The catabolism of phosphatidylinisitol by an EDTA-insensitive phospholipase A1 and calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. A rat brain supernatant and microsomal fraction contained a phospholipase A1 enzyme which hydrolysed phosphatidylinositol at pH 8 in the absence of calcium. Triolein and phosphatidylcholine were not attacked under the same incubation conditions. 2. No evidence could be obtained for a phospholipase A2 in the microsomal preparation, and in the presence of Ca2+ the release of fatty acid observed was due to phosphatidylinositol phosphodiesterase followed by diacylglycerol lipase action. 3. Brain phosphatidylinositol phosphodiesterase showed extensive activity in the alkaline range (7-8.5) as well as at pH 5-5.5. The activity at higher pH values required higher calcium concentrations and disappeared on purification of the soluble enzyme by ammonium sulphate fractionation. 4. In general the ratio between inositol 1,2-(cyclic)phosphate and inositol 1-phosphate produced by phosphodiesterase action decreased with increasing pH.     

Fatty acid stimulation of membrane phosphatidylinositol hydrolysis by brain phosphatidylinositol phosphodiesterase.

The hydrolysis of membrane-bound phosphatidylinositol in rat liver microsomal fraction by the soluble phosphatidylinositol phosphodiesterase from rat brain was markedly stimulated by oleic acid or arachidonic acid. The stimulation did not require added calcium, although it was abolished by EDTA. Lysophosphatidylcholine also totally suppressed the stimulation. A possible role for the fatty acid content of a membrane in controlling phosphatidylinositol turnover is suggested.     

The association between phosphatidylinositol phosphodiesterase activity and a specific subunit of microtubular protein in rat brain

1. Supernatant proteins from rat brain were separated into two fractions containing phosphatidylinositol phosphodiesterase activity by chromatography on DEAE-Sephadex A-50. 2. The first fraction sediments in linear sucrose density gradients in two bands corresponding to molecular weights of 66000 and 36000. There was presumptive evidence that the lighter protein constituted the monomeric form of the enzyme. The second fraction sediments predominantly as a single protein of molecular weight 86000. 3. Treatment of rat brain supernatant with [(3)H]colchicine abolished the second DEAE-Sephadex peak and removed the lighter protein from the first peak. These proteins emerged in the same position as the protein binding [(3)H]colchicine at high salt concentration; phospholipase activity was recovered from linear sucrose density gradients in positions corresponding to molecular weights 88000 and 43000, together with an aggregate of molecular weight 140000. Electrophoresis on sodium dodecyl sulphate-urea-polyacrylamide gels of this fraction revealed only three proteins: the alpha and beta-subunits of microtubular protein, of molecular weights 56000 and 52000 respectively, and a protein of molecular weight 38000. 4. A sample of microtubular protein from mouse, labelled in vivo with [(3)H]proline and (32)P(i), was added to rat brain supernatant together with an equal amount of the same microtubular protein treated with cyclic AMP and [gamma-(32)P]ATP and the mixture subsequently characterized by ion-exchange chromatography. Some phospholipase activity characteristic of the second peak from DEAE-Sephadex was associated with one fraction of added microtubular protein. This fraction was identified on the basis of the (3)H:(32)P ratio as the beta subunit of the protein treated with ATP and cyclic AMP. The subunit of added microtubular protein untreated with nucleotides was not associated with phospholipase activity.     

The phosphatidylinositol kinase of rat brain

1. The presence of a phosphatidylinositol kinase in homogenates of adult rat brain was shown by using labelled ATP or labelled phosphatidylinositol. 2. The kinase was activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+), Cu(2+), K(+), Na(+) and F(-). 3. The detergents sodium deoxycholate, Cutscum and Triton X-100 markedly stimulated the reaction; sodium taurocholate, Tween-20 and cetyltrimethyl-ammonium bromide were less effective. 4. The activity of the enzyme was dependent on SH groups. 5. The subcellular distribution of the kinase in brain resembled that of Na(+)-plus-K(+)-stimulated adenosine triphosphatase and 5'-nucleotidase.     

Heterogeneity of immunoreactive prolactin in the rat brain

Three immunoreactive prolactin proteins (24 Kd, 16 Kd, and 12 Kd) were identified in the rat brain using sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blot analyses. In male and female brains, the primary prolactin protein has a molecular weight of 24 Kd which is similar to that of pituitary prolactin. Two additional proteins with apparent molecular weights of 16 Kd and 12 Kd were also identified and were found in greater concentrations in the brain than in the pituitary, and were more predominant in the female brain. In addition, brain extracts proteolytically modify the 24K dalton PRL resulting in the formation of two fragments with apparent molecular weights of 16 and 8 Kd. These data indicate that the prolactin identified in the rat brain is similar to pituitary prolactin, and suggests, that like other PRL target tissues the brain may have the capacity to proteolytically modify prolactin.     

Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase

Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP: phosphatidate cytidylyltransferase and CDPdiacylglycerol: inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7–10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP: phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP:phosphatidate cytidylyltransferase and CDPdiacylglycerol:inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7-10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP:phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

The hydrolysis of phosphatidylinositol monolayers at an air/water interface by the calcium-ion-dependent phosphatidylinositol phosphodiesterase of pig brain.

