Changes in Inositol 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate Mass Accumulations in Cultured Adrenal Chromaffin Cells in Response to Bradykinin and Histamine

In previous studies it has been shown that both bradykinin and histamine increase the formation of 3H-labeled inositol phosphates in adrenal chromaffin cells prelabelled with [3H]inositol and that both these agonists stimulate release of catecholamines by a mechanism dependent on extracellular calcium. Here, we have used mass assays of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] to investigate changes in levels of these two candidates as second messengers in response to stimulation with bradykinin and histamine. Bradykinin increased the mass of Ins(1,3,4,5)P4 despite the failure in earlier studies with [3H]inositol-labelled cells to observe a bradykinin-mediated increase in content of [3H]InsP4. Bradykinin elicited a very rapid increase in level of Ins(1,4,5)P3, which was maximal at 5-10 s and then rapidly decreased to a small but sustained elevation at 2 min. The bradykinin-elicited Ins(1,3,4,5)P4 response increased to a maximum at 30-60 s and at 2 min was still elevated severalfold above basal levels. Histamine, which produced a larger overall total inositol phosphate response in [3H]inositol-loaded cells, produced significantly smaller Ins(1,4,5)P3 and Ins(1,3,4,5)P4 responses compared with bradykinin. The bradykinin stimulation of Ins(1,4,5)P3 accumulation was partially dependent on a high (1.8 mM) extracellular Ca2+ concentration, whereas the Ins(1,3,4,5)P4 response was almost completely lost when the extracellular Ca2+ concentration was reduced to 100 nM. Changes in the inositol polyphosphate second messengers are compared with the time course of bradykinin-stimulated increases in free intracellular Ca2+ concentrations and noradrenaline release.     

Neurotransmitter and Depolarization-Stimulated Accumulation of Inositol 1,3,4,5-Tetrakisphosphate Mass in Rat Cerebral Cortex Slices

[32P]Inositol 1,3,4,5-tetrakisphosphate ([32P]Ins(1,3,4,5)P4) binds to a rat cerebellar membrane site with high affinity (KD = 2.8 +/- 0.6 nM) and low capacity (Bmax = 176 +/- 34 fmol/mg of protein). Evidence for a low-affinity site (KD = 164 +/- 48 nM) was also apparent. The high-affinity site displayed marked specificity for the Ins(1,3,4,5)P4 isomer, compared with several other inositol polyphosphates, and has been used as the basis of a radioreceptor assay for Ins(1,3,4,5)P4 in extracts of rat cerebral cortex slices. The resting Ins(1,3,4,5)P4 concentration (1.89 +/- 0.11 pmol/mg of protein) in the slices was rapidly and dramatically increased by carbachol and quisqualate. K+ depolarization of cerebral cortex slices also stimulated Ins(1,3,4,5)P4 accumulation, with at least 50% of the response being sensitive to atropine, a result indicating that muscarinic receptor stimulation by released acetylcholine contributes significantly to the K+ effect.     

Muscarinic Cholinoceptor-Stimulated Synthesis and Degradation of Inositol 1,4,5-Trisphosphate in the Rat Cerebellar Granule Cell

Abstract: A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [³H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP₅) and inositol hexakisphosphate (InsP₆), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 m M ) caused an immediate increase in level of Ins(1,4,5)P₃ (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP₅ and InsP₆ was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P₃ and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P₃ metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P₃ 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidylinositol 4-phosphate cannot be excluded.     

