Cerebellar Receptors Research Articles

Modulation of NMDA receptors in the cerebellum. 1. Properties of the NMDA receptor that modulate its function

NMDA receptors modulate important cerebral processes such as synaptic plasticity, long-term potentiation, learning and memory, etc. NMDA receptors in cerebellum have specific characteristics that make their function and modulation different from those of NMDA receptors in other brain areas. In this and the accompanying review we summarize the information available on the modulation of NMDA receptors in cerebellum. We review the properties of the NMDA receptor that modulate its function: subunit composition, post-translational modifications and synaptic localization. NMDA receptors are heteromeric ligand-gated ion channels assembled from two families of subunits, NR1 and NR2. There are at least eight splicing variant isoforms of the NR1 subunit and four types of NR2 subunits: NR2A, NR2B, NR2C and NR2D. NMDA receptors with different subunit composition or different splice variants of NR1 subunit have different properties. The expression of the different subunits and splicing variants varies during development. Two special characteristics of NMDA receptors in cerebellum that do not occur in other brain areas are the enrichment in the NR2C subunit and in the splice variant NR1b. As a consequence of these and other factors the pharmacology of NMDA receptors is also different in cerebellum than in other brain areas. The function and localization of NMDA receptors is also modulated by postranslational modifications including phosphorylation, glycosylation and nytrosylation. NMDA receptors are phosphorylated in serines of both NR1 and NR2 subunits and in tyrosines of NR2 subunits. Another factor modulating NMDA receptors function is the synaptic localization. The trafficking and clustering of NMDA receptors is modulated by phosphorylation and by interaction with other proteins. The signaling pathways and physiological modulators regulating NMDA receptor function as well as the role of these receptors in motor learning and coordination are reviewed in an accompanying article.

Modulation of NMDA receptors in the cerebellum. II. Signaling pathways and physiological modulators regulating NMDA receptor function

NMDA receptors in cerebellum have specific characteristics that make their function and modulation different from those of NMDA receptors in other brain areas. The properties of the NMDA receptor that modulate its function: Subunit composition, post-translational modifications and synaptic localization are summarized in an accompanying article. In this review we summarize how different signaling molecules modulate the function of NMDA receptors. The function of the receptors is modulated by the co-agonists glycine and serine and this modulation is different in cerebellum than in other areas. The NMDA receptor also has binding sites for polyamines that regulate its function. Other signaling molecules that modulate NMDA receptors function are: cAMP, neurotrophic factors such as BDNF, FGF-2 or neuregulins. These and other molecules allow an interplay between NMDA receptors and other receptors for neurotransmitters that may in this way modulate NMDA receptor function. This has been reported, for example, for metabotropic glutamate receptors. The expression and function of NMDA receptor is also modulated by synaptic activity, allowing an adaptation of the receptors function to the external inputs. NMDA receptors modulate important cerebral processes. NMDA receptors in different brain areas seem to modulate different processes. Cerebellar NMDA receptors play a special role in the modulation of motor learning and coordination. This is also briefly reviewed.

Regional Heterogeneity of 5-HT1A Receptors in Human Cerebellum as Assessed by Positron Emission Tomography

