Inositol incorporation into phosphoinositides in retinal horizontal cells of Xenopus laevis: enhancement by acetylcholine, inhibition by glycine

The absorption of light by photoreceptor cells leads to an increased incorporation of [2-3H]inositol into phosphoinositides of horizontal cells in the retina of Xenopus laevis in vitro. We have identified several retinal neurotransmitters that are involved in regulating this response. Incubation with glycine, the neurotransmitter of an interplexiform cell that has direct synaptic input onto horizontal cells, abolishes the light effect. This inhibition is reversed by preincubation with strychnine. Acetylcholine added to the culture medium enhances the incorporation of [2-3H]inositol into phosphoinositides in horizontal cells when retinas are incubated in the dark. This effect is inhibited by preincubation with atropine. However, atropine alone does not inhibit the light-enhanced incorporation of [2-3H]inositol into phosphoinositides in the retina. gamma-Aminobutyric acid, the neurotransmitter of retinal horizontal cells in X. laevis, as well as dopamine and norepinephrine, have no effect on the incorporation of [2-3H]inositol into phosphoinositides. These studies demonstrate that the light-enhanced incorporation of [2-3H]inositol into phosphoinositides of retinal horizontal cells is regulated by specific neurotransmitters, and that there are probably several synaptic inputs into horizontal cells which control this process.     

Phosphoinositide Metabolism in the Retina: Localization to Horizontal Cells and Regulation by Light and Divalent Cations

Isolated retinas from Xenopus laevis incorporated greater amounts of [3H]inositol and 32Pi into phosphoinositides when incubated in light than did control retinas incubated in the dark. Inositol was primarily incorporated into phosphatidylinositol (83-86%), while phosphate labeled the polyphosphoinositides (72-79%). The incorporation of radioactive glycerol, serine, choline, or ethanolamine into retinal lipids was unaffected by light. Following incubation with [3H]inositol, the cell type involved in the light response was identified by light and electron microscope autoradiography to be the horizontal cell. These results are consistent with a classic phosphatidylinositol effect in the retina. An interesting feature of this response is that the stimulus (light) is received in the photoreceptor cell and the effect is manifest in the horizontal cell.     

Light- and cytidine-dependent phosphatidylinositol synthesis in photoreceptor cells of the rat

Incorporation of [3H]inositol into phosphatidylinositol (Pl) in isolated rat retinas is enhanced by light and by the addition of cytidine to the incubation media. In retinas preincubated with [3H]inositol in dark, [3H]inositol was chased into Pl in light by addition of unlabeled cytidine and was chased out of Pl in light by addition of unlabeled cytidine plus inositol. Autoradiograms of retinas show a heavy density of silver grains over photoreceptor cell inner segments (with chase-in) and a loss of labeling (with chase-out). Exogenous cytidine and inositol were shown to enhance not only the turnover of Pl within photoreceptor cells but the synthesis of Pl as well; in media supplemented with these precursors, approximately 50% of [14C]glycerol and 25% of [32Pi]incorporated into lipid in light were associated with Pl. These results suggest that availability of both cytidine and inositol may play a role in the light-dependent changes in Pl metabolism within photoreceptor cells.     

Cytidine metabolism in photoreceptor cells of the rat

During brief (30-min) incubations, isolated rat retinas accumulated [3H]cytidine, converted it to cytidine triphosphate (CTP), and incorporated it into RNA and cytidine diphosphate-diacylglyceride (CDG), a phospholipid precursor of phosphatidylinositol (Pl). Labeled CTP, RNA, and CDG contents were found to be two- to three-fold higher in photoreceptor cells than in cells of the inner retina. Autoradiograms showed that, within photoreceptor cells, silver grains representing RNA were concentrated over the nuclei in dark and light, while silver grains representing CDG were concentrated over the inner segments only after incubation in dark. The formation of labeled CTP and the synthesis of RNA were enhanced in light, while labeled CDG levels became reduced in light concurrent with an increase in the incorporation of labeled inositol into Pl. The 3H-labeled CDG content, however, was increased two- to fourfold in light in the presence of actinomycin D, and autoradiograms show a heavy concentration of silver grains over the inner segments of photoreceptor cells. These findings establish a role for cytidine nucleotides in photoreceptor cell metabolism and in light-dependent increases in RNA and Pl synthesis. Furthermore, the observations indicate that a competition may exist in light for cytidine or CTP and suggest that availability of cytidine for CDG synthesis may have a regulatory role in Pl metabolism within the photoreceptor cells.     