Muscarinic Receptor-Mediated Increase in Extracellular Inositol 1,4,5-Trisphosphate Levels in the Rat Hippocampus: An In Vivo Microdialysis Study

Abstract: The extracellular concentration of inositol 1,4,5-trisphosphate (IP₃) has been monitored in the ventral hippocampus of the anesthetized rat by using a microdialysis technique coupled to a radioreceptor assay. Three hours after the implantation of the cannula, basal extracellular concentration of IP₃ (corrected for a 9% recovery) was 71 n M (0.39 pmol/60-µl fraction) and remained stable for at least 5 h. Local infusion of carbachol for 60 min caused a significant concentration-related increase in extracellular IP₃ levels (0, 24, and 57% at 1, 50, and 100 µ M , respectively). Acetylcholine (100 µ M ) and muscarine (100 µ M ) increased IP₃ outflow by 40 and 42%, respectively. The effect of carbachol was fully prevented by coinfusion of 10 µ M pirenzepine and reduced by 1 µ M tetrodotoxin indicating that the carbachol response is mediated by neuronal muscarinic receptors. These data demonstrate the feasibility of using microdialysis and a radioreceptor assay to measure IP₃ in the extracellular space. This approach could prove useful for the study of the in vivo operation of muscarinic and, by extension, a number of receptors coupled to phosphoinositide turnover.     

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5-trisphosphate also displays such variations, activities of inositol 1,4,5-trisphosphate 5'-phosphatase and 3'-kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5'-phosphatase activity was relatively high at birth (approximately 50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3'-kinase activity was low at birth and reached approximately 50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3'-kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5'-phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5- to 10-fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50-60% of the total inositol 1,4,5-trisphosphate 5'-phosphatase activity present in whole adult rat brain. The localization of the enriched 5'-phosphatase activity within the cerebellum was examined. Application of a histochemical lead-trapping technique for phosphatase indicated a concentration of inositol 1,4,5-trisphosphate 5'-phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell-deficient mutant mice, in which a marked decrement of cerebellar 5'-phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5-trisphosphate depends on both brain region and stage of development.     

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.     

Lithium Enhances Accumulation of [3H]Inositol Radioactivity and Mass of Second Messenger Inositol 1,4,5-Trisphosphate in Monkey Cerebral Cortex Slices

We previously reported that lithium, in the presence of acetylcholine, increased accumulations of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in brain cortex slices from the guinea pig, rabbit, rat, and mouse. In the mouse and rat, the Li(+)-induced increases required supplementation of the medium with inositol. This probably relates to the following facts: (a) Brain cortices of the mouse and rat contain in vivo concentrations of inositol half of that of the guinea pig. (b) Incubated rat brain cortex slices are depleted of inositol by 80%. (c) The slices require 10 mM inositol supplementation to restore in vivo concentrations. We now show that in monkey brain cortex slices, therapeutic concentrations of Li+ increase accumulation of inositol 1,4,5-trisphosphate. The inositol 1,3,4,5-tetrakisphosphate level is not increased. Neither inositol nor an agonist is required. The same effects are seen whether inositol 1,4,5-trisphosphate is quantified by the [3H]inositol prelabeling technique or by mass assay, although mass includes a pool of inositol 1,4,5-trisphosphate that is metabolically inactive. Thus, in a therapeutically relevant model for humans, Li+ increases inositol 1,4,5-trisphosphate levels in brain cortex slices, as was previously seen in lower mammals at non-rate-limiting concentrations of inositol.     

Characterisation and Distribution of Inositol Polyphosphate and Ryanodine Receptors in the Rat Brain

