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.     

Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µ M TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µ M ). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.     

Activation of D1 and D2 Dopamine Receptors Inhibits Protein Kinase C Activity in Striatal Synaptoneurosomes

Abstract: The effects of D₁ and D₂ dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D₁ agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D₂ agonists [LY 171555, (+)-3-(3-hydroxyphenyl)- N - n -propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (−)-3-(3-Hydroxyphenyl)- N - n -propylpiperidine had a D₂ antagonist profile. Apomorphine showed a biphasic dose-response change; i.e., it decreased particulate PKC activity at the D₂ receptor at low concentrations (0.1 µ M ) and increased it at the D₁ receptor at higher concentrations (10 µ M ). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D₂ agonists, but it reversed the changes in PKC activity induced by the D₁ agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1) D₁ and D₂ dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D₁ drugs in the presence of calcium is mediated indirectly via a transneuronal effect.     

Signal Flows from Two Phospholipase C-Linked Receptors Are Independent in PC12 Cells

Abstract: Bradykinin (BK) receptor and P₂-purinergic receptor are known to be coupled to phospholipase C (PLC) in PC12 cells. To study the interaction between these two PLC-linked receptors, the presence of both receptors on individual cells was demonstrated by sequential Ca²⁺ spikes caused by BK and ATP in a single fura-2-loaded cell. BK- and ATP-induced catecholamine (CA) secretions were desensitized within 5 min. However, in the sequential experiment, the BK-induced homologous desensitization of CA secretion did not block the ATP-induced secretion, and vice versa. Each agonist-induced an increase in inositol 1,4,5-trisphosphate (IP₃) production and intracellular free Ca²⁺ concentration also led to homologous desensitization. However, there was no heterologous desensitization between the two agonists. When the cells were treated with both BK and ATP simultaneously, the amounts of CA secretion, IP₃ production, internal Ca²⁺ mobilization, and Ca²⁺ influx were all additive. We also found that both IP₃-induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ influx from extracellular space were able to release [³H]norepinephrine, and the secretion induced by both agonists was exactly additive in the absence or presence of extracellular Ca²⁺. The data suggest that the CA secretions caused by BK or ATP may have separate secretory pathways even though they activate identical second messenger pathways.     

New pathways of phagocyte activation: the coupling of receptor-linked phospholipase D and the role of tyrosine kinase in primed neutrophils

Abstract Protein kinase C (PKC) appears to have a central role in the O₂⁻ response of neutrophils following stimulation of membrane receptors. The second messenger, diacylglycerol (DG), that activates PKC is derived from membrane phospholipids via activation of phosphatidylinositol 4,5-bisphosphate (PIP₂)-phospholipase C (PLC) and phospholipase D (PLD), with the latter pathway being more prominent in primed cells. In resting cells receptor coupling of PLD is through a G-protein. Priming brings a cytoplasmic tyrosine kinase into the transducer sequence which, through protein phosphorylation, increases the efficiency of coupling between membrane receptors and PLD. Phosphatidic acid (PA), the initial product of the PLD pathway, also appears to act as a second messenger by directly activating the NADPH oxidase responsible for generating O₂⁻. Interconversion of PA and DG by phosphatidate phosphohydrolase and DG kinase determines which of these second messengers has the dominant role.     

Role of Protein Kinase C (PKC) in Agonist-Induced μ-Opioid Receptor Down-Regulation

