Tyrosine phosphorylation of PNS myelin P(0) occurs in the cytoplasmic domain and is maximal during early development.

P(0), the major protein of PNS myelin, is considered to play a critical role in the compaction and stabilization of myelin lamellae. The protein undergoes extensive posttranslational modifications, including phosphorylation at multiple serine moieties in the cytoplasmic region. Recently, we demonstrated that P(0) is phosphorylated on one or more tyrosine residues in rat nerve homogenates after incubation. In this study, we show that P(0) phosphorylated on tyrosine is also present in the intact animal. The proportion of P(0) molecules phosphorylated on tyrosine is highest during the first postnatal week, a period that coincides with the most rapid period of myelin deposition in the PNS. A peptide that constitutes the cytoplasmic domain was isolated from purified P(0) and shown by immunochemical and chemical means to be phosphorylated on the tyrosine corresponding to Y(191) in the intact protein. No evidence was obtained supporting the possibility that P(0) is phosphorylated on other tyrosine residues. The sequence of amino acids surrounding Y(191) resemble known substrate phosphorylation sites for some nonreceptor cytoplasmic tyrosine kinases, as well as tyrosine-based recognition signals associated with clathrin vesicle-mediated cndocytosis.

Treatment with inverse agonists enhances baseline atrial contractility in transgenic mice with chronic beta2-adrenoceptor activation.

In this study, we investigate whether chronic treatment with beta-adrenoceptor (betaAR) ligands with inverse agonist activity enhances myocardial beta2AR-mediated atrial tension more than neutral antagonists in transgenic mice (TG35). These mice exhibit chronic adrenoceptor activation because they possess a greater number of constitutively active receptors than wild type mice due to cardiac-specific overexpression of human betaARs. TG35 and wild type mice were chronically treated for 90 h with three inverse agonists, ICI-118,551, propranolol, and carvedilol, and one neutral antagonist, alprenolol. After 96 h, we compared the basal and isoprenaline-stimulated (10 microM) increase in atrial tension in treated or untreated TG35 mice and wild type mice. In parallel, to determine the effect of chronic betaAR ligand treatment on the amounts of G protein receptor kinase-2 (GRK-2) and G proteins, we performed Western blotting on myocardial cytosolic and membrane proteins. Atria from the TG35 mice treated with inverse agonists showed increases in the baseline tension compared to those from alprenolol/vehicle-treated mice. ICI-118,551 and propranolol treatment restored the elevated myocardial G-inhibitory protein (Gialpha) levels to that of wild type. Also, treatment with inverse agonists upregulated G-stimulatory protein (Gsalpha) levels and GRK2 above those levels in vehicle-treated TG35 or wild type mice. The increased baseline atrial tension was reversed by the addition of ICI-118,551. Overall, our data suggests that inverse agonists enhance baseline atrial tension more than neutral antagonists. Based on this, we propose that upregulation of the active conformation of the beta2ARs, Gsalpha protein and restoration of Gialpha as three possible mechanisms to explain this enhanced receptor activity. Therefore, the favourable effects of some ligands used in pathological conditions involving chronic adrenoceptor activation may be due to the inverse agonist activity of the ligand.

Redefining the HIV-infectious window period in the chimpanzee model: evidence to suggest that viral nucleic acid testing can prevent blood-borne transmission.