Two measures used in brain imaging are binding potential (BP) and the specific to nonspecific equilibrium partition coefficient (V(3)''). V(3)'' determined using the 5-HT(1A) ligand [(11)C]WAY-100635 is sensitive to changes in the free and nonspecific binding of the ligand in the reference region (V(2)). Healthy female volunteers have higher 5-HT(1A) BP but not V(3)'' compared with men, because V(2) is higher in women. While there could be several explanations for this observation, we hypothesized that women have more 5-HT(1A) receptors in the cerebellum. We explore the cerebellum to define a subregion that more accurately represents the free and nonspecific binding, potentially allowing the use of V(3)''. A quantitative autoradiogram in human brain using [(3)H]WAY-100635 identified a cerebellar subregion devoid of 5-HT(1A) receptors. In vivo 5-HT(1A) receptors were evaluated using [(11)C]WAY-100635 in 12 healthy women and 13 healthy men. Each subject had a metabolite-corrected arterial input function. The autoradiogram demonstrates the lowest concentration of 5-HT(1A) receptors in the cerebellar white matter (CW) and highest concentration in the cerebellar vermis (CV). The CW volume of distribution (V(T)) is lower than CV. Cerebellar white matter is adequately modeled by a one-tissue compartmental model, while a two-tissue model is necessary to model CV or the total cerebellum (CT). Women have a higher CW V(T) compared with men, suggesting a difference in V(2). Use of CW improves identifiability and time stability of BP in cortical regions. Cerebellar white matter might be a better reference region for use in future 5-HT(1A) studies using [(11)C]WAY-100635. With CW as a reference region, V(3)'' cannot be used to detect differences in 5-HT(1A) receptors between men and women, suggesting the need for arterial input functions to determine BP.

Expression of groups I and II metabotropic glutamate receptors in the rat brain during aging

Age-dependent changes in the expression of group I and II metabotropic glutamate (mGlu) receptors were studied by in situ hybridization, Western blot analysis and immunohistochemistry. Male Fisher 344 rats of three ages (3, 12 and 25 months) were tested. Age-related increases in mGlu1 receptor mRNA levels were found in several areas (thalamic nuclei, hippocampal CA3) with parallel increases in mGlu1a receptor protein expression. However, a slight decrease in mGlu1a receptor mRNA expression in individual Purkinje neurons and a decline in cerebellar mGlu1a receptor protein levels were detected in aged animals. In contrast, mGlu1b receptor mRNA levels increased in the cerebellar granule cell layer. Although mGlu5 receptor mRNA expression decreased in many regions, its protein expression remained unchanged during aging. Compared to the small changes in mGlu2 receptor mRNA levels, mGlu3 receptor mRNA levels showed substantial age differences. An increased mGlu2/3 receptor protein expression was found in the frontal cortex, thalamus, hippocampus and corpus callosum in aged animals. These results demonstrate region- and subtype-specific, including splice variant specific changes in the expression of mGlu receptors in the brain with increasing age.

Delta opioid receptor localization in the rat cerebellum

Opioid receptors have been localized to a number of brain regions in rats as well as in other species. In situ hybridization has demonstrated the presence of mRNA for the delta receptor subtype in adult rat cerebellar cortex and in several deep nuclei, but there are no reports on localization of the delta receptor protein in cerebellar regions. In the present study, both quantitative immunohistochemistry and Western blots reveal the presence of delta receptors in the adult rat cerebellum, using a specific affinity-purified antibody. Purkinje cells and processes, as well as cells in the granule cell layer, were positively stained with the antibody. Quantitation of confocal microscopy images illustrated a lower relative level of delta receptor immunoreactivity in cerebellar cortical neurons as compared to neurons in hippocampal regions, striatum and cerebral cortex. Stimulation of delta receptors with a selective agonist, DPDPE, in frozen sections of rat brain, induced a significant increase in binding of [ 35S]-GTPγS in the cerebellar cortex as compared to basal binding levels, thereby demonstrating coupling of the receptor subtype to G-protein. Functional implications for the delta receptor in the cerebellum are discussed, particularly in light of evidence for the presence of a cerebellar opioid receptor for the endogenous opioid methionine enkephalin during early postnatal life.