Abstract : Agonist-induced down-regulation of opioid receptors appears to require the phosphorylation of the receptor protein. However, the identities of the specific protein kinases that perform this task remain uncertain. Protein kinase C (PKC) has been shown to catalyze the phosphorylation of several G protein-coupled receptors and potentiate their desensitization toward agonists. However, it is unknown whether opioid receptor agonists induce PKC activation under physiological conditions. Using cultured SH-SY5Y neuroblastoma cells, which naturally express μ- and δ-opioid receptors, we investigated whether μ-opioid receptor agonists can activate PKC by measuring enzyme translocation to the membrane fraction. PKC translocation and opioid receptor densities were simultaneously measured by ³H-phorbol ester and [³H]diprenorphine binding, respectively, to correlate alterations in PKC localization with changes in receptor binding sites. We observed that μ-opioid agonists have a dual effect on membrane PKC density depending on the period of drug exposure. Exposure for 2-6 h to [ d -Ala², N -Me-Phe⁴, Gly-ol]enkephalin or morphine promotes the translocation of PKC from the cytosol to the plasma membrane. Longer periods of opioid exposure (>12 h) produce a decrease in membrane-bound PKC density to a level well below basal. A significant decrease in [³H]diprenorphine binding sites is first observed at 2 h and continues to decline through the last time point measured (48 h). The opioid receptor antagonist naloxone attenuated both opioid-mediated PKC translocation and receptor down-regulation. These results demonstrate that opioids are capable of activating PKC, as evidenced by enhanced translocation of the enzyme to the cell membrane, and this finding suggests that PKC may have a physiological role in opioid receptor plasticity.     

P2Y1 receptor-mediated glutamate release from cultured dorsal spinal cord astrocytes

P2 receptors have been implicated in the release of neurotransmitter and proinflammatory cytokines by the response to neuroexcitatory substances in astrocytes. In the present study, we examined the mechanisms of ADP and adenosine 5'-O-2-thiodiphosphate (ADPbetaS, ADP analogue) on glutamate release from cultured dorsal spinal cord astrocytes by using confocal laser scanning microscopy and HPLC. Immunofluorescence activity showed that P2Y₁ receptor protein is expressed in cultured astrocytes. ADP and ADPbetaS-induced [Ca²⁺]_i increase and glutamate release are mediated by P2Y₁ receptor. Ca²⁺ release from IP₃-sensitive calcium stores and protein kinase C (PKC) activation is important for glutamate release from astrocytes. Furthermore, P2Y₁ receptor-evoked glutamate release is regulated by volume-sensitive Cl⁻ channels and anion co-transporter, which open up the possibility that P2Y₁ receptor activation causes the increase of cell volume. Release of glutamate by ADPbetaS was abolished by 5-nitro-2 (3-phenyl propy lamino)–benzoate plus furosemide but was unaffected by botulinum toxin A. These observations indicate that P2Y₁ receptor-evoked glutamate may be mediated via volume-sensitive Cl⁻ channel but not via exocytosis of glutamate containing vesicles. We speculate that P2Y₁ receptors-evoked glutamate efflux, occurring under pathological condition, may modulate the activity of synapses in spinal cord.     

Effect of Heat Shock on Intracellular Calcium Mobilization in Neuroblastoma × Glioma Hybrid Cells

Abstract: The effect of heat shock on agonist-stimulated intracellular Ca²⁺ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma × glioma hybrid cells (NG 108–15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5°C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca²⁺], rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca²⁺]_i rise was abolished and the [Ca²⁺]_i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca²⁺-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP₃) production was not altered by heat shock at 12 h after heat shock, whereas IP₃ receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP₃ receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IPs-mediated intracellular Ca²⁺ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca²⁺]_i rise.     

The membrane effects of 17beta-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E₂); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E₂ directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E₂ activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E₂-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E₂ on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E₂ on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10⁻¹⁰ to 10⁻⁷ M E₂ in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [³H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E₂ in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A₂ [PLA₂]), and melittin (an activator of PLA₂). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [³H]thymidine incorporation was decreased by E₂. The effects of E₂ on all parameters were blocked by chelerythrine. Treatment of the cultures with E₂ produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E₂-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E₂ on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E₂. Inhibition of tyrosine kinase and PLA₂ had no effect on the activation of PKC by E₂. The PLA₂ activator also had no effect on PKC activation by E₂. E₂ stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E₂ on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

The role of inositol lipids in the activation of monocytes by interleukin-1 and bacterial endotoxin

