Rapid accumulation and sustained turnover of inositol phosphates in cerebral-cortex slices after muscarinic-receptor stimulation.

The rapid kinetics of [3H]inositol phosphate accumulation and turnover were examined in rat cerebral-cortex slices after muscarinic-receptor stimulation. Markedly increased [3H]inositol polyphosphate concentrations were observed to precede significant stimulated accumulation of [3H]inositol monophosphate. New steady-state accumulations of several 3H-labelled products were achieved after 5-10 min of continued agonist stimulation, but were rapidly and effectively reversed by subsequent receptor blockade. The results show that muscarinic-receptor activation involves phosphoinositidase C-catalysed hydrolysis initially of polyphosphoinositides rather than of phosphatidylinositol. Furthermore, prolonged carbachol stimulation is shown not to cause receptor desensitization, but to allow persistent hydrolysis of [3H]phosphatidylinositol bisphosphate and permit sustained metabolic flux through the inositol tris-/tetrakis-phosphate pathway.     

Differential effects of lithium on agonist-stimulated inositol polyphosphate formation in rat cerebral cortex slices: Selective actions on muscarinic cholinergic responses

Recent data indicate that BMY 7378 demonstrates high affinity, selectivity and low intrinsic activity at hippocampal 5-HT_1A receptors, suggesting that BMY 7378 may represent the first selective 5-HT_1A functional antagonist. The present study examined the agonist and antagonist properties of BMY 7378 at spinal cord 5-HT_1A receptors. In electrophysiological studies, iontophoretic administration of either the 5-HT_1A agonist 8-OH-DPAT (43.8 ± 5.4 nA) or BMY 7378 (46.3 ± 5.2 nA) significantly inhibited the firing rate of wide-dynamic-range dorsal horn units indicating that BMY 7378 demonstrates significant intrinsic activity at spinal cord 5-HT_1A receptors. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 ejection current (20–100 nA) which antagonized the 8-OH-DPAT induced inhibition of dorsal horn unit activity. In behavioral studies in the spinal rat, 8-OH-DPAT increased the animals' sensitivity to noxious levels of mechanical stimulation (ED₅₀ = 269 ± 24 nmol/kg) as did BMY 7378 (ED₅₀ = 295 ± 70 nmol/kg) with no statistically significant difference in the maximal response (Y_max) observed. Concomitant BMY 7378 and 8-OH-DPAT administration identified no BMY 7378 dose (10–1100 nmol/kg) which blocked the hyperalgesic effect of 8-OH-DPAT, rather, each drug produced similar effects which were additive. Further, the 5-HT_1A agonist effects of BMY 7378 were blocked by the 5-HT_1A antagonist, spiperone. Therefore, both the electrophysiologic and reflex data indicate that BMY 7378 possesses significant intrinsic activity at spinal cord 5-HT_1A receptors, and like 8-OH-DPAT is a partial agonist at these receptors. Differences in BMY 7378 intrinsic activity at spinal cord as opposed to hippocampal 5-HT_1A receptors are discussed in terms of regional differences in G-proteins coupled to 5-HT_1A receptors in these two CNS regions.     

Reduced inositol polyphosphate accumulation and inositol supply induced by lithium in stimulated cerebral cortex slices.

The ability of lithium to interfere with phosphoinositide metabolism in rat cerebral cortex slices has been examined by monitoring the accumulation of CMP-phosphatidate (CMP-PtdOH) and the reduction in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 levels. A small accumulation of [14C]CMP-PtdOH was seen in slices prelabelled with [14C]cytidine and stimulated with carbachol (1 mM) or Li+ (1 mM). However, simultaneous addition of both agents for 30 min produced a 22-fold accumulation, with Li+ producing a half-maximal effect at a concentration of 0.61 +/- 0.19 mM. Kinetic studies revealed that the effects of carbachol and Li+ on CMP-PtdOH accumulation occurred with no initial lag apparent under these conditions and that preincubation with myo-inositol (10 or 30 mM) dramatically attenuated CMP-PtdOH accumulation. myo-Inositol could also attenuate the rate of accumulation of CMP-PtdOH when added 20 min after carbachol and Li+; these effects were not observed when equimolar concentrations of scyllo-inositol were added. Use of specific radioreceptor assays allowed the mass accumulations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 to be monitored. Following a lag of 5-10 min, Li+ resulted in a marked reduction in the accumulation of both inositol polyphosphates resulting from muscarinic-cholinergic stimulation. Preincubation of cerebral cortex slices with myo- (but not scyllo-) inositol delayed, but did not prevent, the reduction in the accumulation of Ins(1,4,5)P3 or Ins(1,3,4,5)P4. The results suggest that cerebral cortex, at least in vitro, is very sensitive to myo-inositol depletion under conditions of muscarinic receptor stimulation. The relationship of such depletion to the generation of inositol polyphosphate second messengers is discussed.     