Effect of Light on the Metabolism of Lipids in the Rat Retina

The effect of light on the in vitro incorporation of a variety of radioactive precursors into glycerolipids was tested in isolated retinas of albino rats. There was an increase in the incorporation of [2-3H]myo-inositol, 32Pi, [2-3H]glycerol, and [methyl-3H]choline into retinal phospholipids in light compared to that in darkness. [2-3H]myo-Inositol was incorporated primarily into phosphatidylinositol. 32Pi was incorporated primarily into the phosphoinositides, although there were significant increases in the specific activities of all retinal phospholipids in light compared to those in darkness. Likewise, [2-3H]glycerol incorporation into all retinal phospholipids and diglycerides was greater in light than in the dark. There was no effect of light on the incorporation of [2-3H]ethanolamine into phosphatidylethanolamine or of [3-3H]serine into phosphatidylserine, although these phospholipids were labeled to a greater extent in light with [2-3H]glycerol. There was no effect of light on the incorporation of [3H]palmitic acid into diglycerides and phospholipids, with the exception of phosphatidylinositol. Light also had no effect on the uptake of [2-3H]glycerol, [2-3H]inositol, or [methyl-3H]choline into the retina. We conclude from these studies that light stimulates the phosphoinositide effect in the rat retina. Although some of the results are consistent with a stimulation of de novo synthesis of all lipid classes, our studies with [3H]palmitate, [2-3H]ethanolamine, and [3-3H]serine do not support this conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Manganese Stimulates the Incorporation of [3H]Inositol into a Pool of Phosphatidylinositol in Brain That Is Not Coupled to Agonist-Induced Hydrolysis

Mn2+ greatly increases the incorporation of myo-[3H]inositol into phosphatidylinositol (PI) of brain and other tissues by stimulating the activity of a PI-myo-inositol exchange enzyme. This study examined the ability of norepinephrine (NE) and carbachol to stimulate the hydrolysis of [3H]PI formed in the absence and presence of Mn2+-stimulated [3H]inositol exchange. Rat cerebral cortical slices were incubated with myo-[3H]inositol for 60 min in an N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) buffer without or with MnCl2 (1 mM). The tissue was washed and further incubated with unlabeled myo-inositol and LiCl (10 mM). Prelabeled slices were then incubated with NE (0.1 mM) or carbachol (1 mM) to induce agonist-stimulated [3H]PI hydrolysis. Mn2+ treatment resulted in eight- and sixfold increases in control levels of [3H]PI and [3H]inositol monophosphate [( 3H]IP), respectively. Both NE and carbachol stimulated [3H]IP formation in tissue prelabeled without or with manganese. However, the degree of stimulation (percentage of control values) was greatly attenuated in the presence of Mn2+. In the absence of Mn2+ treatment, NE decreased [3H]PI radioactivity in the tissue to 80% of control values. However, NE did not decrease [3H]PI radioactivity in the Mn2+-treated tissue. These data demonstrate that Mn2+ stimulates incorporation of myo-[3H]inositol into a pool of PI in brain that has a rapid turnover but is not coupled to agonist-induced hydrolysis.     

Light Exposue Activates Retina Ganglion Cell Lysophosphatidic Acid Acyl Transferase and Phosphatidic Acid Phosphatase by a c-fos-Dependent Mechanism

We previously reported that the biosynthesis of phospholipids in the avian retina is altered by light stimulation, increasing significantly in ganglion cells in light and in photoreceptor cells in dark. In the present work, we have determined that light significantly increases the incorporation of [3H]glycerol into retina ganglion cell glycerophospholipids in vivo by a Fos-dependent mechanism because an oligonucleotide antisense to c-fos mRNA substantially blocked the light-dark differences. We also studied in vitro the enzyme activities of phosphatidate phosphohydrolase (PAPase), lysophosphatidate acyl transferase (AT II), and phosphatidylserine synthase from retinas of chickens exposed to light or dark. Higher PAPase I and AT II activities were found in incubations of retinal ganglion cells from animals exposed to light; no increase was observed in preparations obtained from light-exposed animals treated with the c-fos antisense oligonucleotide. No light-dark differences were found in phosphatidylserine synthase activity. These findings support the idea that a coordinated photic regulation of PAPase I and AT II is taking place in retina ganglion cells. This constitutes a reasonable mechanism to obtain an overall increased synthesis of glycerophospholipids in stimulated cells that is mediated by the expression of Fos-like proteins.     