Abstract: The regional distribution of inositol 1,4,5-trisphosphate (InsP₃), inositol 1,3,4,5-tetrakisphosphate (InsP₄), and ryanodine binding sites has been characterised and compared in the rat brain using radioligand binding assays. Cortical [³H]InsP₃ binding indicated similar positional and stereospecificity as observed in other tissues, with 100-fold selectivity for lnsP₃ over InsP₄. Similarly, high-affinity [³²P]InsP₄ binding also showed a high degree of positional specificity, with a 1,000-fold selectivity for InsP₄ over InsP₃. Initial characterisation of [³H]ryanodine binding to cortical membranes demonstrated that specific binding was highly dependent on high salt and micromolar Ca²⁺ concentrations and inhibited by Ca²⁺ levels of >1 mM. [³H]-Ryanodine binding was also enhanced by β,γ-methylene-adenosine 5′-trisphosphate and caffeine and inhibited by magnesium and ruthenium red (K_i= 0.81 μM). However, dantrolene (300 μM) was ineffective on the binding. Therefore, although the results indicate a greater similarity to the binding properties of the Ca²⁺-induced Ca²⁺ release channel isoform present in skeletal, rather than cardiac, muscle, it does not appear to be identical. Detailed binding analysis of ryanodine and polyphosphate sites, with the exception of ruthenium red, indicated no interaction between binding sites. Ruthenium red markedly enhanced the binding of both [³H]InsP₃ and [³²P]InsP₄, an effect most probably due to nonspecific complex formation. Regional binding of InP₃, InsP₄, and ryanodine in the rat brain was of similar affinity for each ligand in each area, but the density profile for each ligand was clearly different. The highest density of InsP₃ sites was in the cerebellum, whereas the highest density of ryanodine sites was in the hippocampus. High-affinity InsP₄ sites showed less regional diversity, with highest densities in the cerebellum, cortex, and hippocampus. However, in each area studied the density of sites followed the order InsP₃ > InsP₄ > ryanodine.     

Chronic Activation of Muscarinic and Metabotropic Glutamate Receptors Down-Regulates Type I Inositol 1,4,5-Trisphosphate Receptor Expression in Cerebellar Granule Cells

Abstract: The ability of receptors coupled to phosphoinositide turnover to evoke accumulation of inositol 1,4,5-trisphosphate (InsP₃) over extended incubation periods, and consequently to affect the level of InsP₃ receptor expression, was studied in cultured cerebellar granule cells. The cholinergic agonist carbachol (CCh; 1 m M ) evoked a biphasic accumulation of InsP₃, a rapid three- to fourfold peak increase over control levels at ∼10 s, decreasing within 1 min to a long-lasting plateau elevation. Using an antibody against the type I InsP₃ receptor, it was demonstrated that >50% down-regulation of type I InsP₃ receptor expression in cerebellar granule cells occurred within 1 h of incubation with 1 m M CCh. Over 24 h, 1 m M CCh caused an ∼85% decrease in type I InsP₃ receptor levels, and significant decreases in immunoreactivity were evident at much lower concentrations of CCh. Direct assessment of total InsP₃ receptor expression using a radioligand binding method also detected down-regulation, but to an apparently lesser extent. 1-Aminocyclopentane-1 S ,3 R -dicarboxylic acid (200 µ M ), an agonist of metabotropic glutamate receptors, evoked a marked decrease in type I InsP₃ receptors after 24 h of incubation. These findings demonstrate that a functional consequence of maintained InsP₃ production in cerebellar granule cells is the down-regulation of InsP₃ receptor expression and that this down-regulation may be a common mechanism of action of phosphoinositide-linked receptors during prolonged stimulation.     

Carbachol, but Not Norepinephrine, NMDA, lonomycin, Ouabain, or Phorbol Myristate Acetate, Increases Inositol 1,3,4,5-Tetrakisphosphate Accumulation in Rat Brain Cortical Slices

Abstract: lonomycin, a Ca²⁺ ionophore, stimulated phosphoinositide breakdown in rat brain cortical slices incubated in the presence of 1.2 m M Ca²⁺, but, unlike muscarinic cholinergic stimulation, it had little effect on inositol 1,3,4,5-tetrakisphosphate accumulation. However, at 2 min, the increase in inositol 1,4,5-trisphosphate due to 10 μ M ionomycin was equivalent to that seen with 1μ M carbachol. Phorbol 12-myristate 13-acetate or high K⁺ (30 μ M ) increased inositol 1,4,5-trisphosphate, but not inositol 1,3,4,5-tetrakisphosphate accumulation. The stimulation of inositol 1,4,5-trisphosphate accumulation due to ionomycin, unlike that seen with carbachol, was abolished in buffer containing 0.2 μ M Ca²⁺. The increase in inositol 1,3,4,5-tetrakisphosphate accumulation in brain slices due to 1 μ M carbachol ranged from 55 to 68% of that for inositol 1,4,5-trisphosphate. Norepinephrine, NMDA, veratridine, and ouabain also increased inositol 1,4,5-tris-phosphate, but had minimal effects on inositol 1,3,4,5-tetrakisphosphate accumulation. These results suggest that there is something unique about the stimulation of inositol 1,3,4,5-tetrakisphosphate accumulation by carbachol, which is also the only one of these agents that is able to activate phosphoinositidase Cβ, in isolated rat brain membranes.     

