Adenosine Receptors Modulate [Ca2+]i in Hippocampal Astrocytes In Situ

Abstract: Cultured astroglia express both adenosine and ATP purinergic receptors that are coupled to increases in intracellular calcium concentration ([Ca²⁺]_i). Currently, there is little evidence that such purinergic receptors exist on astrocytes in vivo. To address this issue, calcium-sensitive fluorescent dyes were used in conjunction with confocal microscopy and immunocytochemistry to examine the responsiveness of astrocytes in acutely isolated hippocampal slices to purinergic neuroligands. Both ATP and adenosine induced dynamic increases in astrocytic [Ca²⁺]_i that were blocked by the adenosine receptor antagonist 8-( p -sulfophenyl)theophylline. The responses to adenosine were not blocked by tetrodotoxin, 8-cyclopentyltheophylline, 8-(3-chlorostyryl)caffeine, dipyridamole, or removal of extracellular calcium. The P_2Y-selective agonist 2-methylthioadenosine triphosphate was unable to induce increases in astrocytic [Ca²⁺]_i, whereas the P₂ agonist adenosine 5'- O -(2-thiodiphosphate) induced astrocytic responses in a low percentage of astrocytes. These results indicate that the majority of hippocampal astrocytes in situ contain P₁ purinergic receptors coupled to increases in [Ca²⁺]_i, whereas a small minority appear to contain P₂ purinergic receptors. Furthermore, individual hippocampal astrocytes responded to adenosine, glutamate, and depolarization with increases in [Ca²⁺]_i. The existence of both purinergic and glutamatergic receptors on individual astrocytes in situ suggests that astrocytes in vivo are able to integrate information derived from glutamate and adenosine receptor stimulation.     

Zinc Allosterically Modulates Antagonist Binding to Cloned D1 and D2 Dopamine Receptors

Abstract: Cations of various size and charge were used as atomic scale probes of D₁ and D₂ dopamine receptors. Those cations that perturbed the binding of D₁- and D₂-selective dopamine receptor antagonists were identified by screening at 5 m M cation. Pseudo-noble-gas-configuration d-transition metals, such as zinc, exerted a complete inhibition of specific binding, whereas most other cations had little or no effect. The nature of zinc's actions was characterized by measuring the radioligand binding properties of [³H]SCH-23390 and [³H]methylspiperone to cloned D_1A and D_2L dopamine receptors in either the presence or absence of Zn²⁺. Zinc exerts a low-affinity, dose-dependent, EDTA-reversible inhibition of the binding of subtype-specific antagonists primarily by decreasing the ligands' affinity for their receptors. The mechanism of zinc inhibition appears to be allosteric modulation of the dopamine receptor proteins because zinc increases the dissociation constant ( K _D) of ligand binding, Schild-type plots of zinc inhibition reach a plateau, and zinc accelerates antagonist dissociation rates. Here we demonstrate the effect of zinc on the binding of D₁- and D₂-selective antagonists to cloned dopamine receptors and show that the inhibition by zinc is through a dose-dependent, reversible, allosteric, two-state modulation of dopamine receptors.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

Influence of P blood group phenotype on susceptibility to urinary tract infection

Abstract Bacterial attachment is an important event in the pathogenesis of urinary tract infection (UTI). Increased receptivity on the host cells has been suggested influence proneness to infection. The dual function of the globoseries of glycolipids both as receptors for attaching E. coli and as P blood group antigens lead us to examine the P blood group phenotype distribution in UTI prone patient populations. A correlation between the P₁ blood group phenotype and susceptibility to UTI was found. Patients with recurrent pyelonephritis had 74/79 (94%), P₁ compared to 75% in healthy controls. In contrast patients with asymptomatic bacteriuria (ABU) had a reduced frequency of P₁, 43/74 (58%). P₁ and P₂ individuals differ in amount and composition of the globoseries of glycolipids on their erythrocytes. A similar difference in other tissues, e.g. uroepithelial cells might explain the association of P₁ with UTI. There was, however, no significant difference in bacterial adherence to uroepithelial cells from P₁ and P₂ individuals. Other mechanisms explaining the increase in P₁ individuals in recurrent pyelonephritis are discussed.     