Retinoic Acid Rapidly Decreases Phosphatidylinositol Turnover During Neuroblastoma Cell Differentiation

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line that can be induced to differentiate along the neuronal pathway by retinoic acid (RA). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H]inositol or [1(3)-3H]glycerol. No changes were observed in both [3H]inositol and [3H]glycerol uptake within 24 h of RA treatment. Decreased incorporation of the metabolic precursor into PI 4-monophosphate and PI 4,5-bisphosphate occurred within 1 h of RA treatment. No changes were seen in the specific radioactivity of the precursor pools up to 1 h of treatment with RA. Analysis of labeled PI metabolites from prelabeled cells indicated a rapid decrease of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol content within 1 min of induction of LAN-1 cell differentiation. These findings constitute the earliest reported events in neuroblastoma cell differentiation.     

Platelet phosphoinositide turnover in streptozotocin-induced diabetes

Increased platelet aggregation and secretion in response to various agonists has been described in both diabetic humans and animals. Alterations in the platelet membrane fatty acid composition of phospholipids and changes in the prostacyclin and thromboxane formation could only partly explain the altered platelet function in diabetes. In the present study, we have examined the role of phosphoinositide turnover in the diabetic platelet function. We report alterations in 2-[³H] myo-inositol uptake, phosphoinositide turnover, inositol phosphate and diacylglycerol (DAG) formation, phosphoinositide mass, and phospholipase C activity in platelets obtained from streptozotocin (STZ)-induced diabetic rats. There was a significant increase in the 2-[³H) myo-inositol uptake in washed platelets from diabetic rats. Basal incorporation of 2-[³H] myo-inositol into phosphatidylinositol 4,5-bisphosphate (PIP₂), phosphatidylinositol 4-phosphate (PIP) or phosphatidylinositol (PI) in platelets obtained from diabetic rats was, however, not affected. Thrombin stimulation of platelets from diabetic rats induced an increase in the hydrolysis of [³²P]PIP₂ but indicated no change in the hydrolysis of [³²P]PIP and [³²P]PI as compared to their basal levels. Thrombin-induced formation of [³H]inositol phosphates was significantly increased in both diabetic as well as in control platelets as compared to their basal levels. This formation of [³H]inositol phosphates in diabetic platelets was greater than controls at all time intervals studied. Similarly, there was an increase in the release of DAG after thrombin stimulation in the diabetic platelets. Based on these results, we conclude that there is an increase in the transport of myoinositol across the diabetic platelet membrane and this feature, along with alterations in the hydrolysis of PIP₂, inositol phosphates and DAG in the diabetic platelets, may play a role in increased phosphoinositide turnover which could explain the altered platelet function in STZ-induced diabetes.     

Optic nerve integrity is required for light to affect retina ganglion cell gangliosides

The labeling of retina ganglion cell and optic tectum gangliosides after an intraocular injection ofN-[³H]acetylmannosamine ([³H]ManNAc) is higher in chickens exposed to light than in those maintained in darkness (1,2). In the present work we studied whether the signal for the higher labeling of ganglion cells in light originates in the photoreceptor layer or comes from the nerve terminal. For this purpose the labeling of ganglion cell gangliosides was determined in light and dark in chickens with one optic nerve severed. The results showed that the effect of light occurred only in the eye normally connected to the optic tectum. In the eye with its optic nerve severed, no difference was observed between the labeling of gangliosides in animals in light and dark, having both groups the labeling values of the normal eyes exposed to light. The results indicate that the information that decreases labeling in darkness or accelerates it in light originates in the nerve terminal.Special Issue dedicated to Prof. Edwardo De Robertis.     

Phospholipase Cγ1 in Bovine Rod Outer Segments: Immunolocalization and Light-Dependent Binding to Membranes

Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ₁, γ₁, and δ₁. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ₁ was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ₁ showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ₁ on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca²⁺]_free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ₁ in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.     

Autoradiographic identification of acetylcholine in the rabbit retina

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.     

