamma(2) subunit mRNAs of the GABA(A) receptor. We demonstrated that neonatal treatment with 6-OHDA temporarily delays the postnatal transcription of the alpha(1) and gamma(2) subunits in the rat prefrontal cortex, as assessed by in situ hybridization histochemistry. The effect was selective for these subunits (the alpha(2), alpha(5), beta(2), and beta(3) subunit mRNAs remained unchanged) and for this region (the mRNA levels in the hippocampus were not changed). The reduction in mRNA levels at early postnatal stages (postnatal day 5, PD5, and PD10) also affected the subunit protein levels, as shown by immunohistochemistry for the alpha(1) subunit, and the formation of GABA(A) receptor-associated picrotoxinin-insensitive TBPS binding sites, as shown by autoradiography. Our findings indicate that without a noradrenergic influence, the maturation of GABAergic interneurons in the frontal cortex is transiently delayed (from PD5 to PD40). However, it is possible that this transient reduction of the expression of certain GABA subunits - caused by depletion of noradrenergic innervation - cannot cause a lasting alteration to the GABAergic function in the prefrontal cortex.

Animals↗
PubMed1999

Attenuated sensitivity to neuroactive steroids in gamma-aminobutyrate type A receptor delta subunit knockout mice.

gamma-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the delta subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the delta subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids.

Animals↗
PubMed1999

Cerebellar GABA(A) receptors and anxiolytic action of diazepam.

Alcohol-sensitive ANT rats have a point mutation in the cerebellum-enriched GABA(A) receptor alpha6 subunit, which makes this subunit and the ANT rats in vivo highly sensitive to benzodiazepine agonists. In the elevated plus maze test of anxiety, diazepam produced a greater anxiolytic response in the ANT rats than in the control, alcohol-insensitive AT rats. The ANT rats were less sensitive to the sedative effect of diazepam in the staircase test of exploration. The results thus suggest that the mutant cerebellar granule cell layer receptors can participate in GABA(A) receptor-activation-induced anxiolysis.

Analysis of Variance↗
PubMed1999

Loreclezole and La3+ differentiate cerebellar granule cell GABA(A) receptor subtypes.

The effects of loreclezole and La3+ on native cerebellar GABA(A) receptors were compared between GABA(A) receptor alpha6 subunit-deficient (alpha6-/-) and wildtype mouse lines, produced through homologous recombination, using t-[35S]butylbicyclophosphorothionate ([35S]TBPS) autoradiography in brain sections. In the alpha6 subunit-deficient mice, the GABA receptor antagonistic ability of La3+ was abolished in the cerebellar granule cell layer, consistent with its opposite actions on alpha6- and of alpha1 subunit-containing receptors. La3+ significantly potentiated the action of GABA in the molecular layer of the alpha6-/- mice, but not in that of the wildtype mice. The potentiation of agonistic GABA inhibition of [35S]TBPS binding by loreclezole in alpha6-/- granule cells was reduced, suggesting an emergence of low-affinity GABA(A) receptors. The present results thus identified two ligands that may be useful in studying functional roles of cerebellar alpha1 and alpha6 subunit-containing GABA(A) receptor subtypes.

Animals↗
PubMed1999

Effects of continuous opioid receptor blockade on alcohol intake and up-regulation of opioid receptor subtype signalling in a genetic model of high alcohol drinking.

Effects of a continuous naloxone infusion via osmotic pumps on alcohol drinking and opioid receptor density and function in the high-drinking AA (Alko, Alcohol) rats were examined. AA rats were trained to drink 10% (v/v) ethanol in a 1-h limited access procedure and implanted with subcutaneous osmotic pumps delivering either saline, a low dose (0.3 mg/kg per hour), or a high dose (3.0 mg/kg per hour) of naloxone for 7 days. The pumps were then removed and alcohol, food and water intakes were measured for another 4 days. Compared with saline, both naloxone doses significantly suppressed 1-h alcohol intake during the 7-day infusion. The suppression was smaller than that by a bolus injection of the same daily dose 15 min before the session, although a complete blockade of morphine-induced antinociception was achieved even with the smaller naloxone infusion. Significant decreases were also seen in daily food and water intake during the first days, but they quickly returned to their previous baselines. After pump removal, rats of both naloxone-treated groups rapidly increased their alcohol drinking and reached the pretreatment baseline, while their food and water intakes significantly surpassed their baselines. Naloxone infusion at 3.0 mg/kg per hour for 7 days significantly decreased 24-h alcohol drinking without affecting alcohol preference. Twenty-four hours after pump removal, autoradiography with [3H]DAMGO, [3H]DPDPE and [3H]U-69,543 revealed an up-regulation of mu-, delta- and kappa-opioid receptor binding sites in many brain areas of these animals. This receptor up-regulation was functional, because receptor coupling to G-protein activation was enhanced by agonist ligands, as revealed by [35S]GTPgammaS autoradiography. A good correlation existed between ligand binding densities and G-protein activation for mu- and kappa-receptors in control and naloxone-treated brain sections. Furthermore, morphine-induced analgesia in a hot-plate test showed a leftward shift in the morphine dose-response curve after naloxone treatment. These results suggest that the usefulness of a chronic opioid antagonist dosing regime could be limited by nonspecific effects of the antagonist on ingestive behaviour, an up-regulation of opioid receptors with high antagonist doses, and the resulting supersensitivity to opioid agonists after the discontinuation of the treatment.

Alcohol Drinking↗