Expression and Regulation of Types I and II Inositol 1,4,5-Trisphosphate Receptors in Rat Cerebellar Granule Cell Preparations

Abstract: Previous studies have shown that as rat cerebellar granule cell cultures differentiate in the presence of 25 m M KCl, they "up-regulate" their ability to form inositol phosphates and release Ca²⁺ from internal stores in response to the activation of phosphoinositidase C-linked muscarinic and metabotropic receptors. Here we show that they simultaneously up-regulate their ability to respond to inositol 1,4,5-trisphosphate (InsP₃) by increasing InsP₃ receptor (InsP₃R) expression. In contrast, if granule cells are maintained at the more physiological KCl concentration of 5 m M , most cells undergo apoptosis, although a significant number survive. The surviving cells, however, express few InsP₃Rs, suggesting that an influx of Ca²⁺ through voltage-dependent channels is required for InsP₃R up-regulation. In addition, we have determined that these cultures express two genetically distinct InsP₃R types, but that only one, the type I receptor, is expressed in granule cells. Type II receptors are also present but are found exclusively in astrocytes, which are a minor contaminant of granule cell cultures. This segregation of InsP₃R types explains a previous observation, showing that the muscarinic agonist carbachol causes the reduction or "down-regulation" of type I but not type II InsP₃Rs.     

Solubilization of rat liver inositol 1,4,5-trisphosphate receptor

High affinity Ins(1,4,5)P₃-binding sites of permeabilized hepatocytes are probably the ligand recognition sites of the receptors that mediate the effects of Ins91,4,5)P₃ on intracellular Ca²⁺ mobilization. We have now solubilized these sites from rat liver membranes in the zwitterionic detergent, CHAPS, and shown that the solubilized bind Ins(1,4,5)P₃ with an affinity (K_d = 7.26 ± 0.52 nM, Hill coefficient H = 1.05 ± 0.06) similar to that of the sites in native membranes (K_d = 6.02 ± 0.02). ATP and a range of inositol phosphates (Ins(2,4,5)P₃ Ins(4,5)P₂, and inositol 1,4,5-trisphosphorothioate) also bound with similar affinities to the native and solubilized sites. Solubilization of the liver InsP₃ receptor will allow its further characterization, purification, and comparison of its properties with those of InsP₃ receptors already purified from cerebellum and smooth muscle.     

Increased Inositol 1,4,5-Trisphosphate Accumulation Correlates with an Up-Regulation of Bradykinin Receptors in Alzheimer's Disease

Abstract: An alteration in signal transduction systems in Alzheimer's disease (AD) would likely be of pathophysiological significance, because these processes control normal brain functions. Previously, a diminished β-adrenergic-mediated cyclic AMP response was found in cultured fibroblasts from AD patients. Because cross-talk between the phosphoinositide and cyclic AMP pathways exists, the phosphoinositide cascade was studied under conditions that were similar to those for studying the cyclic AMP response. Cells from AD patients and age-matched controls responded to bradykinin (BK) and released inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] in a time- and dose-dependent manner. The level of Ins(1,4,5)P₃ increased rapidly and transiently in response to BK, peaked at 5 s, but still remained 116–132% above the basal level by 30 s. Although the temporal patterns were similar in both groups, the Ins(1,4,5)P₃ concentrations in AD fibroblasts were 73 and 89% above levels in the age-matched controls at 5 and 10 s, respectively. Prostaglandin E₁ also increased Ins(1,4,5)P₃ formation, but this response was not different between the two groups. Although K _D (affinity) values for the BK receptor were similar in both control and AD cells, the number of BK receptors ( B _max) was significantly elevated in AD fibroblasts (186.8 ± 0.8 fmol/mg of protein) as compared with control fibroblasts (57.2 ± 15.3 fmol/mg of protein). These results indicate that the elevated Ins(1,4,5)P₃ production in response to BK in AD fibroblasts is positively correlated with an increase in the receptor numbers.     