Dopaminergic amacrine neurons of rat retinas with photoreceptor degeneration continue to respond to light

Exposure of albino rats to continuous light of low intensity (350–700 lux) for 4 months produces massive degeneration of the photoreceptor segments and cell bodies of the outer nuclear layer of the retina. Only a few heterochromatic, receptor cell nuclei remain, and no photoreceptor segments are present. On the other hand, the inner layers of these retinas remain morphologically intact. The inner nuclear layer of the normal rat retina contains a group of amacrine cells which contain the putative neurotransmitter, dopamine (DA). Short term exposure to light (30 or 60 min) markedly stimulates the rate of DA turnover in these cells in normal, previously dark-adapted rats. Such enhancement of the rate of neurotransmitter turnover in the brain has been correlated with an increase in nerve impulse activity. The present study was undertaken to determine if the dopaminergic amacrine cells of the inner nuclear layer were still responsive to light in the retinas of rats whose photoreceptors were previously destroyed by long term exposure to continuous illumination. One week before sacrifice, the animals which had been housed in continuous light for 4 months were returned to normal 14 hr light: 10 hr dark lighting conditions. At the end of this time they and a group of control rats which had been housed in cyclic lighting conditions for the entire 4 months were dark adapted for approximately 15 hr. Then the rate of retinal DA turnover was estimated from the depletion of DA following inhibition of DA synthesis by α methyl para-tyrosine. The turnover of DA in the dark-adapted retinas of the control rats and of experimental rats with photoreceptor degeneration was dramatically enhanced 2–4 fold by short term exposure (up to 1 hr) to light. Since rats are nocturnal and avoid light, we tested the light aversion of another group of rats which had been exposed to light for 4 months and then returned to cyclic lighting conditions for one week. These rats and control animals which had been maintained in cyclic lighting conditions for 4 months both chose the dark side of a light-dark box over 80% of the time. This behavior of the rats with retinal degeneration was taken as a crude indication of their continued ability to detect light. The light-induced increase in DA activity in retinas with photoreceptor degeneration may play a role in the continued ability of these rats to perceive light.     

The polyphosphoinositides,phosphatidylinositol monophosphate and phosphatidylinositol bisphosphate,are present in nuclei isolated from carrot protoplast

Summary Nuclei were isolated from carrot protoplasts and the distribution of [³H]inositol-labeled phospholipids was analyzed by thinlayer chromatography. Phosphatidylinositol (PI), lysophos-phatidylinositol (LPI), phosphatidylinositol monophosphate (PIP), lysophosphatidylinositol monophosphate (LPIP), and phosphatidylinositol bisphosphate (PIP₂) were 55.7%, 12.3%, 5.0%, 11.5%, and 3.6% of the respective [³H]inositol-labeled lipids recovered from the nuclear fraction. While both the plasma membrane and nuclear fraction contained polyphosphoinositides, the distribution of the phosphoinositides and the amount of inositol-labeled lipid were distinct. For example, the nuclear fraction had a higher percentage of LPI and PIP₂ and less PI and LPIP than the plasma membrane fraction. The amount of [³H]inositol-labeled lipid recovered from the nuclear fraction per mg protein was an order of magnitude lower than that recovered from either the plasma membrane of lower phase fraction isolated by aqueous two-phase partitioning, or from whole cells and protoplasts. In addition, when the ratio of the [³H]inositol-labeled lipid was compared to total [¹⁴C]myristate-labeled lipid recovered there was three to ten fold less [³H] relative to [¹⁴C] in the nuclear fraction.These data indicate that while the polyphosphoinositides are a relatively high percentage of the inositol lipid in the nuclear fraction, the inositol lipid was only a small portion of the total lipid in the nuclei. Despite this low concentration of inositol lipid, when [ ³²P]-ATP was added to the isolated nuclei,³²P-labeled PIP and PIP₂ were synthesized. Thus, the carrot nuclei contained PI and PIP kinase as well as the polyphosphoinositides.Abbreviations PI phosphatidylinositol - LPI lysophosphatidylinositol - PIP phosphatidylinositol monophosphate - LPIP lysophosphatidylinositol monophosphate - PIP₂ phosphatidylinositol bisphosphate - DAG diacylglycerol - IP₃ inositol 1,4,5-trisphosphate     

Agonist-Induced, GTP-Dependent Phosphoinositide Hydrolysis in Postmortem Human Brain Membranes