The regulation of the phosphorylation of inositol 1,3,4-trisphosphate in cell-free preparations and its relevance to the formation of inositol 1,3,4,6-tetrakisphosphate in agonist-stimulated rat parotid acinar cells

High performance liquid chromatography analysis of supernatants from acid-quenched [3H]inositol-labeled parotid acinar cells revealed an inositol pentakisphosphate and three inositol tetrakisphosphates. Two of the latter were identified as the 1,3,4,5 and 1,3,4,6 isomers, whereas the third was probably a mixture of unknown proportions of the 3,4,5,6/1,4,5,6 enantiomeric pair. Methacholine (100 microM) produced a 40-50-fold increase in the levels of inositol trisphosphate (mainly the 1,3,4 isomer) and inositol 1,3,4,5-tetrakisphosphate, but inositol 1,3,4,6-tetrakisphosphate only increased 5-fold. Levels of inositol 3,4,5,6/1,4,5,6-tetrakisphosphate and inositol pentakisphosphate were unaffected by agonist stimulation. Thus, in parotid cells, an agonist-induced increase in both inositol trisphosphate and inositol 1,3,4,6-tetrakisphosphate formation does not result in an increase in the rate of formation of inositol pentakisphosphate. Following the addition of 100 microM atropine to methacholine-stimulated parotid cells, the levels of [3H]inositol 1,3,4,5-tetrakisphosphate fell rapidly, returning to basal levels within 5 min. Inositol trisphosphate was metabolized more slowly and was still elevated 20-fold above basal 5 min after the addition of atropine. Inositol 1,3,4,6-tetrakisphosphate was metabolized much more slowly (t1/2 approximately 15 min). Inositol 1,3,4-trisphosphate metabolism was examined in parotid homogenates as well as in 100,000 x g cytosolic and particulate fractions. Inositol 1,3,4-trisphosphate was both dephosphorylated and phosphorylated. Two inositol tetrakisphosphate products were formed, namely the 1,3,4,6 and 1,3,4,5 isomers. Over 90% of both kinase and phosphatase activities were found in the cytosolic fractions. The ratio of activities of kinase to phosphatase decreased as the levels of inositol 1,3,4-trisphosphate substrate were increased from 1 nM to 10 microM. These data led to the conclusion that the kinetic parameters of the inositol 1,3,4-trisphosphate kinases and phosphatases are such that in stimulated cells, dephosphorylation of inositol 1,3,4-trisphosphate is greatly favored. Inositol 1,3,4-trisphosphate kinase activity was potently inhibited by inositol 3,4,5,6-tetrakisphosphate (IC50 = 0.1-0.2 microM), which leads us to propose that inositol 3,4,5,6-tetrakisphosphate is an endogenous inhibitor of the kinase.     

Conversion of glutamate into aspartate in guinea-pig cerebral-cortex slices

1. When guinea-pig cerebral-cortex slices were incubated with [U-(14)C]glutamate as substrate, the specific radioactivities of the citric acid-cycle intermediates were lower than that of the aspartate isolated from the same vessels. 2. Aspartate was significantly labelled when [5-(14)C]glutamate was used as substrate and the aspartate contained almost no label when [1-(14)C]glutamate was present as substrate. 3. When specifically labelled glutamate was used as substrate, the label was found in the isolated aspartate in the position that would be predicted by citric acid-cycle mechanisms. 4. The results are consistent with the theory of ;compartmentation' of amino acid metabolism.     

Stimulation by acetylcholine of phosphatidylinositol labelling. Subcellular distribution in rat cerebral-cortex slices

1. Rat cerebral-cortex slices were incubated with ³²P_i, acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.     

Mechanism of conversion of aspartate into glutamate in cerebral-cortex slices

1. When slices of guinea-pig cerebral-cortex slices are incubated with [U-¹⁴C]-aspartate and non-radioactive glucose as substrates, the specific radioactivities of the citric acid-cycle intermediates are lower than that of the glutamate isolated from the same vessels. 2. Glutamate was significantly labelled when [1-¹⁴C]-aspartate and glucose were present in the incubation medium. These results would not be expected on the basis of simple conversion of aspartate into glutamate through the citric acid cycle, since the C-1 position of oxaloacetate is decarboxylated in the conversion of isocitrate into α-oxoglutarate. 3. It appears that aspartate is converted into glutamate by citric acid-cycle mechanisms; however, the carbon `skeleton' is not immediately condensed with acetyl-CoA to form citrate but first follows the cycle in a reverse direction to fumarate or succinate and then proceeds in the forward direction. 4. The conversion of aspartate into glutamate appears to be compartmentalized.     