Functional Modulation of P2×2 Receptors by Cyclic AMP-Dependent Protein Kinase

Abstract: It is generally believed that protein phosphorylation is an important mechanism through which the functions of voltage- and ligand-gated channels are modulated. The intracellular carboxyl terminus of P_2×2 receptor contains several consensus phosphorylation sites for cyclic AMP (cAMP)-dependent protein kinase (PKA) and protein kinase C (PKC), suggesting that the function of the P_2×2 purinoceptor could be regulated by the protein phosphorylation. Whole-cell voltage-clamp recording was used to record ATP-evoked cationic currents from human embryonic kidney (HEK) 293 cells stably transfected with the cDNA encoding the rat P_2×2 receptor. Dialyzing HEK 293 cells with phorbol 12-myristate 13-acetate, a PKC activator, failed to affect the amplitude and kinetics of the ATP-induced cationic current. The role of PKA phosphorylation in modulating the function of the P_2×2 receptor was investigated by internally perfusing HEK 293 cells with 8-bromo-cAMP or the purified catalytic subunit of PKA. Both 8-bromo-cAMP and PKA catalytic subunit caused a reduction in the magnitude of the ATP-activated current without affecting the inactivation kinetics and the value of reversal potential. Site-directed mutagenesis was also performed to replace the intracellular PKA consensus phosphorylation site (Ser⁴³¹) with a cysteine residue. In HEK 293 cells expressing (S431C) mutant P_2×2 receptors, intracellular perfusion of 8-bromo-cAMP or purified PKA catalytic subunit did not affect the amplitude of the ATP-evoked current. These results suggest that as with other ligand-gated ion channels, protein phosphorylation by PKA could play an important role in regulating the function of the P_2×2 receptor and ATP-mediated physiological effects in the nervous system.     

STUDIES ON NICOTINIC ACETYLCHOLINE RECEPTORS IN MAMMALIAN BRAIN. CHARACTERIZATION OF A MICROSOMAL SUBFRACTION ENRICHED IN RECEPTOR FUNCTION FOR DIFFERENT NEUROTRANSMITTERS

Abstract— A subfraction, derived from the microsomal fraction of rat cerebral cortex, with a buoyant density of 1.112 g μ ml⁻¹ appears to be enriched in receptor sites for a number of potential neurotrans-mitters. These include the cholinergic (nicotinic and muscarinic) and ß-adrenergic receptors. This microsomal subfraction (P₃B₂) has been isolated on a preparative scale by two sequential isopycnic sedimentations in discontinuous sucrose gradients.
We have studied the morphology, enzymatic markers and protein composition of this fraction and have compared them with the properties of other subcellular fractions from the same source. Synaptic plasma membranes resembled P₃B₂ by exhibiting the same high extent of enrichment in receptors. However, the synaptic membranes appear to contain more mitochondrial and presynaptic (axonal and cell surface) membranes than does P₃B₂, and the postsynaptic membranes in the two fractions appear morphologically distinct since P₃B₂ does not contain the characteristic postsynaptic densities. Thus these membranes may be derived from Gray's type II synapses.     

Changes in Orthophosphate, Pyrophosphate and Long-Chain Polyphosphate Levels in Leishmania major Promastigotes Incubated With and Without Glucose

The intracellular levels of orthophosphate (P₁), pyrophosphate (PP₁) and short- and long-chain polyphosphate (Poly P) were measured in Leishmania major promastigotes incubated in a phosphate-free medium. In the absence of exogenous substrate, the levels of both P₁ and PP₁ increased during a 1 h incubation. The increase in both P₁ and PP₁ was prevented when glucose was present, but glycerol prevented the rise in P₁ only. A rise in P₁ and PP₁ was also seen in cells incubated in the absence of exogenous substrate under anaerobic conditions. This was reversed upon addition of glucose plus oxygen. Polyphosphate, here shown to be present in L. major , was measured by means of a polyphosphate glucokinase assay. Short-chain Poly P content did not differ between cells incubated for 1 h in the absence of exogenous substrate or in the presence of glucose or glycerol. Long-chain Poly P content, however, was lower in cells incubated without glucose than in cells incubated with glucose and was also lower in cells incubated for 1 h with glycerol as compared with freshly washed cells. Up to 61% of the increase in P₁ and PP₁ that occurred in promastigotes incubated in the absence of exogenous substrate could have arisen from the concomitant decrease in long-chain Poly P.     

Protective Effect of FTY720 Against Sevoflurane-Induced Developmental Neurotoxicity in Rats