BACKGROUND: Although it is rare, blood-transmitted HIV infection can occur when a donor presents in the window period between HIV-1 exposure and the first appearance of detectable p24 antigen. STUDY DESIGN AND METHODS: To study this seronegative window period, a chimpanzee (X034) was inoculated with 38 median tissue culture infective doses of HIV-1 IIIB; serum and peripheral blood mononuclear cells were obtained one to two times per week for 12 weeks and then biweekly for 12 weeks. Infectivity was monitored by the detection of serum HIV RNA, cell-associated HIV DNA, p24 antigen, and anti-HIV and by co-culture methods. RESULTS: No HIV markers were noted until 5 weeks after inoculation, at which time virus was isolated and HIV RNA and DNA were detected in plasma and cells, respectively. Anti-HIV and HIV p24 antigen were not present until 8 weeks after inoculation. Plasma and cells obtained from Chimpanzee X034 3 or 4 weeks after exposure were then sequentially inoculated into a second chimpanzee (X176); no HIV infection was observed in this animal during serial follow-up for 24 weeks after each inoculation. In contrast, when the fifth-week HIV-1 RNA- and DNA-positive sample was inoculated, Chimpanzee X176 was unequivocally infected with HIV-1. CONCLUSIONS: Nucleic acid testing narrowed the seronegative window by 3 weeks (37%). More important, there was no demonstrable infectivity in either plasma or peripheral blood mononuclear cells obtained before molecular markers were detectable. This suggests that the infectious window may be considerably shorter than the total window as measured from exposure and that nucleic acid testing might not only shorten the seronegative window, but totally prevent transfusion-transmitted HIV infection.

Abnormal myo-inositol and phospholipid metabolism in cultured fibroblasts from patients with ataxia telangiectasia.

Ataxia telangiectasia (AT) is a complex autosomal recessive disorder that has been associated with a wide range of physiological defects including an increased sensitivity to ionizing radiation and abnormal checkpoints in the cell cycle. The mutated gene product, ATM, has a domain possessing homology to phosphatidylinositol-3-kinase and has been shown to possess protein kinase activity. In this study, we have investigated how AT affects myo-inositol metabolism and phospholipid synthesis using cultured human fibroblasts. In six fibroblast lines from patients with AT, myo-inositol accumulation over a 3-h period was decreased compared to normal fibroblasts. The uptake and incorporation of myo-inositol into phosphoinositides over a 24-h period, as well as the free myo-inositol content was also lower in some but not all of the AT fibroblast lines. A consistent finding was that the proportion of 32P in total labeled phospholipid that was incorporated into phosphatidylglycerol was greater in AT than normal fibroblasts, whereas the fraction of radioactivity in phosphatidic acid was decreased. Turnover studies revealed that AT cells exhibit a less active phospholipid metabolism as compared to normal cells. In summary, these studies demonstrate that two manifestations of the AT defect are alterations in myo-inositol metabolism and phospholipid synthesis. These abnormalities could have an effect on cellular signaling pathways and membrane production, as well as on the sensitivity of the cells to ionizing radiation and proliferative responses.

Evening primrose oil treatment corrects reduced conduction velocity but not depletion of arachidonic acid in nerve from streptozotocin-induced diabetic rats.

The effects of evening primrose oil (EPO) treatment, a source of gamma-linolenic acid, on the proportions of arachidonoyl-containing molecular species (ACMS) in sciatic nerve phosphatidylcholine and phosphatidylethanolamine were determined in conjunction with alterations in nerve conduction velocity. Normal and diabetic rats were either untreated or fed a dietary supplement containing isocalorically equivalent amounts of either EPO or corn oil for the duration of the experiment. After 8 weeks of streptozotocin-induced diabetes, nerve conduction velocity was reduced 16% and this deficit was prevented by either EPO or corn oil treatment. Neither EPO nor corn oil supplementation significantly increased the depressed proportions of ACMS. The level of the linoleoyl-containing molecular species, 16:0/18:2, was elevated in the phospholipids from untreated diabetic rats and was further increased by EPO treatment. These results are consistent with decreased activity of the delta6 desaturase that is required for arachidonic acid synthesis in vivo, but suggests that an accompanying deficit in the subsequent delta5 desaturase-catalyzed reaction may be rate-limiting. These findings indicate that maintenance of normal ACMS levels is not required for prevention of diminished nerve conduction velocity and suggest that other factors influenced by an altered polyunsaturated fatty acid pattern, such as metabolites of linoleic acid or gamma-linolenic acid other than arachidonic acid, the energy state of the nerve or the degree of membrane fluidity may contribute to impaired nerve conduction velocity in diabetic neuropathy.

