NF-κB subunits are differentially distributed in cells of lumbar dorsal root ganglia in naïve and diabetic rats.

Previous studies demonstrated that nuclear factor κB (NF-κB) activation is decreased in dorsal root ganglia (DRG) of rats having streptozotocin (STZ)-induced diabetes. DRG contain cell bodies of neurons that convey sensory signals from the periphery. To determine the relationship between diabetes-induced neuropathy and NF-κB expression in DRG, behavioral, immunohistochemical, and biochemical studies were performed on naïve and 3-month diabetic rats. Behavioral studies confirmed that many diabetic rats develop tactile allodynia, or increased sensitivity to light touch, in the hind paws. Immunohistochemical studies on lumbar DRG that receive input from the affected regions revealed that p50 and p65, frequent NF-κB subunit partners, are differentially localized. Intense p65 immunostaining was detected in the cytoplasm of small- and medium-sized neurons as well as in satellite cells. In contrast, p50 was localized in the cytoplasm of virtually all neurons. In many cases, prominent staining was also present in nuclei, a location consistent with transcription factor activation. Immunohistochemical and biochemical studies found that the nuclear to cytoplasmic ratio of p50 expression was significantly reduced in diabetic rats compared to that in naïve animals. Our findings raise the possibility that changes in NF-κB activation in a subset of DRG neurons participates in mediating diabetes-induced sensory neuropathy.

Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen-activated protein kinase and Ca(2+)-independent phospholipase A2.

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen-activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet-derived growth factor (PDGF), a known mitogen in these cells. Both endothelin-1 (ET-1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET-1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET-1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET-1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca(2+)-independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET-1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET-1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.

Endothelins regulate arachidonic acid release and mitogen-activated protein kinase activity in Schwann cells.

Immortalized rat Schwann cells (iSC) express endothelin (ET) receptors coupled to inhibition of adenylyl cyclase and stimulation of phospholipase C (PLC). These effects precede phenotypic changes and increased DNA synthesis. We have investigated the role of ETs in the regulation of arachidonic acid (AA) release and mitogen-activated protein kinases (MAPKs). Both ET-1 and ET-3 increased AA release in iSC. This effect was sensitive to the phospholipase A(2) (PLA(2)) inhibitors E:-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H:-pyran-2-one and arachidonyl-trifluoromethyl ketone but was insensitive to inhibitors of PLC or phospholipase D-dependent diacylglycerol generation. ET-1-dependent AA release was also unaffected by removal of extracellular Ca(2+) and blocking the concomitant elevation in [Ca(2+)](i), consistent with participation of a Ca(2+)-independent PLA(2). Treatment of iSC with ETs also resulted in activation of extracellular signal-regulated kinase, c-Jun-NH(2)-terminal kinase (JNK), and p38 MAPK. A cause-effect relationship between agonist-dependent AA release and stimulation of MAPKs, but not the opposite, was suggested by activation of JNK by exogenous AA and by the observation that inhibition of MAPK kinase or p38 MAPK was inconsequential to ET-1-induced AA release. Similar effects of ETs on AA release and MAPK activity were observed in cultures expanded from primary SC and in iSC. Regulation of these effectors may mediate the control of proliferation and differentiation of SC by ETs during peripheral nerve development and regeneration.

Signal transduction pathways of the human V1-vascular, V2-renal, V3-pituitary vasopressin and oxytocin receptors.

Vasopressin (VP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by stimulation of specific G protein-coupled receptors (GPCRs) currently classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) VP receptors and OT receptors (OTR). The recent cloning of the different members of the VP/OT family of receptors now allows the extensive characterization of the molecular determinants involved in ligand binding and signal transduction pathways coupled to a given VP/OT receptor subtype in stably transfected mammalian cell lines. In this article, we review the present knowledge of the signal transduction pathways coupled to the different VP/OT receptor subtypes and we present new observations derived from the study of each human VP or OT receptor subtype stably expressed in CHO cells.

Molecular pharmacology of human vasopressin receptors.