Metabotropic Glutamate 1α and Adenosine A1 Receptors Assemble into Functionally Interacting Complexes

Recently, evidence has emerged that seven transmembrane G protein-coupled receptors may be present as homo- and heteromers in the plasma membrane. Here we describe a new molecular and functional interaction between two functionally unrelated types of G protein-coupled receptors, namely the metabotropic glutamate type 1alpha (mGlu(1alpha) receptor) and the adenosine A1 receptors in cerebellum, primary cortical neurons, and heterologous transfected cells. Co-immunoprecipitation experiments showed a close and subtype-specific interaction between mGlu(1alpha) and A1 receptors in both rat cerebellar synaptosomes and co-transfected HEK-293 cells. By using transiently transfected HEK-293 cells a synergy between mGlu(1alpha) and A1 receptors in receptor-evoked [Ca(2+)](i) signaling has been shown. In primary cultures of cortical neurons we observed a high degree of co-localization of the two receptors, and excitotoxicity experiments in these cultures also indicate that mGlu(1alpha) and A1 receptors are functionally related. Our results provide a molecular basis for adenosine/glutamate receptors cross-talk and open new perspectives for the development of novel agents to treat neuropsychiatric disorders in which abnormal glutamatergic neurotransmission is involved.

Ligand-binding affinity of the type 1 and 2 inositol 1,4,5-trisphosphate receptors: effect of the membrane environment

The inositol 1,4,5-trisphosphate (InsP 3) receptor is essential for Ca 2+ release from intracellular stores. There are three InsP 3 receptor types which are targets for several types of regulation. Ca 2+, phosphorylation, and protein–protein interactions may contribute to the complex pattern of the Ca 2+ signal in stimulated cells. Furthermore, the 3 receptor types could have different affinities for InsP 3. We compared the affinities of the type 1 receptor from the cerebellum with the liver type 2 receptor both in their membrane environment and after isolation by immunoprecipitation. Measurements of [ 3H]InsP 3 binding in a cytosol-like medium revealed that the K d of the liver receptor (45 ± 5 nM, N = 14) was higher than the K d of the cerebellar receptor (28 ± 3 nM, N = 9). Solubilization and immunopurification of the liver InsP 3 receptor resulted in a 10-fold increase in its affinity for InsP 3. The affinity of the cerebellar receptor did not change under these conditions. Therefore, the extraction of the liver and the cerebellar receptors from their membrane environments induced an inversion of their relative affinities. Treatment of liver membranes with low concentrations of detergents also increased the affinity for InsP 3 binding. These data indicate that the type 1 and the type 2 InsP 3 receptors have different affinities for InsP 3 and that the properties of the type 2 receptor are strongly regulated by hydrophobic interactions within its membrane environment.

Melatonin Influences Human Balance

In order to evaluate a possible correlation between melatonin, the cerebellum and, consequently, human balance, a double-blind pilot study was performed in 5 subjects with random administration of different doses of melatonin. Before and 1 h after a single administration, a complete otoneurological examination was performed. This first pilot study revealed that melatonin had effects on human equilibrium although these effects were not dosage related and were different in individual subjects. On the basis of these results, a second study was performed. Fourteen healthy volunteers were investigated before and 1 h after administration of a single dose of 10 mg melatonin. The otoneurological examination was restricted to the evaluation of: horizontal saccades, horizontal sinusoidal smooth pursuit, eyes open, eyes closed and head retroflexed static posturography. All subjects showed a decrease in posturographic performances, especially in the simplest test (eyes open) and half of them (6 out of 13) showed also impairment of eye movements. These results confirm the role of melatonin in the control of sensorimotor performances, and the cerebellar receptors might be correlated with the control of human balance.

Chronic ethanol administration alters the modulatory effect of 5α-pregnan-3α-ol-20-one on the binding characteristics of various radioligands of GABA A receptors

In this study, we investigated the modulatory effect of 5α-pregnan-3α-ol-20-one, a neurosteroid, on the binding characteristics of [ 3 H ]flunitrazepam (2 nM), [ 3 H ]muscimol (5 nM), and 4 nM [ 35 S ] t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5α-Pregnan-3α-ol-20-one potentiated the binding of [ 3 H ]flunitrazepam and [ 3 H ]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [ 3 H ]flunitrazepam as well as [ 3 H ]muscimol binding ( E max) in the ethanol-dependent rat cerebellum as compared to control group ( p<0.025). Furthermore, 5α-pregnan-3α-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [ 35 S ]TBPS at lower concentrations (⩽100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [ 35 S ]TBPS binding (− E max) by 5α-pregnan-3α-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats ( p<0.025). These observations suggest that the neurosteroid binding site associated with the γ-aminobutyric acid A (GABA A) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.