Comparison of inositol phosphates accumulation induced by different effectors in rat thyroid slices.

Rat thyroid slices were submitted to different effectors and hormones in order to investigate their action on the phosphatidylinositol metabolism. Fluoride and vanadate induced a clear increase of the inositol phosphates with half maximal stimulation at 7 mM and 8 mM respectively. The inositol bisphosphate (IP2) and inositol trisphosphate (IP3) accumulation induced by vanadate was relatively higher than that observed in the case of fluoride stimulation. Carbachol stimulated also the generation of inositol phosphates with half maximal activation at 2.5 x 10(-6) M. In the same conditions, no significant effect on inositol phosphates production could be detected by the action of TSH or TRH.     

Molecular definition of a novel inositol polyphosphate metabolic pathway initiated by inositol 1,4,5-trisphosphate 3-kinase activity in Saccharomyces cerevisiae

The production of inositol polyphosphate (IPs) and pyrophosphates (PP-IPs) from inositol 1,4,5-trisphosphate (I(1,4,5)P3) requires the 6-/3-/5-kinase activity of Ipk2 (also known as Arg82 and inositol polyphosphate multikinase). Here, we probed the distinct roles for I(1,4,5)P3 6- versus 3-kinase activities in IP metabolism and cellular functions reported for Ipk2. Expression of either I(1,4,5)P3 6- or 3-kinase activity rescued growth of ipk2-deficient yeast at high temperatures, whereas only 6-kinase activity enabled growth on ornithine as the sole nitrogen source. Analysis of IP metabolism revealed that the 3-kinase initiated the synthesis of novel pathway consisting of over eleven IPs and PP-IPs. This pathway was present in wild-type and ipk2 null cells, albeit at low levels as compared with inositol hexakisphosphate synthesis. The primary route of synthesis was: I(1,4,5)P3 --> I(1,3,4,5)P4 --> I(1,2,3,4,5)P5 --> PP-IP4 --> PP2-IP3 and required Kcs1 (or possibly Ipk2), Ipk1, a novel inositol pyrophosphate synthase, and then Kcs1 again, respectively. Mutation of kcs1 ablated this pathway in ipk2 null cells and overexpression of Kcs1 in ipk2 mutant cells phenocopied IP3K expression, confirming it harbors a novel 3-kinase activity. Our work provides a revised genetic map of IP metabolism in yeast and evidence for dosage compensation between IPs and PP-IPs downstream of I(1,4,5)P3 in the regulation of nucleocytoplasmic processes.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Movements of radioactive sodium in cerebral-cortex slices in response to electrical stimulation

1. Sodium exchange was measured with ²⁴Na in incubated guinea-pig cerebral-cortex slices maintained under adequate metabolic conditions with a steady content of fluid and ions resembling that of brain in vivo. 2. Evidence was obtained indicating that Na⁺ ions behaved in the inulin space as if they were extracellular, and that their entry into the non-inulin space of unstimulated tissue was about 10 times slower and could be separated, on the basis of complete exchangeability, into two components, a `fast' one, which reacted to electrical stimulation, and a `slow' one, exchanging at a rate of about 8μequiv./g./hr., which was not affected by stimulation. 3. The average rate of sodium turnover in unstimulated slices was 175–275μequiv./g./hr., whereas that for stimulated slices was approx. 4–6 times this, or 1050–1180μequiv./g./hr. The stimulated rate was equivalent to a turnover of 32% of the sodium in the non-inulin space/min., or 3mμequiv./g./impulse. 4. Response to the onset of stimulation appeared to be immediate, but after cessation of stimulation increased sodium movements persisted for several minutes before return to unstimulated values. 5. Calculations based on electrochemical gradients suggested that about one-quarter of the energy available from respiration was required for sodium and potassium transport at maximal rates in both unstimulated and stimulated cerebral-cortex slices.     

Homologous desensitization of substance-P-induced inositol polyphosphate formation in rat parotid acinar cells.