Vasopressin (AVP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by activation of specific G protein-coupled receptors (GPCRs) currently classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) AVP receptors and OT receptors (OTR). The cloning of the different members of the AVP/OT family of receptors now allows the extensive molecular pharmacological characterization of a single AVP/OT receptor subtype in stably transfected mammalian cell lines. The human V1-vascular (CHO-V1), V2-renal (CHO-V2), V3-pituitary (CHO-V3) and oxytocin (CHO-OT) receptors stably expressed in CHO cells display distinct binding profiles for 18 peptide and 5 nonpeptide AVP/OT analogs. Several peptide and nonpeptide compounds have a greater affinity for the V1R than AVP itself. V2R peptide agonists and antagonists tend to be non-selective ligands whereas nonpeptide V2R antagonists are potent and subtype-selective. None of the 22 AVP/OT analogs tested has a better affinity for the human V3R than AVP itself. Several peptide antagonists do not select well between V1R and OTR. These results underscore the need for developing specific and potent analogs interacting specifically with a given human AVP/OT receptor subtype.

The human V3 pituitary vasopressin receptor: ligand binding profile and density-dependent signaling pathways.

The vasopressin (AVP) V3 pituitary receptor (V3R) is a G protein-coupled corticotropic phenotypic marker that is overexpressed in ACTH-hypersecreting tumors. Studies of the agonist/antagonist binding profile and signal transduction pathways linked to the human V3R have been limited because of the scarcity of this protein. To define the signals activated by V3Rs and the eventual changes triggered by developmental or pathological receptor regulation, we developed Chinese hamster ovary (CHO)-V3 cells stably expressing low, medium, or high levels of human V3Rs (binding capacity, <10, 10-25, and 25-100 pmol/mg, respectively). The affinity of the V3R for 21 peptide and nonpeptide AVP analogs was clearly distinct from that exhibited by the human V1R and V2R. AVP triggered stimulation of phospholipase C in CHO-V3 cells (partially sensitive to treatment with pertussis toxin) with a potency directly proportional to receptor density. V3R-mediated arachidonic acid release also was also sensitive to pertussis toxin and more efficacious in cells exhibiting medium than in those with high receptor density. AVP also stimulated the pertussis toxin-insensitive uptake of [3H]thymidine in CHO-V3 cells. The concentration-response curves for this effect were monophasic in cells expressing low and medium levels of V3Rs; on the contrary, a biphasic curve was observed in cells with high V3R density. Coupling of V3R to increased production of cAMP was only observed in CHOV3 high cells, suggesting a negative relationship between increased cAMP production and DNA synthesis. Activation of mitogen-activated protein kinases by V3R was pertussis toxin insensitive, but was dependent on activation of phospholipase C and protein kinase C; both the level and duration of activation were a function of the receptor density. Thus, the human V3R has a pharmacological profile clearly distinct from that of the human V1R and V2R and activates several signaling pathways via different G proteins, depending on the level of receptor expression. The increased synthesis of DNA and cAMP levels observed in cells expressing medium and high levels of V3Rs, respectively, may represent important events in the tumorigenesis of corticotroph cells.

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 ETB-selective agonists ET-3, sarafotoxin 6c and [Ala1,3,11,15]-ET-1 with IC50cor values ranging between 2 and 20 nM. No competition was observed with the ETA 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 [P1 that reached a plateau at approximately 100 nM. The efficacy of [Ala1,3,11,15]-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 [Ala1,3,11,15]-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 ETB) 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.

P2-purigenic receptors regulate phospholipase C and adenylate cyclase activities in immortalized Schwann cells.