Sevoflurane, a common used inhaled anaesthetic, induces neuronal apoptosis in preclinical studies and correlates with functional neurological impairment. We investigated whether FTY720, a known sphingosine-1 phosphate (S1P) receptor agonist, could exert neuroprotective effect against sevoflurane-induced neurotoxicity. Neuroprotective effect of FTY720 was evaluated in vitro in hippocampal neuronal cells from neonatal rats and in vivo in rat pups. In vitro cell apoptosis was determined by flow cytometry after exposure to 3 % sevoflurane for different period of time, or after 6-h exposure to sevoflurane with the presence of FTY720, SEW2871 (selective S1P1 receptor agonist) or combination of FTY720 and VPC23019 (S1P antagonist). Western blot analysis was performed with hippocampal tissue from rat pups exposed to 3 % sevoflurane for 6 h with or without pre-treatment with FTY720 injection. Neurological function tests were also performed with rat pups exposed to 3 % sevoflurane for 6 h with or without pre-treatment with FTY720 injection. FTY720, at nanomolar concentration, significantly prevents sevoflurane-induced neuronal apoptosis. SEW2871 showed similar neuroprotective effect to FTY720, whereas VPC23019 abrogated the neuroprotective effect of FTY720 when given together. Western blots results demonstrated that FTY710 significantly preserved the level of phosphorylated ERK1/2, Bcl-2 and Bax. Although anaesthetic treatment did not affect general health and emotional status, sevoflurane-induced cognitive impairment in rat models. Administration of FTY720 at 1 mg/kg significantly attenuated sevoflurane-induced neurocognitive impairment. Although further studies are needed to evaluate the feasibility of clinical usage of FTY720 as neuroprotective agent, the study provides preclinical experimental evidence for the efficacy of FTY720 against sevoflurane-induced developmental neurotoxicity.     

PROTEIN COMPOSITION OF MYELIN OF THE PERIPHERAL NERVOUS SYSTEM

Abstract— Myelin was purified from the peripheral nervous system (PNS) of several species. The protein composition of these preparations was examined by discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Proteins characteristic of all samples include, in order of increasing mobility: a series of high molecular weight proteins, the major peripheral nerve protein (P₀), two uncharacterized proteins, and two basic proteins (P₁ and P₂). Quantitative results, obtained by densitometry of gels stained with Fast Green showed differences in protein distribution, both between species, and from different types of nerves obtained from the same animal. The relative amounts of P₁ and P₂ proteins were the most variable; e.g. myelin from guinea-pig sciatic nerve had little or no P₂ protein, whereas 15 per cent of the myelin protein of beef posterior intradural root was Pz protein. P₀, P₁ and P₂ proteins from rabbit sciatic nerve and P₀ and P₂ proteins from beef dorsal and ventral intradural roots were purified and their amino acid compositions were determined. Our results indicated that the P₁ protein is very similar in size and amino acid composition to the basic protein of central nervous system myelin, whereas the P₀ and P₂ proteins are unique to the PNS.     

Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

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.     

Mapping pathways downstream of sphingosine 1-phosphate subtype 1 by differential chemical perturbation and proteomics

Sphingosine 1-phosphate subtype 1 (S1P(1)) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S1P(1) down-regulation. Discovery of the in vivo active S1P(1)-selective agonist, SEW2871, has broadened our understanding of minimal requirements for S1P(1) function while highlighting differences regarding agonist effect on S1P(1) fate, because SEW2871 does not degrade S1P(1). To further understand the mechanism of agonist-induced S1P(1) down-regulation, we compared signaling and fate of human S1P(1)-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW2871, and S1P. Although all agonists acutely internalized S1P(1) to late endosomal vesicles and activated GTPgammaS(35) binding and pERK to similar maxima, only AFD-R led to significant S1P(1) down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S1P(1). All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S1P and being 3-4 logs more potent than SEW2871. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S1P(1) and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S1P(1) (AFD-R > S1P > SEW2871) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.     

S1P1 receptor localization confers selectivity for Gi-mediated cAMP and contractile responses

Adult mouse ventricular myocytes express S1P(1), S1P(2), and S1P(3) receptors. S1P activates Akt and ERK in adult mouse ventricular myocytes through a pertussis toxin-sensitive (G(i/o)-mediated) pathway. Akt and ERK activation by S1P are reduced approximately 30% in S1P(3) and 60% in S1P(2) receptor knock-out myocytes. With combined S1P(2,3) receptor deletion, activation of Akt is abolished and ERK activation is reduced by nearly 90%. Thus the S1P(1) receptor, while present in S1P(2,3) receptor knock-out myocytes, is unable to mediate Akt or ERK activation. In contrast, S1P induces pertussis toxin-sensitive inhibition of isoproterenol-stimulated cAMP accumulation in both WT and S1P(2,3) receptor knock-out myocytes demonstrating that the S1P(1) receptor can functionally couple to G(i). An S1P(1) receptor selective agonist, SEW2871, also decreased cAMP accumulation but failed to activate ERK or Akt. To determine whether localization of the S1P(1) receptor mediates this signaling specificity, methyl-beta-cyclodextrin (MbetaCD) treatment was used to disrupt caveolae. The S1P(1) receptor was concentrated in caveolar fractions, and associated with caveolin-3 and this localization was disrupted by MbetaCD. S1P-mediated activation of ERK or Akt was not diminished but inhibition of cAMP accumulation by S1P and SEW2871 was abolished by MbetaCD treatment. S1P inhibits the positive inotropic response to isoproterenol and this response is also mediated through the S1P(1) receptor and lost following caveolar disruption. Thus localization of S1P(1) receptors to caveolae is required for the ability of this receptor to inhibit adenylyl cyclase and contractility but compromises receptor coupling to Akt and ERK.     