Multiple mechanisms of regulation of the inositol 1,4,5-trisphosphate receptor by calcium

Ca 2+ mobilisation by inositol 1,4,5-trisphosphate (InsP 3) is a complex phenomenon which involves positive and negative feedback regulation by cytosolic Ca 2+. It has been shown that Ca 2+ increased the affinity of [ 3H]-InsP 3 binding to liver membranes and inhibited [3H]-InsP3 binding to cerebellar membranes. We investigated the effects of Ca 2+ on the [ 3H]-InsP 3 binding to receptor solubilised and rapidly purified by immunoprecipitation. The InsP 3 binding to the purified liver receptor was insensitive to the addition of Ca 2+, indicating that Ca 2+ did not interact directly with the receptor. The loss of the Ca 2+ effect on liver receptor affinity was reproduced by alkaline treatment of liver membranes, which is known to extract the peripheral membrane proteins. This suggests that Ca 2+ regulates the liver InsP 3 receptor by interacting with a membrane-associated protein. Ca 2+ inhibited the binding of [ 3H]-InsP 3 to purified cerebellar receptors as was found with the membrane fraction. The treatment of the purified cerebellar receptor with media of high ionic strength or at alkaline pH did not abolish the effect of Ca 2+ on the receptor. This indicates that the inhibitory effect of Ca 2+ on [ 3H]-InsP 3 binding to cerebellar membranes occurs either via direct interaction with the receptor or via an integral protein strongly associated with the receptor. In conclusion, the mechanisms of regulation of InsP 3-induced Ca 2+ release by Ca 2+ involve different molecular support in cerebellum and in liver. This may reflect different regulation dependent on the receptor type.

Neurosteroids may differentially affect the function of two native GABAA receptor subtypes in the rat brain

Hippocampal noradrenergic and cerebellar glutamatergic axon terminals are known to possess GABA(A) receptors mediating, respectively, enhancement of noradrenaline (NA) and glutamate release. It has been recently found that the hippocampal receptor is benzodiazepine-sensitive, whereas the cerebellar receptor is insensitive to benzodiazepine agonists. We here tested the effects of neurosteroids on these two native GABA(A) receptors using superfused rat hippocampal and cerebellar synaptosomes. Allopregnanolone (3alpha,5alpha-P), at nanomolar concentrations, potentiated the GABA-induced [3H]-NA release from superfused hippocampal synaptosomes; in the absence of GABA, the steroid was ineffective up to 10 microM. The enhancement by GABA of the K+-evoked [3H]-D-aspartate release from cerebellar synaptosomes also was potentiated by nanomolar 3alpha,5alpha-P; in addition, at 1-10 microM, the steroid increased [3H]-D-aspartate release in the absence of GABA. Both in hippocampus and cerebellum the potentiations of the GABA effects produced by nanomolar 3alpha,5alpha-P were abolished by dehydroepiandrosterone sulphate (DHEAS). Added up to 10 microM, DHEAS could not inhibit the effects of GABA alone. The enhancement of [3H]-D-aspartate release elicited by 3 microM 3alpha,5alpha-P in the absence of added GABA was antagonized completely by bicuculline and picrotoxin and halved by DHEAS. To conclude, 3alpha,5alpha-P, at nanomolar concentrations, behaves as a positive allosteric GABA modulator at both the GABA(A) receptors under study. Low micromolar 3alpha,5alpha-P can directly activate the cerebellar receptor, whereas the hippocampal GABA(A) receptor is insensitive to the neurosteroid alone. DHEAS appears to be a pure antagonist at the neurosteroid allosteric sites. Along with the previously observed differential sensitivity to benzodiazepines, the present data strengthen the idea that the two receptors investigated represent native subtypes of the GABA(A) receptor having distinct pharmacology, neuronal localization and function.