Schwann cells play an important role in both the development and regeneration of peripheral nerves. Proliferation and differentiation of Schwann cells are critically dependent on changes in the levels of cAMP. ATP is a fast excitatory transmitter in the peripheral nervous system, inducing depolarization of the vagus nerve through occupancy of P2-purinergic receptors. In the present study we demonstrate that extracellular ATP stimulates phospholipase C and inhibits adenylate cyclase activities in cultured Schwann cells. Addition of ATP inhibited, in a concentration-dependent manner, forskolin- or isoprenaline-stimulated adenylate cyclase activity. The rank order of potency corresponding to different purinergic receptor agonists was 2-methylthio-ATP > ATP = ADP > or = adenosine 5'-[gamma-thio]triphosphate (ATP[S]) > UTP, consistent with the involvement of a P2y subtype. Adenosine and adenosine 5'-[alpha,beta-methylene]-triphosphate (pp[CH2pA) were ineffective. Preincubation with pertussis toxin completely blocked this inhibitory effect. When Schwann cells were pre-labelled with myo-[3H]inositol and incubated in Hanks' balanced salt solution containing Ca2+ and Mg2+, addition of ATP[S] resulted in a concentration-dependent increase in the release of InsP with a concomitant increase in intracellular free [Ca2+] ([Ca2+]i). Under these conditions, the effects of both ATP and UTP were of lower magnitude. Removal of Ca2+ and Mg2+ from the assay medium resulted in a significant increase in the effects of ATP[S], ATP and UTP. The decreased response observed in the presence of both bivalent cations (1.2 mM Ca2+ and 1 mM Mg2+) could not be explained either by increased degradation of ATP by Ca2+/Mg2+-dependent nucleotidases or by cation influx. The rank order of potency for the effects of agonists on phospholipase C activity was ATP[S] = adenosine 5'[gamma-imido]triphosphate > ATP -UTP > ADP, indicating the involvement of a P(2U) receptor subtype in this response. Adenosine, AMP and pp[CH2]pA were ineffective. These results demonstrate that immortalized Schwann cells express P(2U) and P(2Y) purinoceptors, which are coupled to stimulation of phospholipase C and inhibition of adenylate cyclase, respectively. Our observations unveil signal-transduction pathways that may be used by ATP to regulate proliferation and differentiation of Schwann cells, and ultimately to influence nerve homeostasis.

Signal transduction alterations in peripheral nerves from streptozotocin-induced diabetic rats.

We have previously determined the presence of muscarinic receptors and the expression of several G proteins in homogenates and myelin fractions from rat sciatic nerves. In the present study we investigated whether changes in several signal transduction pathways in peripheral nerves might be responsible for some of the biochemical abnormalities (e.g., phosphoinositide metabolism) present in sciatic nerves from streptozotocin-induced diabetic rats. Sciatic nerves from 5 week diabetic rats that were prelabelled with [3H]-myo-inositol displayed a significant increase in the basal release of inositol mono- and bis-phosphate, while carbamylcholine-stimulated release was significantly smaller. Basal- and forskolin-stimulated adenylyl cyclase activity was significantly decreased in sciatic nerve homogenates from diabetic animals. However, we were unable to detect any significant differences in the levels of cAMP in intact nerves or in nerve segments that were incubated in the presence or absence of forskolin. ADP-ribosylation experiments showed that in sciatic nerves from experimentally diabetic rats there was a significant increase in the ADP-ribosylation catalyzed by cholera and pertussis toxins. Measurements of the levels of alpha-subunits of G proteins revealed that the expression of Gq/11 alpha, Gs alpha, and Gi-3 alpha was increased by 30 to 50%. These results indicate that during the course of experimental diabetes, peripheral nerves exhibit an abnormal production of inositol phosphates and cAMP, together with an abnormal expression and/or function of G proteins. One of the consequences of such alterations is the diminished release of inositol phosphates triggered by muscarinic agonists in diabetic sciatic nerves.

Decreased polyphosphoinositide metabolism accompanies myelinated fiber loss in human peripheral neuropathies.

The distribution of incorporated 32P in phospholipids of sural nerve biopsy samples from patients with several peripheral neuropathies was measured. Both the absolute amount and the proportion of isotope in polyphosphoinositides was decreased in nerves that displayed substantial (> 50%) depletion of myelinated fibers as compared to nerves that exhibited minimal depletion. The results suggest that diminished metabolism of these substances is an indicator of myelin loss, and are consistent with the conclusion that polyphosphoinositide turnover in human nerve is nearly entirely localized to the myelin sheath.

Identification of G protein subtypes in peripheral nerve and cultured Schwann cells.

In this study, we investigated the expression of various G proteins in whole sciatic nerves, in myelin and nonmyelin fractions from these nerves, and in membranes of immortalized Schwann cells. In myelin, nonmyelin, and Schwann cell membranes we detected two 39-40-kDa pertussis toxin substrates that were resolved on separation on urea-gradient gels. Two cholera toxin substrates with apparent molecular masses of 42 and 47 kDa were present in nerve and brain myelin and in Schwann cell membranes. In these membranes, a third 45-kDa cholera toxin substrate, which displayed the highest labeling, was also present. Immunoblotting with specific antisera allowed the identification of G(o) alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Gq/G11 alpha, and the two isoforms of Gs alpha in nerve homogenates, nerve, and brain myelin fractions. In Schwann cell membranes we identified G(o) alpha, Gi2 alpha, Gi3 alpha, and proteins from the Gq family, but no immunoreactivity toward anti-Gi1 alpha antiserum was detected. In these membranes, anti-Gs alpha antibody recognized the three cholera toxin substrates mentioned above, with the 45-kDa band displaying the highest immunoreactivity. Relative to sciatic nerve myelin, the Schwann cell membranes revealed a significantly higher expression of Gi3 alpha and the absence of Gi1 alpha. The different distribution of G proteins among the different nerve compartments might reflect the very specialized function of Schwann cells and myelin within the nerve.