Changes of inositol phosphates during decapitation-induced lateral bud break of Malus domestica

An increase in phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylglycerol (PG), and phosphatidylinositol (PI) occurred during bud break induced by decapitation. Inositol-1-phosphate [Ins(l)P₁], inositol-1,4-bisphosphate [Ins(1,4)P₂], and inositol-1,4,5-triphosphate [Ins(1,4,5)P₃] were found in apple buds and increased progressively following decapitation. Ins(1)P₁ and Ins(1,4)P₂ peaked 48 h after decapitation and Ins(1,4,5)P₃ peaked 72 h after decapitation during the metabolic transition when buds emerged from dormancy. Ins(1,4)P₂ and Ins(1,4,5)P₃ levels declined there after. The lateral buds on shoots with intact terminals and decapitated shoots treated with indole-3-acetic acid (IAA) in the terminals tip remained dormant and there were no significant changes in phospholipid and inositol phosphate contents.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.     

Activation of Adenosine A2 Receptors Stimulates Phosphoinositide Metabolism in Rat Peripheral Nerve

Abstract: The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [³H]-cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [³H]cytidine monophosphate phosphatidic acid ([³H]-CMP-PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5'- N -ethylcarboxamidoadenosine (NECA), a nonselective analogue, and two different A₂-selective analogues, N ⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and 2- p -(2-carboxyethyl)phenethylamino-NECA (CGS 21680), but not by N ⁶-cyclopentyladenosine, an A₁-selective analogue. The stimulatory action of CGS 21680 was blocked by the A₂-selective adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 1,3-dipropyl-7-methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP-PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A₂ subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine-receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.     

Two Distinct ATP Receptors Activate Calcium Entry and Internal Calcium Release in Bovine Chromaffin Cells

Abstract: ATP, an established neurotransmitter, causes elevation of cytosolic Ca²⁺ and catecholamine secretion when applied to chromaffin cells in the intact adrenal gland. The ATP-induced rise in Ca²⁺ is due both to release from internal stores and to entry across the plasma membrane. The latter source of Ca²⁺ causes secretion; the primary role of Ca²⁺ released from internal stores remains undetermined. In this article, we have studied the nucleotide specificity for activating the two types of Ca²⁺ increases. The agonist potency order for the increase in fluorescence from fura-2-loaded chromaffin cells due to release of Ca²⁺ from internal stores is ATP = UTP > ADP > 2-methylthio-ATP, α,β-methylene ATP, identifying the receptor as a P_2U purinoceptor. The potency order for secretion is 2-methylthio-ATP > ATP > α,β-methylene ATP, ADP, UTP, placing the receptor in the P_2Y subtype. Thus, two distinct receptors are responsible for Ca²⁺ release and secretion. Agonists were more effective in the absence of extracellular Mg²⁺, suggesting that ATP uncomplexed with divalent cations binds preferentially to both receptors. The low response of both receptors to ADP distinguishes them from the ATP receptor on these cells that inhibits voltage-dependent Ca²⁺ current and secretion.     

Ultrastructural Localization of P2 Protein in Actively Myelinating Rat Schwann Cells

Abstract: The myelin specific protein, P₂, was localized immunocytochemically in electron micrographs of 4-day-old rat peripheral nerve by a preembedding technique. P₂ staining was restricted to Schwann cells that had established a one-to-one relationship with an axon. P₂ antiserum produced a diffuse staining throughout the entire cytosol of myelinating Schwann cells. In addition, the cytoplasmic side of Schwann cell plasma membranes and the membranes of cytoplasmic organelles that were exposed to cytosol were stained by P₂ antiserum. This cytoplasmic localization of P₂ protein is similar to that described for soluble or peripheral membrane proteins that are synthesized on free ribosomes. P₂ antiserum stained the cytoplasmic side of Schwann cell membranes that formed single or multiple loose myelin spirals around an axon. In the region of the outer mesaxon, P₂ antiserum stained the major dense line of compact myelin. These results demonstrate that P₂ protein is located on the cytoplasmic side of compact myelin membranes and are consistent with biochemical studies demonstrating P₂ to be a peripheral membrane protein.     

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.     

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X₇ receptors, it is still unclear whether neuronal functions can be attributed to P2X₇ receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X₇ receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non-neuronal cells and allows exposure of 'nude' release-regulating pre-synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X₇ receptors. Together, our findings suggest that (i) P2X₇ receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X₇ receptors are localized on glutamatergic nerve terminals; (iii) P2X₇ receptors play a significant role in ATP-evoked glutamate efflux, which involves Ca²⁺-dependent vesicular release; and (iv) the P2X₇ receptor itself constitutes a significant Ca²⁺-independent mode of exit for glutamate.