Cerebellar gamma-aminobutyric acid type A receptors: pharmacological subtypes revealed by mutant mouse lines.

The vast molecular heterogeneity of brain gamma-aminobutyric acid type A (GABAA) receptors forms the basis for receptor subtyping. Using autoradiographic techniques, we established the characteristics of cerebellar granule cell GABAA receptors by comparing wild-type mice with those with a targeted disruption of the alpha6 subunit gene. Cerebellar granule cells of alpha6(-/-) animals have severe deficits in high affinity [3H]muscimol and [3H]SR 95531 binding to GABA sites, in agonist-insensitive [3H]Ro 15-4513 binding to benzodiazepine sites, and in furosemide-induced increases in tert-[35S]butylbicyclophosphorothionate binding to picrotoxin-sensitive convulsant sites. These observations agree with the known specific properties of these sites on recombinant alpha6beta2/3gamma2 receptors. In the presence of GABA concentrations that fail to activate alpha1 subunit-containing receptors, methyl-6,7-dimethoxy-4-ethyl-beta-carboline (30 microM), allopregnanolone (100 nM), and Zn2+ (10 microM) are less efficacious in altering tert-[35S]butylbicyclophosphorothionate binding in the granule cell layer of the alpha6(-/-) than alpha6(+/+) animals. These data concur with the deficiency of the cerebellar alpha6 and delta subunit-containing receptors in the alpha6(-/-) animals and could also account for the decreased affinity of [3H]muscimol binding to alpha6(-/-) cerebellar membranes. Predicted additional alterations in the cerebellar receptors of the mutant mice may explain a surplus of methyl-6,7-dimethoxy-4-ethyl-beta-carboline-insensitive receptors in the alpha6(-/-) granule cell layer and an increased diazepam-sensitivity in the molecular layer. These changes may be adaptive consequences of altered GABAA receptor subunit expression patterns in response to the loss of two subunits (alpha and delta) from granule cells.

Pharmacological heterogeneity of NMDA receptors: characterization of NR1a/NR2D heteromers expressed in Xenopus oocytes

The pharmacology of recombinant NR1a/NR2D NMDA receptors expressed in Xenopus oocytes was examined and compared to the pharmacology of NR1a/NR2A, NR1a/NR2B and NR1a/NR2C heteromers. The NR1/NR2D heteromer showed a pharmacological profile distinct from each of the other NR1/NR2 heteromers. This unique pharmacological profile was characterized by a relatively lower affinity for the agonist homoquinolinate and the antagonists 2-amino-5-phosphonopentanoate ( d-AP5) and ( R, E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid ( d-CPPene) but not for the antagonists (±)-4-(4-phenylbenzoyl) piperazine-2,3-dicarboxylic acid (PBPD) and α-amino-5-(phosphonomethyl)[1,1′-biphenyl]-3-propanoic acid (EAB515). NR2D-containing receptors displayed a pharmacological profile most similar to that observed for receptors containing the genetically related NR2C subunit. These findings parallel observations obtained for native NMDA receptors in the medial thalamus (presumed to contain NR2D subunits) and forebrain (presumed to contain NR2A and NR2B subunits). Thus, only compounds that discriminate between either NR2A- or NR2B-containing heteromers and NR2D-containing hetermers also discriminate between forebrain and medial thalamic NMDA receptors. While the pharmacology of the NR1a/NR2D receptor shows many parallels to the medial thalamic NMDA receptor, some differences were observed. Certain compounds which discriminate between medial thalamic and cerebellar (presumed to contain NR2C subunits) receptors (e.g., homoquinolinate, d-CPPene) did not show a similar selectivity for NR1a/NR2D receptors relative to NR1/NR2C receptors. Co-expression of NR1a, NR2B and NR2D subunits in Xenopus oocytes resulted in the formation of heteromeric complexes with unique pharmacological properties, suggesting the co-existence of these two distinct NR2 subunits in the same receptor complex.