Abstract: Membranes prepared from postmortem human brain were used to measure the activities of three components of the phosphoinositide second messenger system. [³H] Phosphatidylinositol ([³H] PI) hydrolysis was stimulated by directly activating phospholipase C with calcium, by activating guanine nucleotide-binding proteins (G proteins) with guanosine-5′-O-(3-thiotriphosphate) (GTPγS) or with AIF₄, and by receptors activated with several agonists (in the presence of GTPγS), including (in order of increasing magnitudes of responses) carbachol, pilocarpine, histamine, trans-1-aminocyclopentyl-1, 3-dicarboxylic acid (a selective excitatory amino acid metabotropic receptor agonist), serotonin, and ATP. G_q/11 was identified as the G protein most likely to mediate [³H] PI hydrolysis in human brain membranes based on the findings that this process was not impaired by pretreatment with pertussis toxin and it was inhibited by antibodies specific for the α-subunit of G_q/11 but not by antibodies for G_o or G₁₁. The effects of postmortem delay on [³H] PI hydrolysis were examined by studying tissues obtained 6–21 h postmortem. A slight increase in basal [³H] PI hydrolysis was associated with increased postmortem time, suggesting a slow loss of the normal inhibitory control of phospholipase C. GTPγS- stimulated [³H] PI hydrolysis was unaffected by postmortem times within this range, but carbachol-induced [³H] PI hydrolysis tended to decrease with increasing postmortem times. These results demonstrate that the entire phosphoinositide complex remains functional and experimentally detectable in postmortem human brain membranes. This method provides a means to study the function, regulation, effects of diseases, and responses to drugs of the phosphoinositide system in human brain.     

The effect of ischaemia-reperfusion on [3H]inositol phosphates and Ins(1,4,5)P3 levels in cardiac atria and ventricles — a comparative study

In this study incorporation of [³H]inositol into inositol phosphates and phosphoinositides as well as tissue Ins(1,4,5)P₃ levels of the atria and ventricles of isolated, perfused rat hearts were compared. Although the incorporation of [³H]inositol into the phosphoinositides of atria and ventricles was similar, significantly higher (2–3 fold) incorporation rates into inositol phosphates were observed in atrial tissue. Using a D-myo-[³H]Ins(1,4,5)P₃ assay system, the Ins(1,4,5)P₃ levels observed in atria from perfused rat hearts were also significantly higher than those obtained under the same experimental circumstances in the ventricles.Since previous studies on whole hearts showed inhibition of the phosphatidylinositol (PI) pathway during ischaemia with an immediate significant stimulation upon reperfusion [12, 20], the effects of ischaemia and 1 min postischaemic reperfusion were also examined separately in atria and ventricles. The results showed that 20 min of global ischaemia significantly depressed Ins(1,4,5)P₃ levels as well as incorporation of [3H]inositol into ventricular InSP₂ and InSP₃. Reperfusion caused an immediate (within 1 min) increase in Ins(1,4,5)P₃ levels and also [³H]inositol incorporation into all three cytosolic inositol phosphates in the ventricles. However, the effect of ischaemia and reperfusion on Ins(1,4,5)P₃ levels as well as the incorporation of [³H]inositol into the inositol phosphates were less prominent in the atria. It therefore appears that the differential responses of the atria and the ventricles to an oxygen deficiency [41] are also reflected in the differences in PI metabolism during ischaemia-reperfusion.     

Modulation of carbachol-stimulated inositol phospholipid hydrolysis in rat cerebral cortex

Cortical slices from rat brain were used to study carbachol-stimulated inositol phospholipid hydrolysis. Omission of calcium during incubation of slices with [³H]inositol increased its incorporation into receptor-coupled phospholipids. Carbachol-stimulated hydrolysis of [³H]inositol phospholipids in slices was dose-dependent, was affected by the concentrations of calcium and lithium present and resulted in the accumulation of mostly [³H]inositol-l-phosphate. Incubation of slices withN-ethylmaleimide or a phorbol ester reduced the response to carbachol. Membranes prepared from cortical slices labeled with [³H]inositol retained the receptor-stimulated inositol phospholipid hydrolysis reaction. The basal rate of inositol phospholipid hydrolysis was higher than in slices and addition of carbachol further stimulated the process. Addition of GTP stimulated inositol phospholipid hydrolysis, suggesting the presence of a guanine nucleotide-binding protein coupled to phospholipase C. Carbachol and GTP-stimulated inositol phospholipid hydrolysis in membranes was detectable following a 3 min assay period. In contrast to slices, increased levels of inositol bisphosphate and inositol trisphosphate were detected following incubation of membranes with carbachol. These results demonstrate that agonist-responsive receptors are present in cortical membranes, that the receptors may be coupled to phosphatidylinositol 4,5-bisphosphate, rather than phosphatidylinositol, hydrolysis and that a guanine nucleotide-binding protein may mediate the coupling of receptor activation to inositol phospholipid hydrolysis in brain.     

