Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.     

Effect of Hyperglycemia and Its Prevention by Insulin Treatment on the Incorporation of 32P into Polyphosphoinositides and Other Phospholipids in Peripheral Nerve of the Streptozotocin Diabetic Rat

The influence of varying doses of streptozotocin and preventive insulin treatment on phospholipid metabolism in sciatic nerve in vitro from diabetic rats was studied. Animals were given 30, 45, and 60 mg/kg injections of streptozotocin and 10 weeks later nerves were removed and incubated in the presence of [32P]-orthophosphate. The quantity of isotope incorporated into phosphatidylinositol-4,5-bisphosphate (PIP2) was progressively greater with increasing drug dosage, whereas uptake of label into other phospholipids was unchanged. Rats were made diabetic and within 72 h were implanted with long-acting, insulin-containing osmotic minipumps and the incorporation of [32P]orthophosphate into phospholipids of intact and epineurium-free nerves was examined 8 weeks later. For whole nerve, increased labeling in nerves from diabetic animals occurred only in PIP2 and phosphatidylinositol-4-phosphate (PIP) and was completely prevented by insulin treatment. Isotope incorporation into polyphosphoinositides was also markedly elevated (greater than or equal to 100%) in desheathed diabetic nerves, but not in nerves from insulin-treated animals. Other phospholipids in epineurium-free nerves displayed some rise in isotope uptake, but the increases were not prevented by insulin treatment and appeared unrelated to hyperglycemia. Morphological examination of nerves extended previous findings that prolonged insulin treatment produces axonal degeneration. These observations indicate that abnormal nerve polyphosphoinositide metabolism is at least in part a consequence of hyperglycemia. The metabolic alterations may be intimately involved in reduced nerve conduction velocity, which is characteristic of diabetic neuropathy.     

Muscarinic Cholinergic Receptor-Mediated Phosphoinositide Metabolism in Peripheral Nerve

Few receptor-mediated phenomena have been detected in peripheral nerve. In this study, the ability of the muscarinic cholinergic receptor agonist carbamylcholine to enhance phosphoinositide (PPI) breakdown in sciatic nerve was investigated by measuring the accumulation of inositol phosphates. Rat sciatic nerve segments were prelabeled with myo-[3H]inositol and then incubated either with or without carbamylcholine in the presence of Li+. [3H]Inositol monophosphate ([3H]IP) accumulation contained most of the radioactivity in inositol phosphates, with [3H]inositol bisphosphate ([3H]IP2) and [3H]inositol trisphosphate ([3H]IP3) accounting for 7-8% and 1-2% of the total, respectively. In the presence of 100 microM carbamylcholine, [3H]IP accumulation increased by up to 150% after 60 min. The 50% effective concentration for the response was determined to be 20 microM carbamylcholine and stimulated IP generation was abolished by 1 microM atropine. Enhanced accumulation of IP2 and IP3 was also observed. Determination of the pA2 values for the muscarinic receptor antagonists atropine (8.9), pirenzepine (6.5), AF-DX 116 (11-[[2-[(diethylamino)methyl]-1-piperidinyl] acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepin-6-one) (5.7), and 4-diphenylacetoxy-N-methylpiperidinemethiodide (4-DAMP) (8.6) strongly suggested that the M3 muscarinic receptor subtype was predominantly involved in mediating enhanced PPI degradation. Following treatment of nerve homogenates and myelin-rich fractions with pertussis toxin and [32P]NAD+, the presence of an ADP-ribosylated approximately 40-kDa protein could be demonstrated. The results indicate that peripheral nerve contains key elements of the molecular machinery needed for muscarinic receptor-mediated signal transduction via the phosphoinositide cycle.     

A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme

A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4- glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast. The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan. The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression. The recombinant enzyme was purified to apparent homogeneity by anion- exchange and gel permeation chromatography. The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC. The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase. XEG hydrolyzes its substrates with retention of the anomeric configuration. The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein. The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls. It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.      