Furosemide interactions with brain GABAA receptors.

1. The loop diuretic furosemide is known to antagonize the function of gamma-aminobutyric acid type A (GABAA) receptors. The purpose of the present study was to examine the direct interaction of furosemide with the GABAA receptors by autoradiography and ligand binding studies with native rat and human receptors and with recombinant receptors composed of rat subunits. 2. Autoradiography with [35S]-t-butylbicyclophosphorothionate ([35S]-TBPS) as a ligand indicated that furosemide (0.1-1 mM) reversed the 5 microM GABA-induced inhibition of binding only in the cerebellar granule cell layer of rat brain sections. In all other regions studied, notably also in the hippocampal and thalamic areas, furosemide failed to antagonize GABA. Furosemide 1 mM decreased [35S]-TBPS binding only in a limited number of brain regions, but facilitation of the GABA-inhibition of the binding was much more widespread. 3. In well-washed rat cerebellar, but not cerebrocortical, membranes, furosemide enhanced the [35S]-TBPS binding over basal level in the absence of added GABA. The GABAA antagonist, SR 95531, and the convulsant, Ro 5-4864, blocked this furosemide-induced increase. Both interactions with the furosemide enhancement are likely to be allosteric, since furosemide affected the binding of [3H]-SR 95531 and [3H]-Ro 5-4864 identically in the cerebellar and cerebrocortical membranes. Maximal GABA-antagonism induced by furosemide in cerebellar membranes was further increased by SR 95531 but not by Ro 5-4864, indicating additive antagonism only for SR 95531. In human cerebellar receptors, only GABA antagonism by furosemide, but not the enhancement without added GABA, was observed. 4. In recombinant GABAA receptors, furosemide antagonism of GABA-inhibition of [35S]-TBPS binding depended only on the presence of alpha 6 and beta 2/3 subunits, irrespective of the presence or absence of gamma 2 or delta subunits. 5. In alpha 6 beta 3 gamma 2 receptors, clozapine reversed the enhancement of [35S]-TBPS binding by furosemide in the absence of GABA. However, it failed to affect the GABA-antagonism of furosemide, suggesting that the enhancement of basal binding and the GABA antagonism might represent two different allosteric actions of furosemide. 6. In conclusion, the present results indicate that furosemide is a subtype-selective GABAA antagonist with a mode of action not shared by several other antagonists, which makes furosemide a unique compound for development of potential GABAA receptor subtype-specific and -selective ligands.

2242 Functional expression of NMDA receptors in cerebellum and their contribution to eyeblink classical conditioning

2242 Functional expression of NMDA receptors in cerebellum and their contribution to eyeblink classical conditioning

The Elusive Nature of Cerebellar Somatostatin Receptors: Studies in Rat, Monkey and Human Cerebellum

The pharmacological profile and localization of somatostatin (SRIF) receptors were determined in rat, monkey and human cerebellum. In rat cerebellar cortex, low ss1/sst4, intermediate sst2 and very high sst3 receptor mRNA levels were found, sst1 mRNA was also expressed in the deep cerebellar nuclei. [125I]Tyr3-octreotide binding sites in cerebellar membranes correlated with recombinant sst2, but not with sst5 or sst3 receptors and were found in the molecular layer of the cerebellum. [125I]CGP 23996 (in Na+-buffer) binding in rat cerebellum correlated with sst1 or sst4, but not with sst2, sst3 or sst5 receptor binding. Similar data were obtained in rhesus monkey cerebellum. mRNAs for all five receptors were found in the granule cell layer of the human cerebellum and/or in the dentate nucleus. [125I]Tyr3-octreotide binding was strong in the molecular layer and correlated with that of recombinant sst2 receptors, but not with sst3 or sst5 receptors. [125I]CGP 23996 (in Mg++-buffer) binding was heterogeneous (about 75%. to sst2 and 25% to sst1 and/or sst4 receptors). The molecular and granular layers were equally and the dentate nucleus strongly labeled. Thus. SRIF receptors of the sst2, sst1 and/or sst4 subtype are present in the rat, monkey and human cerebellum. In the latter two species, the sst2 type appears to be predominant. Surprisingly, the high expression of sst3 receptor mRNA is not supported by radioligand binding data in any of the species studied. The reason for this discrepancy remains to be elucidated.