Depletion of phospholipid arachidonoyl-containing molecular species in a human Schwann cell line grown in elevated glucose and their restoration by an aldose reductase inhibitor.

In experimental diabetic neuropathy, defective arachidonic acid metabolism characterized by a decrease in the proportion of glycerophospholipid arachidonoyl-containing molecular species (ACMS) occurs and has been implicated in the pathogenesis of the disorder. In this study, we evaluated the suitability of a tumor-derived human Schwann cell line (NF1T) as a model to investigate the mechanism underlying the loss of ACMS. NF1T cells grown in 30 versus 5.5 mM glucose undergo a marked reduction in ACMS in phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol, in a manner resembling that of diabetic nerve. The depletion of ACMS can be reversed on transferring the cells from 30 mM glucose to medium containing physiological levels of glucose. Cells maintained in 5.5 mM glucose plus 25 mM mannitol or sorbitol did not exhibit decreased ACMS levels, indicating that osmotic effects were not responsible for ACMS depletion. However, growth in 25 mM fructose elicited a reduction of ACMS similar to that produced by 30 mM glucose. Excessive glucose flux through the polyol pathway has been implicated in the neural and vascular abnormalities associated with diabetes. Therefore, we examined the effects of polyol pathway inhibitors, including two aldose reductase inhibitors, zopolrestat and sorbinil, and a sorbitol dehydrogenase inhibitor (SDI), CP166,572, on ACMS levels in NF1T cells cultured in elevated glucose concentrations. At 200 microM, zopolrestat fully and sorbinil partially corrected ACMS depletion. The SDI at concentrations up to 100 microM failed to affect diminished ACMS levels. Neither zopolrestat nor the SDI restored ACMS levels reduced in the presence of elevated fructose concentrations. These findings suggest that enhanced flux through the polyol pathway and, in particular, elevated aldose reductase activity may play a significant role in the reduction of ACMS levels in the cells brought about by elevated glucose levels.

Induction of neutralizing antibodies to T-cell line-adapted and primary human immunodeficiency virus type 1 isolates with a prime-boost vaccine regimen in chimpanzees.

Five chimpanzees were immunized by administration of one or more intranasal priming doses of one to three recombinant adenoviruses containing a gp160 insert from human immunodeficiency virus type 1 (HIV-1) MN (HIV-1MN) followed by one or more boosts of recombinant HIV-1SF2 gp120 delivered intramuscularly with MF59 adjuvant. This regimen resulted in humoral immune responses in three of five animals. Humoral responses included immunochemically active anti-H1V-1 antibodies (Abs) directed to recombinant gp120 and neutralizing Abs reactive with T-cell-line-adapted HIV-1MN and HIV-1SF2. In addition, neutralizing activity was detected to the two homologous primary isolates and to two of three heterologous primary isolates which, like the immunizing strains, can use CXCR4 as a coreceptor for infection. The three animals with detectable neutralizing Abs and a fourth exhibiting the best cytotoxic T-lymphocyte response were protected from a low-dose intravenous challenge with a cell-free HIV-1SF2 primary isolate administered 4 weeks after the last boost. Animals were rested for 46 weeks and then rechallenged, without a boost, with an eightfold-higher challenge dose of HIV-1SF2. The three animals with persistent neutralizing Abs were again protected. These data show that a strong, long-lived protective Ab response can be induced with a prime-boost regimen in chimpanzees. The data suggest that in chimpanzees, the presence of neutralizing Abs correlates with protection for animals challenged intravenously with a high dose of a homologous strain of HIV-1, and they demonstrate for the first time the induction of neutralizing Abs to homologous and heterologous primary isolates.

Rubidium uptake and accumulation in peripheral myelinated internodal axons and Schwann cells.