1. The activity of Ca2+-dependent phosphatidylinositol phosphodiesterase (EC 3.1.4.10) of pig brain against [32P]phosphatidylinositol monolayers at an air/water interface has been measured. As the monolayer pressure was increased a sharp cut-off of enzymic hydrolysis occurred at 33 X 10(-3) N/m. 2. The addition of either phosphatidic acid, phosphatidylglycerol or oleyl alcohol increased the film pressure at which cut off occurred, as well as increasing the rate of hydrolysis at lower pressures. 3. The rate of hydrolysis, but not the cut-off pressure, was markedly increased by oleic acid and slightly increased by phosphatidylethanolamine. 4. Phosphatidylcholine, palmitoylcholine and octadecylamine decreased the cut-off pressure, as well as the enzymic activity below this pressure. 5. Stearic acid and stearyl alcohol had no effect on either the cut-off pressure or the activity. 6. All activators decreased the length of the lag phase before enzyme activity began, and phosphatidylcholine increased it. 7. These results are compared with the stimulatory and inhibitory effects of various amphiphiles observed previously with phosphatidylinositol dispersions [Irvine, Hemington & Dawson (1979) Eur. J. Biochem. 99, 525-530], and their possible relevance to the control of the phosphatidylinositol phosphodiesterase in vivo are discussed.     

Soluble phosphatidylinositol phosphodiesterase in normal and denervated fast and slow muscles of the rat.

Phosphatidylinositol phosphodiesterase activity was determined in cytosol prepared from rat slow (soleus) and fast (extensor digitorum longus) muscles. The substrate was prepared by incubation of sarcoplasmic reticulum with myo-[2-3H]inositol. The enzyme hydrolysed both membrane-bound and extracted phosphatidylinositol. The activity determined with the isolated phospholipid exhibited an optimum at pH 5.5. Ca2+ ions stimulated the activity. The enzyme specific activity was higher in cytosol prepared from soleus muscle than in that from extensor digitorum longus muscle. After section of the motor nerve, the activity of the enzyme increased in both muscles up to 36 h and then declined. A function for this enzyme in the control of acetylcholine sensitivity in muscle is discussed.     

A family of calcium-dependent potassium channels from rat brain

By incorporating rat brain plasma membrane vesicles into planar lipid bilayers, we have found and characterized four types of Ca2(+)-activated K+ channels. The unitary conductances of these channels are 242 +/- 14 pS, 236 +/- 16 pS, 135 +/- 10 pS, and 76 +/- 6 pS in symmetrical 150 mM KCI buffers. These channels share a number of properties. They are all activated by depolarizing voltages, activated by micromolar concentrations of internal Ca2+ with a Hill coefficient for Ca2+ activation of between 2 and 3, noninactivating under our assay conditions, blocked by low millimolar concentrations of TEA from the outside, apamin-insensitive, and very selective for K+ over Na+ and Cl-. Three of the four channels are also blocked by nanomolar concentrations of charybdotoxin. One of the high conductance Ca2(+)-activated K+ channels is novel in that it is not blocked by charybdotoxin and exhibits gating kinetics highlighted by long closed times and long open times. This family of closely related Ca2(+)-activated K+ channels may share structural domains underlying particular functions.     

Bombesin-like peptides stimulate phosphatidylinositol turnover in rat brain slices

The ability of bombesin (BN)-like peptides to stimulate phosphatidylinositol turnover in rat brain slices was investigated. BN (1 μM) significantly stimulated inositol-1-phosphate (IP₁) but not inositol-4,5-biphosphate (IP₂) or inositol-1,4,5-trisphosphate (IP₃) production using frontal cortex slices in the presence of LiCl (7.5 mM); BN had no effect on cAMP or cGMP levels. BN and the structurally-related gastrin releasing peptide (GRP) elevated IP₁ levels in a dose-dependent manner. Similarly, nanomolar concentrations of the GRP fragment (Ac-GRP^20–27) significantly elevated IP₁ levels, whereas micromolar concentrations of the inactive GRP^1–16 did not. BN significantly elevated IP₁ levels in those brain regions enriched in BN receptors such as the olfactory bulb, hippocampus, striatum, thalamus and frontal cortex, whereas IP₁ levels were not significantly increased in areas which have a low density of BN receptors such as the cerebellum, medulla/pons and midbrain. These data suggest that CNS BN receptors may utilize phosphatidylinositol as a second messenger.     

Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.     

An unusual phosphodiesterase activity towardsP-nitrophenylphosphorylcholine present in rat brain membranes

A phosphodiesterase activity present in rat brain membranes has been examined utilizing p-nitrophenylphosphorylcholine as the substrate. This enzyme activity has a pH optimum of 8.5, is stimulated by a variety of free fatty acids, requires either Zn⁺² or Ca⁺² and is relatively stable to heating at 75°C for 7.5 minutes. These properties appear to distinguish this particular activity from those previously reported for alkaline phosphatase, nonspecific phosphodiesterase, phosphodiesterases I and II, lecithinase, and sphingomyelinase.     

Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotide phosphodiesterase and calcium-dependent regulator in porcine brain.

Abstract— Cyclic nucleotide phosphodiesterase activity (calcium-dependent and calcium-independent) and CDR (calcium-dependent regulator protein of phosphodiesterase) are present in all ten brain regions examined, with the specific activity of both being highest in areas predominating in grey matter and lowest in areas consisting largely of white matter. Fractionation of cerebral cortex into neuronal and glial perikarya shows that cyclic nucleotide phosphodiesterase and CDR are present at approximately equal levels in both cell types. Subcellular fractionation reveals that calcium-sensitive enzyme specific activity, as well as CDR are highly localized in the 100,000 g supernatant fraction. Regional, cellular and subcellular distribution studies indicate that the distribution of CDR closely parallels the distribution of enzyme specific activity, suggesting that the levels of cyclic nucleotide phosphodiesterase and CDR may be synchronized.