6-Deoxy-6-hydroxymethyl scyllo-inositol 1,2,4-trisphosphate: A potent agonist at the inositol 1,4,5-trisphosphate receptor

The synthesis of racemic 6-deoxy-6-hydroxymethyl scyllo-inositol 1,2,4-trisphosphate is described. This compound is a highly potent agonist at the platelet d- myo-inositol 1,4,5-trisphosphate receptor, and it binds to the rat cerebellar receptor with an affinity equal to that of the natural ligand. These results suggest that the 5″-hydroxymethyl group of adenophostin A may contribute to its unusual potency.

Characterization of [125I]-endothelin-1 and [125I]-BQ3020 binding to rat cerebellar endothelin receptors.

1. We performed radioligand binding experiments on rat cerebellar homogenates using [125I]-endothelin-1 ¿[1251]-ET-1¿ and [125I]-BQ3020 to examine the pharmacology of endothelin receptors in rat brain. Saturation experiments demonstrated a single population of binding sites with high affinity for both radioligands ([125I]-ET-1, pKd = 8.94 +/- 0.17; [125I]-BQ3020, pKd = 9.18 +/- 0.14 nM; mean +/- s.e.mean). However, [125I]-BQ3020 only recognised approximately one third the number of endothelin receptors measured with [125I]-ET-1. 2. Saturation binding experiments with [125I]-PD151242 revealed high affinity binding to a single population of ETA receptors in the cerebellar homogenates (pKd = 9.95 +/- 0.14; Bmax = 30 +/- 15 fmol mg-1 protein). 3. Competition experiments were performed with ligands that are either non-selective for endothelin receptor subtypes. The rat cerebellar endothelin receptor displayed a high affinity for endothelin-1 (ET-1), endothelin-3 (ET-3) and sarafotoxin-S6c (STX-6c) although the affinity for ET-3 was slightly higher than the affinity for ET-1 using both radioligands. The selective ETA antagonists, BQ123, BMS-182,874 and JKC-301 all displayed low affinities at the endothelin receptors. In contrast the selective ETB agonists, IRL1620 and [Ala1,3,11,15]ET-1 and the selective ETB antagonist, BQ-788 had moderate affinities at the endothelin receptor, in the low nanomolar range. The ETB agonist, BQ3020, had approximately 10 fold higher affinity than IRL1620 and [Ala1,3,11,15]ET-1 at the rat cerebellar endothelin receptors. The non-selective antagonists, Ro-46,2005, Ro-47,0203 and PD-142,893 displayed moderate affinities at the cerebellar receptor. 4. Since [125I]-BQ3020 recognises only a fraction of the [125I]-ET-1 binding sites, the majority of the endothelin receptors in the cerebellum cannot be classed as ETB. Although [125I]-PD151242 was able to detect ETA receptors in the rat cerebellar homogenates, the small population of ETA receptors (2% of the total endothelin population as measured with [125I]-ET-1) could not account for the non-ETB receptor population. We conclude that the rat brain cerebellar receptor has a profile similar to the ETB1 receptor as it has a high affinity for ET-1, ET-3, STX-6c and was moderately sensitive to PD-142,893. However, as the ETB ligands BQ-788, IRL1620 and [Ala1,3,11,15]ET-1 have only a moderate affinity for the rat cerebellar endothelin receptor and since ET-3 has a higher affinity as compared to ET-1, our findings suggest that the rat cerebellum contains predominately ETc receptors.