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.

Correction of altered metabolic activities in sciatic nerves of streptozocin-induced diabetic rats. Effect of ganglioside treatment.

The effect of ganglioside administration to nondiabetic and streptozocin-induced diabetic rats on sciatic nerve Na(+)-K(+)-ATPase, polyphosphoinositide (PPI) turnover, and protein phosphorylation was investigated. Gangliosides were injected (10 mg/kg body wt i.p.) for 10 or 30 days beginning 20 days after induction of diabetes. Na(+)-K(+)-ATPase activity was reduced nearly 50% in diabetic nerve and was restored to normal by both ganglioside treatments. The elevated levels of fructose and sorbitol and depressed content of myoinositol in diabetic nerve were unaffected by 30 days of ganglioside treatment, indicating that the restoration of Na(+)-K(+)-ATPase activity is not dependent on normal concentrations of these compounds. In the same nerves, 32P incorporation into phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate increased 73-76 and 39-53%, respectively, in diabetic compared with nondiabetic tissue. Ganglioside administration abolished the elevated labeling of PPIs after 30 days but was ineffective after only 10 days. Neither ganglioside regimen was able to reverse enhanced phosphorylation of the major peripheral nerve myelin protein P0. The finding that gangliosides can more quickly correct the effects of diabetes on Na(+)-K(+)-ATPase activity than on PPI turnover suggests that the mechanisms underlying these two phenomena are not closely related and are distinct from the sequence of events responsible for altered myelin protein phosphorylation.

Acrylamide administration alters protein phosphorylation and phospholipid metabolism in rat sciatic nerve.

The effects of ACR on protein phosphorylation and phospholipid metabolism were assessed in rat sciatic nerve. After 5 days of ACR administration (50 mg/kg/day) an increase in the incorporation of 32P into phosphatidylinositol-4,5-bisphosphate, phosphatidylinositol-4-phosphate, and phosphatidylcholine was detected in proximal sciatic nerve segments. In contrast, no changes in phospholipid metabolism were observed in distal segments. After 9 days of ACR treatment when neurotoxicological symptoms were clearly apparent, a generalized increase in radiolabel uptake into phospholipids was noted exclusively in proximal nerve regions. ACR-induced increases in phospholipid metabolism were toxicologically specific since comparable administration of MBA (108 mg/kg/day X 5 or 9 days) produced only minor changes. ACR intoxication was also associated with a rise in sciatic nerve protein phosphorylation. After 9 days of ACR treatment, phosphorylation of beta-tubulin, P0, and several unidentified proteins (38 and 180 kDa) was increased in distal segments. In contrast, chronic administration of MBA caused increases in phosphorylation of beta-tubulin and the major myelin proteins of proximal nerve segments. In cell free homogenates prepared from sciatic nerves of treated and control rats, MBA caused an increase in phosphorylation of major myelin proteins similar to its effect in intact proximal nerve segments. The most striking effect observed in nerve homogenates of ACR-treated rats was a marked decrease in phosphorylation of an 80-kDa protein. Addition of ACR (1 mM) to homogenates of normal nerve had no effect on protein phosphorylation. Our results indicate that changes in the phosphorylation of phospholipids and proteins in sciatic nerve might be a component of the neurotoxic mechanism of ACR.

Hexanedione effects on protein phosphorylation in rat peripheral nerve.

Rats were treated with either 2,5-hexanedione (2,5-HD), 1,6-hexanediol (1,6-HDIOL), or saline for 7, 15 or 24 days. Protein phosphorylation was measured in proximal and distal sciatic nerve segments following incubation with [32P]orthophosphate. In proximal segments, 2,5-HD administration caused selective time-dependent increases in isotope incorporation in a 55 kDa protein, tentatively identified as tubulin, and a 180 kDa protein. Enhanced phosphorylation was highest at 24 days when motor function was most impaired. Administration of 1,6-HDIOL produced no consistent phosphorylation changes. Animals intoxicated with 3,4-dimethyl-2,5-hexanedione for 12 days showed proximal region increases in phosphorylation of the 55 and 180 kDa proteins and the major myelin proteins, Po and Pr.