Electroconvulsive Shock Reduces Inositol 1,4,5-Trisphosphate 3-Kinase mRNA Expression in Rat Dentate Gyrus

Abstract: We investigated the expression of inositol 1,4,5-trisphosphate (InsP₃) 3-kinase mRNA after a single electroconvulsive shock (ECS) with in situ hybridization histochemistry in rat brain. At 6 h after ECS, the expression was markedly decreased in the dentate gyrus, and the decrease was maintained until 9 h with a slight recovery. The InsP₃ 3-kinase mRNA content returned to basal levels after 12 h. We could not detect any apparent changes in the expression of InsP₃ 3-kinase mRNA in the CA1–CA3 areas of hippocampus, the striatum, and the cerebral cortex at any time point examined. In the temporal pattern, the reduction of the expression in the dentate gyrus was preceded by the induction of c- fos after ECS. These observations suggest that the InsP₃ 3-kinase might be one of the genes whose expression can be altered by ECS.     

Agonist-Evoked Ca2+ Mobilization from Stores Expressing Inositol 1,4,5-Trisphosphate Receptors and Ryanodine Receptors in Cerebellar Granule Neurones

Abstract: The mechanisms involved in Ca²⁺ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-d -aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca²⁺ concentration ([Ca²⁺]_i) response to CCh (to 45 ± 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 ± 6% of the control). CCh-evoked [Ca²⁺]_i responses were inhibited by preincubation with thapsigargin (10 µM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 µM), ryanodine (10 µM), or dantrolene (25 µM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca²⁺]_i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 µM) evoked a much diminished increase in [Ca²⁺]_i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 ± 14%; ACPD, 27 ± 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP₃) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca²⁺ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca²⁺]_i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP₃ receptors, appear to be crucial to the mAChR-mediated [Ca²⁺]_i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP₃-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP₃ receptors may not contribute significantly to NMDA-evoked elevation of [Ca²⁺]_i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca²⁺ stores is proposed.     

Ca2+ Release from Inositol 1,4,5-Trisphosphate-Sensitive Ca2+ Store by Antidepressant Drugs in Cultured Neurons of Rat Frontal Cortex

Abstract: The ability of antidepressant drugs (ADs) to increase the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) was examined in primary cultured neurons from rat frontal cortices using the Ca²⁺-sensitive fluorescent indicator fura-2. Amitriptyline, imipramine, desipramine, and mianserin elicited transient increases in [Ca²⁺]_i in a concentration-dependent manner (100 μM to 1 mM). These four AD-induced [Ca²⁺]_i increases were not altered by the absence of external Ca²⁺ or by the presence of La³⁺ (30 μM), suggesting that these ADs provoked intracellular Ca²⁺ mobilization rather than Ca²⁺ influx. All four ADs increased inositol 1,4,5-trisphosphate (IP₃) contents by 20–60% in the cultured cells. The potency of the IP₃ production by these ADs closely correlated with the AD-induced [Ca²⁺]_i responses. Pretreatment with neomycin, an inhibitor of IP₃ generation, significantly inhibited amitriptyline- and imipramine-induced [Ca²⁺]_i increases. In addition, by initially perfusing with bradykinin (10 μM) or acetylcholine (10 μM), which can stimulate the IP₃ generation and mobilize the intracellular Ca²⁺, the amitriptyline responses were decreased by 76% and 69%, respectively. The amitriptyline-induced [Ca²⁺]_i increases were unaffected by treatment with pertussis toxin. We conclude that high concentrations of amitriptyline and three other ADs mobilize Ca²⁺ from IP₃-sensitive Ca²⁺ stores and that the responses are pertussis toxin-insensitive. However, it seems unlikely that the effects requiring high concentrations of ADs are related to the therapeutic action.     