To study mechanisms of K+ transport in peripheral nerve, uptake of rubidium (Rb+), a K+ tracer, was characterized in rat tibial nerve myelinated axons and glia. Isolated nerve segments were perfused with zero-K+ Ringer's solutions containing Rb+ (1-20 mM) and x-ray microanalysis was used to measure water content and concentrations of Rb, Na, K, and Cl in internodal axoplasm, mitochondria, and Schwann cell cytoplasm and myelin. Both axons and Schwann cells were capable of removing extracellular Rb+ (Rb+(o)) and exchanging it for internal K+. Uptake into axoplasm, Schwann cytoplasm, and myelin was a saturable process over the 1-10 mM Rb+(o) concentration range, although corresponding axoplasmic uptake rates were higher than respective glial velocities. Mitochondrial accumulation was a linear function of axoplasmic Rb+ concentrations, which suggests involvement of a nonenzymatic process. At 20 mM Rb+(o), a differential stimulatory response was observed; i.e., axoplasmic Rb+ uptake velocities increased more than fivefold relative to the 10 mM rate, and glial cytoplasmic uptake rose almost threefold. Finally, Rb+(o) uptake rate into axons and glia was completely inhibited by ouabain (2-4 mM) exposure or incubation at 4 degrees C. These results suggest that Rb+ uptake into peripheral nerve internodal axons and Schwann cells is mediated by Na+,K+-ATPase activity and implicate the presence of axonal- and glial-specific Na+ pump isozymes.

Long-term protection of chimpanzees against high-dose HIV-1 challenge induced by immunization.

A combination AIDS vaccine approach consisting of priming with adenovirus-HIV-1MN gp160 recombinants followed by boosting with HIV-1SF2 gp120 was evaluated in chimpanzees. Long-lasting protection, requiring only three immunizations, was achieved against a low-dose challenge with the SF2 strain of HIV-1 and a subsequent high-dose SF2 challenge administered 1 year later without an intervening boost. Notably, neutralizing antibody responses against both clinical and laboratory isolates developed in three chimpanzees and persisted until the time of high-dose challenge. The possibility that cytotoxic T-lymphocytes contribute to low-dose protection of a chimpanzee lacking neutralizing antibodies is suggested. Our results validate the live vector priming/subunit booster approach and should stimulate interest in assessing this combination vaccine approach in humans.

CD8+ cell-mediated immune responses: relation to disease resistance and susceptibility in lentivirus-infected primates.

Immune responses mediated by CD8+ lymphocytes have been correlated with protection from HIV infection and disease progression in humans and nonhuman primates. The CD8+ cell population is heterogeneous in terms of biological function and phenotype. We have undertaken a review of the current state of knowledge of subtypes of CD8+ cells and their role in immune responses directed to HIV and related primate lentiviruses. Differences in the pathogenesis of lentivirus infections in various primate hosts were examined and the possible roles of the various subpopulations of CD8+ lymphocytes in the resistance and/or susceptibility to lentivirus-related disease were compared.

[Vaccination of pigeons against Salmonella infections]. / Zur Schutzimpfung gegen die Taubensalmonellose.

The efficiency of the live vaccine Zoosal T with a double marker mutant of Salmonella Typhimurium was tested on conditioned pigeons. For challenge infection we used a pigeon specific variation copenhagen strain in a defined state of virulence. The reduction of mortality and the persistence of Salmonella in organs were evaluated. An oral booster enhances the protection due to vaccination.

Effects of hypoxia and severity of diabetes on Na,K-ATPase activity and arachidonoyl-containing glycerophospholipid molecular species in nerve from streptozotocin diabetic rats.