Maximal concentrations of substance P and methacholine induced a rapid increase in [3H]inositol trisphosphate ([3H]IP3) formation. After about 1 min, the [3H]IP3 in the substance-P-treated cells ceased to increase further, whereas in the methacholine-treated cells [3H]IP3 continued to increase. Addition of methacholine to the substance-P-treated cells caused a rapid increase in [3H]IP3, whereas a second addition of a 10-fold excess of substance P had no effect. Pretreatment of cells with substance P, followed by removal of the substance P by washing, resulted in a decreased response to a second application of substance P. A similar protocol involving pretreatment with methacholine had no effect on subsequent responsiveness to substance P. Analysis of [3H]substance P binding to substance-P-treated cells indicated that the number of receptors for substance P was decreased, but the affinity of the receptors for substance P was unaffected. After substance P pretreatment, a prolonged incubation (2 h) restored responsiveness of the cells to substance P, measured as [3H]IP3 formation, and restored the number of binding sites to control values. These findings indicate that, in the rat parotid gland, substance P induces a homologous desensitization of its receptor, which involves a slowly reversible down-regulation or sequestration of substance-P-binding sites.     

Characterization of agonist-stimulated incorporation of myo-[3H]inositol into inositol phospholipids and [3H]inositol phosphate formation in tracheal smooth muscle.

The effects of the muscarinic agonist carbachol, histamine and bradykinin on incorporation of [3H]inositol into the phosphoinositides and the formation of [3H]InsPs were examined in bovine tracheal smooth-muscle (BTSM) slices labelled with [3H]inositol. These agonists result in substantial and dose-related increases in the incorporation of [3H]inositol into the phospholipids. Carbachol and histamine stimulated the incorporation of [3H]inositol into the phospholipids to the same degree, despite histamine being only 35% as effective as carbachol on [3H]InsP accumulation. Histamine and carbachol, at maximal concentrations, were non-additive with respect to both the stimulated incorporation of [3H]inositol and [3H]InsP formation. For carbachol this effect on incorporation was found to occur to a similar extent in PtdInsP and PtdInsP2 as well as PtdIns. The initial effect of carbachol on [3H]inositol incorporation was rapid (maximal by 10 min); however, with prolonged stimulation large secondary declines in PtdInsP and PtdInsP2 labelling were observed, with depletion of the much larger PtdIns pool only evident in the presence of Li+. Lowering buffer [Ca2+] increased the incorporation of [3H]inositol under basal conditions, but did not attenuate the subsequent agonist-stimulated incorporation effect. The large changes in specific radioactivity of the phosphoinositides, and consequently the [3H]InsP products, after carbachol stimulation resulted in the apparent failure of atropine to reverse the [3H]InsP response completely. Labelling muscle slices with [3H]inositol in the presence of carbachol or labelling for longer periods (greater than 6 h) prevented subsequent carbachol-stimulated effects on incorporation without significantly altering the dose-response relationship for carbachol-stimulated [3H]InsP formation and resulted in steady-state labelling conditions confirmed by the ability of atropine to reverse fully the [3H]InsP response to carbachol. This study demonstrates the profound effects of a number of agonists on [3H]inositol incorporation into the phospho- and polyphosphoinositides in BTSM with important consequent changes in the specific radioactivity of these lipids and the resulting [3H]InsP products. In addition, a selective depletion of PtdInsP and PtdInsP2 over PtdIns has been demonstrated with prolonged muscarinic-receptor stimulation.     

Hydrolysis of chyle cholesterol esters with cell-free preparations of rat liver.

1. The effect of pH on the hydrolysis of chylomicron and chylomicron remnant cholesterol ester with rat liver homogenate was examined. The hydrolysis had three pH optima, at pH 4.5, at pH 6.0-6.5 and at pH 8.5. At the two upper pH optima extensive cholesterol ester hydrolysis occurred without simultaneous degradation of the triacylglycerol portion. 2. Similarly, microsomes (at pH 6.5-8.0) and 100 000 X g supernatant (at pH 7.5-8.5) efficiently hydrolyzed the cholesterol ester but not the triacylglycerol of chylomicron remnants. 3. With the same substrate no enrichment of neutral cholesterol esterase activity was seen in isolated plasma membranes. 4. At pH 4.5 lysosomes efficiently hydrolyzed both the cholesterol ester and the triacylglycerol portion of chylomicron remnants. 5. Three conclusions are drawn: (a) the study provides evidence against the existence of a plasma membrane-bound enzyme-hydrolyzing chylomicron cholesterol ester before or during its penetration into the cell; (b) enzymes of the cell sap and possibly of the endoplasmic reticulum can degrade cholesterol ester of chylomicron remnants without preceeding hydrolysis of the triacylglycerol core; and (c) lysosomal enzymes can degrade both the cholesterol ester and the triacylglycerol portion of chylomicron remnants if these are taken up as whole particles by endocytosis.     