Abstract The effect of interleukin-1 (IL-1) and bacterial endotoxin (lipopolysaccharide, LPS) on the activation of phosphoinositidase C (PIC) and on prostaglandin E₂ release was studied in monocytes (Mø). Both IL-1α and IL-1β increased the release of PGE₂ in a concentration-dependent manner, with EC₅₀s of 0.48 nM and 0.12 nM, respectively. Intact Mø were prelabelled with [³H]inositol and the formation of inositol phosphates (IPs) was estimated by ion exchange chromatography. PIC activity was estimated directly by measuring the conversion of [³H]phosphatidylinositol-4,5,-bisphosphate to aqueous soluble radioactivity by Mø homogenates. IL-1α (5.8 nM) increased the accumulation of IPs within 1–4 minutes and increases in IP₃ and IP₄ occured before the increase in IP₁₊₂ whereas LPS only increased the IPs level after at least 30 min. IL-1α increased PIC activity in Mø homogenates within 15 min with an EC₅₀ of 0.58 nM and IL-1β (0.1 nM) also increased activity. Neither IL-1α nor IL-1β affected the PIC activity of membrane or cytosolic fractions. LPS decreased activity in all fractions. These data indicate that IL-1, but not LPS, can directly lead to an increased activity of PIC which may be involved in eicosanoid formation in Mø.     

Abnormality of α1Adrenergic Receptors in the Frontal Cortex of Epileptic Rats

Abstract: We found that the binding of [³H]prazosin, a selective ligand for α₁-adrenergic recognition sites, is significantly lower in the frontal cortex of the genetically epilepsy-prone rats (GEPRs), as compared with normal Sprague-Dawley rats. Scatchard analysis reveals a decrease in the B _max of [³H]prazosin binding with no change in the apparent K _D, suggesting that there are fewer α₁-adrenergic recognition sites in the frontal cortex of the GEPR. This abnormality is associated with a reduced capacity of norepinephrine (NE) to stimulate [³H]inositol monophosphate ([³H]IP₁) formation in frontal cortex slices prelabeled with [³H]inositol. No significant differences in [³H]prazosin binding as well as NE-stimulated [³H]IP₁ formation have been observed in other brain regions including hippocampus, corpus striatum, and inferior colliculus. These results indicate that a deficit in the α₁-adrenergic receptor system in the frontal cortex may play a role in the seizure process in the GEPR.     

Inositol 1,4,5-trisphosphate formation in leaves of winter oilseed rape plants in response to freezing, tissue water potential aed abscisic acid

Time courses of formation of inositol 1,4,5-trisphosphate (IP₃) were followed in the leaves of non-acclimated and cold (2°C)-acclimated winter oilseed rape ( Brassica napus L. var. oleifera ) plants, subjected to different freezing temperatures or to polyethylene glycol 8000 (PEG) and abscisic acid (ABA) treatments. Changes in water potential (Ψ_w) and in ABA level in the frost- and PEG-treated tissues were also determined. Results obtained indicate that temperatures sligthly higher than LT₅₀ induced a transient and substantial increase in IP₃ level, both in non-acclimated and cold-acclimated tissues. At comparable freezing temperature (–5°C) the response of cold-acclimated leaves was lower than that of non-acclimated ones. The PEG-depedent decrease in Ψ_w to –0.9 MPa or ABA (0.1 m M ) treatment gave rise to a transient increase in IP₃ content in non-acclimated tissues only. Collectively, the data indicate that cold acclimation of plants may lead to lower cell responsiveness to the factors studied in terms of induction of IP₃ formation. Changes in the IP₃ content, observed in the present experiments, support our previous suggestion that non-killing freezing temperatures may induce the phosphoinositide pathway, both in non-acclimated and cold-acclimated tissues. Lowering of tissue water potential to some threshold value or a high exogenous ABA supply may mimic the freezing-dependent reaction in the non-acclimated leaves.     