The pathogenesis of experimental diabetic neuropathy is associated with the development of endoneurial hypoxia. Exposure of normal rats to hypoxic conditions has previously been shown to reduce nerve conduction velocity. To study the biochemical effects of hypoxia further, streptozotocin-induced diabetic and age-matched nondiabetic rats were maintained in air containing 10% oxygen for nine weeks. As compared to nondiabetic rats kept in room air, sciatic nerve Na,K-ATPase activity was decreased 38% in nondiabetic, hypoxic rats and tended to be lower in diabetic animals maintained in a normoxic environment. However, the enzyme activity was unchanged in diabetic, hypoxic rats, suggesting the existence of an undefined compensatory interaction between these two conditions. Arachidonoyl-containing molecular species (ACMS) of phosphatidylcholine and phosphatidylethanolamine were substantially depleted in nerves from diabetic rats. Hypoxia alone also caused a lesser depletion of some but not all of these ACMS. However, the two conditions together did not produce a further decrease, consistent with the concept that the same mechanism is responsible for loss of ACMS in hypoxia and diabetes. To examine the effects of severity of diabetes on these parameters, groups of rats were injected with either 50 mg/kg or 100 mg/kg streptozotocin. The latter group was maintained by administration of minimal insulin doses and the experiment was terminated after 3 weeks. Serum glucose in rats that received the high dose of drug averaged 12% higher than in the low dose group. As compared to nondiabetic rats, Na,K-ATPase activity was reduced 32-36%, but there was no difference in activity between the two diabetic groups. However, there was a greater loss of ACMS in the more severely hyperglycemic rats. In rats that received comparable streptozotocin doses, measurement of ACMS depletion after 3, 9 and 32 weeks of diabetes revealed the loss is progressive with time. Thus, glycerophospholipid ACMS is a sensitive index of the severity and duration of experimental diabetic neuropathy.

Tyrosine phosphorylation of myelin protein PO.

Po (M(r) 30 kDa), the major protein component of peripheral nervous system (PNS) myelin, is known to be phosphorylated by protein kinase C on serine residues at multiple sites. This study was conducted to assess whether other amino acids might be phosphorylated in the protein. Segments of rat sciatic nerve were incubated with 32P in either the presence or absence of phorbol ester. Labeled Po was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial acid hydrolysis. Upon separation of the hydrolysis products by either thin-layer electrophoresis or thin-layer chromatography, a radioactive spot was detected which comigrated with authentic phosphotyrosine. In other experiments, nerves were incubated with the tyrosine phosphatase inhibitors vanadate or vanadyl hydroperoxide (pervanadate). When the nerve homogenate proteins were separated on gels and probed with a monoclonal antibody to phosphotyrosine on Western blots, a positive immune reaction was obtained for a protein species which migrated with the same mobility as PO on Coomassie Blue-stained gels. In the absence of 2-mercaptoethanol, this immunoreactive band displayed increased mobility on gels which is characteristic of the migration pattern of Po. The same immunostaining results were obtained using a purified peripheral myelin fraction prepared from nerve homogenates. Furthermore, the positions of immunoreactive bands produced by anti-Po and antiphosphotyrosine antibodies coincided on the same immunoblot of myelin proteins and purified Po. These data indicate that one or more tyrosyl residues in Po can be phosphorylated in intact sciatic nerve.

Angiotensin-converting enzyme activity in retinas of streptozotocin-induced and Zucker diabetic rats. The effect of angiotensin II on Na+,K(+)-ATPase activity.