Intracellular calcium stores and inositol 1,4,5-trisphosphate receptor in rat liver cells

The D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor was localized by immunofluorescence experiments in situ in liver cryosections. Two anti-Ins(1,4,5)P3 receptor antibodies (against the 14 C-terminal residues of the type 1 receptor or against the entire cerebellar receptor) weakly decorated the whole cytoplasm, and a more intense labelling was observed at the periphery of the hepatocytes, particularly beneath the canalicular and the sinusoidal domains of the plasma membrane (PM). Antibodies against calreticulin, the Ca2+ pump (SERCA2b) or endoplasmic reticulum (ER) membranes homogeneously labelled the cytoplasm and the subplasmalemmal area. These data indicate that the ER can be divided into at least two specialized subregions: one is located throughout most of the cytoplasm and contains markers of the rough ER (RER), calreticulin, SERCA2b and a low density of Ins(1,4,5)P3 receptor, and the other is confined to the periphery of the cells and contains calreticulin, Ca2+ pump, RER markers and a high density of Ins(1,4,5)P3 receptor. A membrane fraction enriched in Ins(1,4,5)P3 receptor and in markers of the PM was immuno-adsorbed with the antibody against the C-terminal end of the Ins(1,4,5)P3 receptor and pelleted with Sepharose protein A. The immuno-isolated material was enriched in Ins(1,4,5)P3 receptor, but none of the markers of the ER or of the PM could be detected. This suggests that the Ins(1,4,5)P3 receptor is localized on discrete domains of the ER membrane beneath the canalicular and the sinusoidal membranes, where it was found at higher densities than the other markers.

Inositol 1,4,5-Trisphosphate Slowly Converts Its Receptor to a State of Higher Affinity in Sheep Cerebellum Membranes

Incubation of cerebellar microsomes with d-myo-inositol 1,4,5-trisphosphate (InsP3) (0.01 1 microM), at 4 or 20 degrees C in a cytosolic-like medium devoid of Ca2+ and Mg2+, followed by InsP3 removal, induced an increase in InsP3 binding determined with 1 nm [3H]InsP3. At 20 degrees C, and pH 7.1, maximal stimulation (1.5 2. 5-fold) was obtained with 1 mum InsP3, and the EC50 was 60 +/- 5 nm. Several lines of evidence suggested that the activating site is identical with the InsP3 binding site: (i) activation and binding exhibited the same inositol phosphate specificity; (ii) addition of decavanadate, a competitive inhibitor of [3H]InsP3 binding, to the preincubation mixture, prevented the activating effect of InsP3; (iii) the concentration of InsP3 giving half-maximal activation was close to that giving half-maximal InsP3 binding. The time course of activation was found to be much slower than that of binding. While a t1/2 less than 0.4 s has been measured recently at neutral pH and 20 degrees C for binding of 0.5 nm [3H]InsP3 (Hannaert-Merah, Z., Coquil, J.-F., Combettes, L., Claret, M., Mauger, J.-P., and Champeil, P. (1994) J. Biol. Chem. 269, 29642-29649), a 20-s preincubation with 1 microM InsP3 was required to half-maximally stimulate binding. Under the present conditions, the InsP3-induced binding increase was only partially reversible. However, this effect was not blocked by antiproteases suggesting that it did not involve proteolysis. Taking advantage of the marked difference in the kinetics of InsP3 binding and InsP3-dependent activation, we performed binding experiments on a short period (3 s) to determine the effect of InsP3 pretreatment on the binding parameters. The data showed that this treatment increased the affinity of the receptor without changing the number of binding sites (control: KD = 107 nm, Bmax = 28 pmol/mg of protein; after preincubation with 1 microM InsP3: KD = 53 nm, Bmax = 32 pmol/mg of protein). The two states of the receptor bound InsP3 with a Hill coefficient close to 1 on a 3-s scale. In agreement with the effect of InsP3 pretreatment, equilibrium binding experiments performed on 10-min incubations revealed an apparent positive cooperative behavior (apparent Hill coefficient = 1.6; apparent KD = 66 nm). These results report a new regulatory process of the InsP3 receptor in cerebellum occurring independently of Ca2+ and on a relatively long time scale.