Immortalized Schwann Cells Express Endothelin Receptors Coupled to Adenylyl Cyclase and Phospholipase C

Endothelins (ETs) are potent regulators of renal, cardiovascular and endocrine functions and act as neurotransmitters in the CNS. Here we report that immortalized Schwann cells express receptors for ETs and characterize some of the cellular events triggered by their activation. Specific binding of [¹²⁵I]-ET-1 to Schwann cell membranes was inhibited by ET-1 and the ET_B-selective agonists ET-3, sarafotoxin 6c and [A1a^1,3,11,15]-ET-1 with IC_50cor values ranging between 2 and 20 nM. No competition was observed with the ET_A receptor-selective antagonist BQ123. Incubation of [³H]-inositol pre-labeled Schwann cells with ET-1, ET-3 or sarafotoxin 6c elicited a concentration-dependent increase in the release of IP₁ that reached a plateau at approximately 100 nM. The efficacy of [Ala^1,3,11,15]-ET-1 (a linear peptide analog of ET-1) was half of that corresponding to ET-1. These stimulatory effects were partially blocked by pre-incubation with pertussis toxin. When Schwann cells were incubated in the presence of 100 nM ET-1 or ET-3 there was a significant inhibition of basal and isoproterenol-stimulated cAMP levels. The inhibitory effects of sarafotoxin 6c and [Ala^1,3,11,15]-ET-1 on isoproterenol-stimulated cAMP levels were similar to that observed with ET-1. Pre-incubation with pertussis toxin completely prevented this effect. These observations indicate that immortalized Schwann cells express receptors for ET peptides (predominantly ET_B) coupled to modulation of phospholipase C and adenylyl cyclase activities. The actions of ETs on Schwann cells provide a novel example of the influence of vascular factors on nerve function.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Insulin Reverses Enhanced Incorporation of 32P into Polyphosphoinositides in Peripheral Nerve of the Streptozotocin Diabetic Rat

The ability of insulin treatment to reverse altered phosphoinositide metabolism in sciatic nerve from streptozotocin diabetic rats was studied. Diabetes was induced in rats by means of a single injection of streptozotocin. Enhanced incorporation of 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) was detectable as early as 8 days following intravenous injection of streptozotocin and was maximal after 4 weeks. Hormone treatment was initiated at this time by daily injections of protamine zinc insulin followed by the implantation of long-acting insulin osmotic minipumps, and 4 weeks later sciatic nerves were removed and incubated in the presence of [32P]orthophosphate. The increased labeling of PIP2 was completely reversed by hormone administration. In contrast, insulin (0.1 and 1.0 mU/ml) added to the incubation medium failed to reverse the altered pattern of 32P incorporation into PIP2. The uptake of 32P into PIP2 was greater than 80% higher into the proximal than into the distal portion of normal sciatic nerve when these were incubated separately. This metabolic difference was abolished in diabetic rats, although the incorporation into both segments was still significantly higher than in controls. These results strengthen the association of altered nerve PIP2 metabolism with the diabetic state and are consistent with the concept that experimental diabetic neuropathy is a distal axonopathy.     

Hyperglycemia triggers abnormal signaling and proliferative responses in Schwann cells

Peripheral neuropathy is a serious diabetic complication. Delayed nerve regeneration in diabetic animal models suggests abnormalities in proliferation/differentiation of Schwann cells (SC). We recently reported that endothelins (ETs) regulate proliferation and phenotype in primary and immortalized SC (iSC). We now investigated changes in the effects of ETs on SC proliferation and signaling in nerve segments from streptozotocin-induced diabetic rats and in iSC exposed to high glucose. Cultured explants from diabetic rats displayed a delay in the time-course of [³H]-thymidine incorporation as well as enhanced sensitivity to endothelin-1 (ET-1) or insulin. iSC cultured in high (25 mM) glucose-containing media also exhibited higher [³H]-thymidine incorporation, along with an enhanced activation of p38 mitogen-activated protein kinase and phospholipase C in response to ET-1 or platelet-derived growth factor as compared to controls (5.5 mM glucose). These studies support an extra-vascular role of ETs in peripheral nerves and SC. The increased sensitivity to ET-1 in nerves and iSC exposed to high glucose may contribute to abnormal SC proliferation characterizing diabetic neuropathy.