PURPOSE: To investigate whether serum and/or retinal angiotensin-converting enzyme (ACE) activity might correlate with the decrease in sodium potassium adenosine triphosphatase (Na,K-ATPase) activity in the retina of experimentally diabetic rats. METHODS: Insulin-dependent diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (STZ) in male Sprague-Dawley rats. Male Zucker fatty diabetic (ZDF/Gmifa) rats were used as models of non-insulin-dependent diabetes mellitus. ACE activity in the serum and retina of diabetic rats (1 through 5 months) and age-matched control animals was measured by radioimmunoassay using benzoyl-gly-gly-gly as substrate. The activity of total Na,K-ATPase was determined spectrophotometrically. The alpha 1 and alpha 3 isozymes of Na,K-ATPase were distinguished pharmacologically by their differential sensitivity to ouabain and were measured in the retina. RESULTS: Serum ACE activity was significantly increased in rats with STZ-induced diabetes at 3 weeks through 4 months of diabetes (28% to 32%) but was significantly decreased in ZDF rats after 2 to 5 months of diabetes (-9% to -16%). The activity of ACE in retinas obtained from the same groups of STZ and ZDF rats was significantly reduced at all time points examined in both models (-43% and -55%, respectively). The effect of angiotensin II (AngII) on the activity of Na,K-ATPase in retinas from normal rats was also studied in vitro. AngII significantly lowered the activities of total Na,K-ATPase (-16%) and its alpha 1 and alpha 3 isozymes. The inhibitory effect of AngII was abolished completely by losartan (0.1 microM), a specific antagonist of the AT1 receptor-subtype of AngII, and by nordihydroguaiaretic acid (50 microM), which at this concentration inhibits the lipoxygenase and cytochrome P-450-dependent pathways of arachidonic acid metabolism. The inhibitory effect of AngII on the Na,K-ATPase activity was not altered significantly by NG-iminoethyl ornithine (10 microM), an irreversible nitric oxide synthase inhibitor. CONCLUSIONS: The authors suggest that systemic ACE probably is not involved in the mechanisms responsible for the reduced activity of Na,K-ATPase in diabetes. Although AngII inhibits retinal Na,K-ATPase by a mechanism possibly involving arachidonic acid metabolites, it is unlikely that AngII contributes to the decreased Na,K-ATPase activity because of its reduced formation by retinal ACE in diabetes. The possible importance of reduced retinal ACE activity in diabetes warrants further investigation.

Receptor-mediated phosphoinositide metabolism in peripheral nerve and cultured Schwann cells.

Peripheral nerve possesses muscarinic cholinergic receptors, predominantly of the M3 subtype, that stimulate phosphoinositide metabolism. Evidence suggests that one site of this response is the myelin sheath. Purified peripheral nerve myelin contains several heterotrimeric GTP-binding proteins. Furthermore, carbachol and guanosine-5'-(3-O-thio)triphosphate-stimulated hydrolysis of exogenous phosphatidylinositol-4,5-bis-phosphate that is blocked by atropine can be reconstituted in a purified peripheral myelin-rich fraction. Nerve phosphoinositide turnover is also stimulated by adenosine analogs and blocked by adenosine receptor antagonists in a pattern consistent with the presence of adenosine A2 receptors in the tissue. Receptor-mediated phosphoinositide metabolism has also been studied in a human tumor-derived Schwann cell line (NF1T) derived from a neurofibromatosis-1 patient. By the same experimental criteria, NF1T cells also appear to contain adenosine A2 receptors which upon activation stimulate phosphoinositide turnover. However, phosphoinositide metabolism in these cells is not increased by either carbachol or ATP. Our findings taken together with other reports suggest that Schwann cells may possess a variety of receptors which regulate phosphoinositide metabolism.

An immunovirological study of central nervous system involvement during HIV-1 infection of chimpanzees.

Chimpanzees infected with human immunodeficiency virus type 1 (HIV-1) are used to model acquired immunodeficiency syndrome (AIDS). Since the central nervous system (CNS) is involved in AIDS, we performed an immunovirological study in 18 chimpanzees inoculated up to 87 months prior to the study (mean, 45 months) with HIV-1 and 8 uninfected controls. Serum and cerebrospinal fluid (CSF) IgG and albumin levels of infected chimpanzees never exceeded those of controls. The CSF/serum albumin ratio was elevated in 1 of 18 infected chimpanzees compared to controls; however, all animals had an elevated ratio indicating a more open blood-brain barrier relative to humans. The intrathecal IgG production index was elevated in only 1 of 18 infected chimpanzees compared to controls. Identical serum and CSF IgG bands were found by isoelectric focusing in 2 of 8 controls and in 1 of 18 infected chimpanzees. None of these bands reacted with recombinant HIV-1 p24gag or gp 120env. HIV-1 was isolated from the peripheral blood of 4 of 18 infected chimpanzees but never from the paired CSF samples. Anti-HIV-1 antibody was detected by a enzyme-linked immunosorbent assay in 18 of 18 paired serum and CSF samples and by Western blot in 18 of 18 serum and 13 of 18 CSF samples from infected chimpanzees without a difference in pattern. Polymerase chain reaction analysis on brain tissue of one animal was negative for HIV-1 sequences. Our results demonstrate that, unlike human infection, chimpanzees inoculated with HIV-1 show no evidence of isolatable virus in the CSF and no evidence of intrathecal anti-HIV-1 antibody synthesis up to several years after experimental infection. The lack of CNS involvement may contribute to the delay or suppression of clinical disease in infected chimpanzees.