Calcium Influx Through AMPA Receptors and Through Calcium Channels Is Regulated by Protein Kinase C in Cultured Retina Amacrine-Like Cells

Abstract: The functional modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors by protein kinase C (PKC) was investigated in cultures enriched in retinal amacrine-like cells. The kainate-evoked [Ca²⁺]_i increase is due to Ca²⁺ entry through open AMPA receptor channels, because it was blocked by the active isomer of a 2,3-benzodiazepine (LY 303070), an AMPA receptor antagonist. The AMPA receptor response to kainate was potentiated by phorbol 12-myristate 13-acetate, which specifically stimulates PKC, and it was decreased by bisindolylmaleimide I, a selective inhibitor of PKC, as well as by PKC down-regulation. The results indicate not only that the AMPA receptor activation has a PKC requirement, but also that PKC amplifies maximal receptor activation by 100 µ M kainate. The effect of PKC activation or inhibition on voltage-gated Ca²⁺-channel activity was also investigated. Activation of PKC caused inhibition of Ca²⁺ channels, and the same effect was produced by inhibition of PKC, whereas the inactive analogue of the phorbol ester did not affect channel activity. Our results show an important role for PKC in regulating the function of both AMPA receptors and Ca²⁺ channels in cultured retina cells.     

Type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors promote the differentiation of granule cell precursors in the postnatal cerebellum

During postnatal development of the cerebellum, granule cell precursors (GCPs) proliferate in the external granular layer (EGL), exit the cell cycle, differentiate, and migrate from the EGL to the internal granular layer. In the present study, we report that type 2 and 3 inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R2 and IP₃R3) regulate the differentiation of GCPs after postnatal day 12 (P12). 5-Bromodeoxyuridine labeling experiments revealed that in mutant mice lacking both of these receptors (double mutants) a greater number of GCPs remain undifferentiated after P12. Consequently, the EGL of the double mutants is thicker than that of control mice at this age and thereafter. In addition, granule cells remain in the EGL of the double mutants at P21, an age when migration has concluded in wild-type mice. Whereas differentiation of GCPs was reduced in the double mutants, the absence of IP₃R2 and IP₃R3 did not affect the doubling time of GCPs. We conclude that intracellular calcium release via IP₃R2s and IP₃R3s promotes the differentiation of GCPs within a specific interval of postnatal development in the cerebellum.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.     

Involvement of Calcium Influx in Muscarinic Cholinergic Regulation of Phospholipase C in Cerebellar Granule Cells

Abstract: Inositol phosphate accumulation on carbachol stimulation of rat cerebellar granule cells shows a marked dependence on factors affecting cytosolic Ca²⁺ concentration ([Ca²⁺]_c). After 5 min, potassium depolarisation caused a modest accumulation of inositol phosphates but augmented the response to carbachol by a factor of 2–3. These effects of potassium were dependent on an extracellular source of calcium and could be partially blocked by specific (nifedipine) and nonspecific (verapamil) calcium channel blockers. Measurements of [Ca²⁺]_c under a range of stimulatory conditions demonstrated a close correlation between the elevation of [Ca²⁺]_c and agonist-stimulated phospholipase C (PLC) activity. The maximal potentiation of carbachol-stimulated inositol phosphate accumulation was achieved using 20 m M KCl, which increased [Ca²⁺]_c from ∼20 to ∼75 n M , indicating the involvement of relatively low threshold Ca²⁺ channels and the high sensitivity of the relevant PLC to small changes in [Ca²⁺]_c. By contrast, increases in [Ca²⁺]_c induced by the Ca²⁺ ionophore ionomycin were associated with more modest and less potent effects on agonist-stimulated PLC. These results demonstrate a cooperative interaction between a receptor/G protein-regulated PLC and voltage-stimulated elevations of [Ca²⁺]_c, which may function to integrate ionotropic and metabotropic signalling mechanisms in cerebellar granule cells.     

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.