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.

Alteration of phosphoinositide metabolism, protein phosphorylation, and carbohydrate levels in sciatic nerve from Wistar fatty diabetic rats.

Sciatic nerve from the Wistar fatty diabetic (FD) rat, a prospective model for non-insulin-dependent diabetes mellitus, was investigated to determine the content of carbohydrates and to measure the incorporation of 32P into phosphoinositides and proteins. This strain has been shown to develop structural abnormalities in nerves and to exhibit reduced conduction velocity. Males became diabetic between the ages of 8 and 10 wk and were maintained together with lean sibling controls until the animals were either 22 or 44 wk old. Throughout this period, FD rats displayed moderate hyperglycemia. The carbohydrate profile of FD rat sciatic nerve exhibited modest increases in glucose, fructose, and sorbitol levels and significantly reduced myo-inositol concentrations, which were comparable at both ages. When nerves from 22-wk-old animals were incubated with [32P]orthophosphate and incorporation of radioactivity into phospholipids was measured, an increase in isotope uptake into phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-4-phosphate in the distal portions of tissue from the FD rat was observed. This effect was more pronounced in nerves from 44-wk-old rats. Phosphorylation of the major myelin protein P0 was 70% higher in the most distal portion of FD sciatic nerve from 22-wk-old animals. A comparable rise in phosphorylation of P0 as well as the large (P1) and small (Pr) myelin basic proteins occurred in nerves from 44-wk-old rats. In these animals, an approximately 50% decrease in the uptake of 32P into P0 and P1 in the most proximal region of FD nerve was also apparent.(ABSTRACT TRUNCATED AT 250 WORDS)

Phospholipid metabolism and protein phosphorylation in sciatic nerve from genetically diabetic (db/db) mouse.

The incorporation of [32P]orthophosphate into phospholipids and proteins of sciatic nerve from genetically diabetic (db/db) and littermate control (db/m) C57BL/KsJ mice was studied. Nerves from animals of ages 12, 16, 22, 26, and 38 wk were incubated in vitro. Among phospholipids, the uptake of isotope into phosphatidic acid was higher at nearly all ages examined. Phosphorylation of several proteins, including the major myelin glycoprotein, P0, and the small myelin basic proteins Pr + P2, was significantly enhanced in nerves from both 12- and 38-wk-old diabetic mice. The altered pattern of protein phosphorylation, but not that of phospholipid metabolism, was similar to changes observed in sciatic nerve from streptozocin-induced diabetic rats. The relationship of the results to reported levels of myo-inositol, sorbitol, and Na+-K+-ATPase activity and to functional abnormalities in nerves of db/db mice is discussed. The findings suggest that caution should be exercised in reaching conclusions concerning which biochemical alterations observed in different animal models of diabetic neuropathy are invariably associated with the development of this disorder.

Altered protein phosphorylation in sciatic nerve from rats with streptozocin-induced diabetes.

The effect of experimental diabetes on the phosphorylation of proteins in the rat sciatic nerve was studied. Nerves from animals made diabetic with streptozocin were incubated in vitro with [32P]orthophosphate and divided into segments from the proximal to the distal end, and proteins from each segment were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The principal labeled species were the major myelin proteins, P0, and the basic proteins. After 6 wk of diabetes, the incorporation of isotope into these proteins rose as a function of distance along the nerve in a proximal to distal direction and was significantly higher at the distal end compared with incorporation into nerves from age-matched controls. The overall level of isotope uptake was similar in nerves from diabetic animals and weight-matched controls. The distribution of 32P among proteins also differed in diabetic nerve compared with both control groups in that P0 and the small basic protein accounted for a greater proportion of total label incorporated along the entire length of nerve. In contrast to intact nerve, there was no significant difference in protein phosphorylation when homogenates from normal and diabetic nerve were incubated with [32P]-gamma-ATP. The results suggest that abnormal protein phosphorylation, particularly of myelin proteins, is a feature of experimental diabetic neuropathy and that the changes are most pronounced in the distal portion of the nerve.