Phosphorylation of myelin protein: recent advances.

Since it was first described 25 years ago, phosphorylation has come to be recognized as a widespread and dynamic post-translation modification of myelin proteins. In this review, the phosphorylation characteristics of myelin basic protein, protein zero (P0), myelin-associated glycoprotein and 2'3' cyclic nucleotide 3'-phosphodiesterase are summarized. Emphasis is placed on recent advances in our knowledge concerning the protein kinases involved and the sites of phosphorylation in the amino acid sequences, where known. The possible roles of myelin protein phosphorylation in modulating myelin structure, the process of myelin assembly and mediation of signal transduction events are discussed.

An aldose reductase inhibitor but not myo-inositol blocks enhanced polyphosphoinositide turnover in peripheral nerve from diabetic rats.

Experimental diabetic neuropathy, whether chemically induced or present in several spontaneously diabetic animal models, is characterized by sorbitol accumulation and myo-inositol depletion and usually also by enhanced turnover of the monoesterified moieties of polyphosphoinositides, particularly phosphatidylinositol-4,5-bisphosphate (PIP2). This study examined the relationship of these alterations by assessing the effects of myo-inositol and the aldose reductase inhibitor, sorbinil, supplied as dietary supplements, on sorbitol and myo-inositol concentrations and incorporation of 32P into polyphosphoinositides in sciatic nerve from rats killed 8 weeks after induction of diabetes with streptozotocin. Nerves from diabetic rats killed after 8 weeks of disease exhibited 52% to 76% greater PIP2 labeling, markedly elevated sorbitol levels, and 30% less myo-inositol when compared with age-matched normal rats. Incorporation of isotope into PIP2 in nerves from animals fed a myo-inositol supplement, added to either a high-sucrose diet or standard rat chow beginning immediately after induction of diabetes, remained substantially elevated, whereas myo-inositol levels were corrected to normal. Essentially the same results were obtained when rats were fed the myo-inositol-containing diet beginning 4 weeks after streptozotocin injection. In contrast, PIP2 labeling in nerves from diabetic rats that received the sorbinil-supplemented diet for either 4 or 8 weeks was not different from that in controls. myo-Inositol levels in these animals were also restored to normal, whereas sorbitol levels remained elevated, albeit reduced by approximately 30%. These results indicate that myo-inositol administration is unable to completely counteract the impact of diabetes on the turnover of monoesterified phosphate groups in PIP2. In contrast, sorbinil can correct this abnormality, but this beneficial effect is not dependent on the presence of normal sorbitol concentrations.

Activation of adenosine A2 receptors stimulates phosphoinositide metabolism in rat peripheral nerve.

The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [3H]-cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [3H]cytidine monophosphate phosphatidic acid [3H]-CMP-PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5'-N-ethylcarbox-amidoadenosine (NECA), a nonselective analogue, and two different A2-selective analogues, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and 2-p-(2-carboxyethyl)phenethylamino-NECA (CGS 21680), but not by N6-cyclopentyladenosine, an A1-selective analogue. The stimulatory action of CGS 21680 was blocked by the A2-selective adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 1,3-dipropyl-7-methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP-PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A2 subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine-receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.