Reduced Striatal [3H]Inositol 1,4,5-Trisphosphate Binding in Huntington''s Disease

Specific [3H]inositol 1,4,5-trisphosphate [( 3H]InsP3) binding was studied in regions of postmortem brain from 15 patients with Huntington's disease (HD) and 13 nonneurological controls. Single-point binding analyses, using 5.0 nM InsP3, showed statistically significant reductions in specific [3H]InsP3 binding in the caudate (-71%) and putamen (-75%) of HD patients compared with controls. Frontal and occipital cortical [3H]InsP3 binding was not significantly different between HD and controls, a finding suggesting that the reduced [3H]InsP3 binding parallels the brain regional specificity of the neuropathological changes in HD. Scatchard analyses of data from [3H]InsP3 competition binding assays performed on caudate nucleus revealed that the reductions found using single-point binding assays were due to a decrease in both binding density (-57%) and affinity (-50%) in HD brain compared with controls. The concomitant changes in InsP3 receptor density and affinity in HD brain suggest that these alterations may be produced by processes in addition to cell loss. These results suggest the possibility that disturbances in InsP3 receptor function, possibly resulting in altered intracellular calcium flux and homeostasis, occur in HD and may participate in the pathogenesis of this neurodegenerative disorder.     

Muscarinic Receptor-Mediated Inositol 1,4,5-Trisphosphate Formation in SH-SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP₃) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ～60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca²⁺ with EGTA or inhibition of Ca²⁺ channels with Ni²⁺ showed that the plateau phase was dependent upon Ca²⁺ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca²⁺ from intracellular stores revealed that Ca²⁺ from this source was capable of supporting the peak phase of the InsP₃ response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca²⁺ concentration (20–100 nM) and suggests that under normal conditions, InsP₃ formation is enhanced by increases in cytosolic free Ca²⁺ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li⁺ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP₃ accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca²⁺ concentration amplify InsP₃ formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP₃ accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca²⁺ or protein kinase C activity.     

Inhibition of Bradykinin-Induced Cytosolic Ca2+ Elevation by Muscarinic Stimulation Without Attenuation of Inositol 1,4,5-Trisphosphate Production in Human Neuroblastoma SK-N-BE(2)C Cells

Abstract: Cross talk between two phospholipase C (PLC)-linked receptor signalings was investigated in SK-N-BE(2)C human neuroblastoma cells. Sequential stimulation with two agonists at 5-min intervals was performed to examine the interaction between muscarinic and bradykinin (BK) receptors. Pretreatment of cells with a maximal effective concentration (5 µ M ) of BK did not affect the subsequent carbachol (CCh)-induced [Ca²⁺]_i rise, but CCh (1 m M ) pretreatment completely abolished the BK-induced [Ca²⁺]_i rise without inhibition of BK-induced inositol 1,4,5-trisphosphate (IP₃) generation. Thapsigargin (1 µ M ) pretreatment abolished the subsequent BK- and CCh-induced [Ca²⁺]_i rise, though it did not affect agonist-induced IP₃ generation. However, the addition of atropine at plateau phases of CCh-induced [Ca²⁺]_i rise and IP₃ production caused a rapid decline to the basal levels and then restored the [Ca²⁺]_i rise by BK. Treatment of cells with both CCh and BK at the same time showed additive effects in IP₃ production. However, the [Ca²⁺]_i rise induced by both agonists in the presence or absence of extracellular Ca²⁺ was the same as the responses triggered by CCh alone. The results suggest that each receptor or receptor-linked PLC activity is not influenced by pretreatment with the other agonist but IP₃-sensitive Ca²⁺ stores are shared by signal pathways from both receptors.