Stress-induced Synthesis of Phosphatidylinositol 3-Phosphate in Mycobacteria

Phosphoinositides play key roles in regulating membrane dynamics and intracellular signaling in eukaryotic cells. However, comparable lipid-based signaling pathways have not been identified in bacteria. Here we show that Mycobacterium smegmatis and other Actinomycetes bacteria can synthesize the phosphoinositide, phosphatidylinositol 3-phosphate (PI3P). This lipid was transiently labeled with [³H]inositol. Sensitivity of the purified lipid to alkaline phosphatase, headgroup analysis by high-pressure liquid chromatography, and mass spectrometry demonstrated that it had the structure 1,2-[tuberculostearoyl, octadecenoyl]-sn-glycero 3-phosphoinositol 3-phosphate. Synthesis of PI3P was elevated by salt stress but not by exposure to high concentrations of non-ionic solutes. Synthesis of PI3P in a cell-free system was stimulated by the synthesis of CDP-diacylglycerol, a lipid substrate for phosphatidylinositol (PI) biosynthesis, suggesting that efficient cell-free PI3P synthesis is dependent on de novo PI synthesis. In vitro experiments further indicated that the rapid turnover of this lipid was mediated, at least in part, by a vanadate-sensitive phosphatase. This is the first example of de novo synthesis of PI3P in bacteria, and the transient synthesis in response to environmental stimuli suggests that some bacteria may have evolved similar lipid-mediated signaling pathways to those observed in eukaryotic cells.     

The effect of ischaemia and reperfusion on sarcolemmal inositol phospholipid and cytosolic inositol phosphate metabolism in the isolated perfused rat heart

In this study the mass of polyphosphoinositides as well as the turnover of [³H]inositol phospholipids and [³H]inositol phosphates during ischaemia and short periods of reperfusion were studied in the isolated perfused rat heart. Since the phosphoinositides located within the sarcolemma are precursors for release of inositoltrisphosphate (InsP₃) and diacylglycerol, sarcolemmal membranes (rather than whole tissue) isolated at the end of the experimental procedure, were used. Hearts were prelabelled with [³H]inositol and subsequently perfused with 10 mM LiCI to block the phosphatidylinositol (PI) pathway. The results showed that 20 min of global ischaemia depressed the amount of [³H]inositol present in both sarcolemmal phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂), as well as in the cytosolic [³H]inositol phosphates, [³H]InsP₂ and [³H]InsP₃. The mass of the sarcolemmal inositol phospholipids remained unchanged during ischaemia. Reperfusion caused an immediate (within 30 sec) increase in the amount of [³H]inositol in sarcolemmal PI, PI-4-P and PI-4,5-P₂. PI-4-P levels showed a transient increase after 30 seconds postischaemic reperfusion, while the mass of the other sarcolemmal inositol phospholipids, PI and PI-4,5-P₂, remained unchanged. [³H]Insp, [³H]InsP₂ and [³H]InsP₃ also increased significantly in comparison to ischaemic hearts after only 30 sec postischaemic reperfusion.In summary, the results obtained indicate inhibition of the PI pathway during ischaemia with an immediate significant stimulation upon reperfusion. In view of the capacity of InsP3 to mobilize Ca²⁺ the possibility exists that stimulation of this pathway during reperfusion may play a role in the intracellular Ca²⁺ overload, characteristic of postischaemic reperfusion.     

Phosphatidylinositol 4,5-bisphosphate: Light-mediated breakdown in the vertebrate retina

Isolated frog ($\text{Rana}\text{Pipiens}$) retinas were labeled in the dark with either [³²P]PO₄-orthophosphate or $\text{myo}$-[2-³H]inositol for 2.5–4 hrs. After washing the retinas with cold buffer, they were exposed to brief flashes of light (5 secs or 15 secs) and their rod outer segments isolated. Upon separation of labeled phospholipids, a specific decrease in label in phosphatidylinositol 4,5-bisphosphate was observed, whereas there was no significant effect on the labeling of phosphatidylinositol 4-phosphate, phosphatidylinositol, or phosphatidic acid. These results are indicative of a light-activated phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in frog rod outer segments.