Automated isocratic high-performance liquid chromatography of inositol phosphate isomers.

Isomers of inositol phosphates from biological samples can be analysed by anion-exchange h.p.l.c., by using isocratic elution with phosphate buffers. The method involves the preliminary processing of the extracted samples with conventional soft-gel anion-exchange resins, including the commonly used Dowex resins, followed by direct analysis with h.p.l.c. of a portion of relevant fractions. Run times (up to 20 min) and collected fraction numbers (up to 24) are minimal, so that if the method is used in conjunction with automated h.p.l.c. injection a high throughput of samples is achieved.     

Structural and functional characterization of an inositol polyphosphate receptor from cerebellum.

An inositol polyphosphate receptor has been purified from bovine cerebellum which consists of three different polypeptides with Mr of 111,000, 102,000, and 52,000. Negative staining electron microscopy reveals globular-like structures 10-13 nm in diameter. The receptor has a Stokes radius of 400,000 daltons as determined by molecular sieve high performance liquid chromatography. The receptor preparation binds inositol 1,3,4,5-tetrakisphosphate, inositol hexaphosphate (or phytol), and inositol 1,4,5-trisphosphate (IP4, IP6, and IP3, respectively) with submicromolar affinity (0.19, 0.15, and 0.54 microM, respectively) at conditions approximating physiological ionic strength and pH. The purified receptor preparation, when reconstituted into planar bilayers, displays ion channel activity, preferentially permeable to K+. Permeability ratios of the channel are PK+/PNa+ approximately 5 and PK+/PCl approximately 19. In symmetrical 100 mM KCl, the channel is characterized by long open times (minutes) with a conductance of 7.2 picosiemens. The channel is selectively modulated by IP4. That is, at 1 microM IP4, the mean open time decreased substantially to rapid flicker behavior and the channel is completely closed at 10 microM IP4. IP6 and IP3 did not modulate the channel under similar conditions. Thus, the channel appears to be an IP4-modulated K+ channel.     

Underexpression of the 43 kDa inositol polyphosphate 5-phosphatase is associated with cellular transformation.

The 43 kDa inositol polyphosphate 5-phosphatase (5-phosphatase) hydrolyses the second messenger molecules inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. We have underexpressed the 43 kDa 5-phosphatase by stably transfecting normal rat kidney cells with the cDNA encoding the enzyme, cloned in the antisense orientation into the tetracycline-inducible expression vector pUHD10-3. Antisense-transfected cells demonstrated a 45% reduction in Ins(1,4,5)P3 5-phosphatase activity in the total cell homogenate upon withdrawal of tetracycline, and an approximately 80% reduction in the detergent-soluble membrane fraction of the cell, as compared with antisense-transfected cells in the presence of tetracycline. Unstimulated antisense-transfected cells showed a concomitant 2-fold increase in Ins(1,4,5)P3 and 4-fold increase in Ins(1,3,4,5)P4 levels. The basal intracellular calcium concentration of antisense-transfected cells (170 +/- 25 nM) was increased 1.9-fold, compared with cells transfected with vector alone (90 +/- 25 nM). Cells underexpressing the 43 kDa 5-phosphatase demonstrated a transformed phenotype. Antisense-transfected cells grew at a 1.7-fold faster rate, reached confluence at higher density and demonstrated increased [3H]thymidine incorporation compared with cells transfected with vector alone. Furthermore, antisense-transfected cells formed colonies in soft agar and tumours in nude mice. These studies support the contention that a decrease in Ins(1,4,5)P3 5-phosphatase activity is associated with cellular transformation.     

Identification of the inositol isomers present in Tetrahymena

The inositol isomer composition of phosphoinositides, polyphosphoinositols, phosphatidylinositol-linked glycans, and glycosyl phosphatidylinositol-anchored proteins of logarithmic phase Tetrahymena vorax was determined by GC-MS analysis of trimethylsilylimadazole derivatives. The most abundant inositol found was the myo-isomer; however, appreciable percentages of scylloinositol were present in the free inositol pool, phosphatidylinositol-linked glycan fraction, and glycosyl phosphatidylinositol-anchored protein fraction. Trace quantities of chiro- and neo-inositols also were present.