Loss of Cnot6l Impairs Inosine RNA Modifications in Mouse Oocytes.

Mammalian oocytes must degrade maternal transcripts through a process called translational mRNA decay, in which maternal mRNA undergoes translational activation, followed by deadenylation and mRNA decay. Once a transcript is translationally activated, it becomes deadenylated by the CCR4-NOT complex. Knockout of CCR4-NOT Transcription Complex Subunit 6 Like (Cnot6l), a deadenylase within the CCR4-NOT complex, results in mRNA decay defects during metaphase I (MI) entry. Knockout of B-cell translocation gene-4 (Btg4), an adaptor protein of the CCR4-NOT complex, results in mRNA decay defects following fertilization. Therefore, mechanisms controlling mRNA turnover have significant impacts on oocyte competence and early embryonic development. Post-transcriptional inosine RNA modifications can impact mRNA stability, possibly through a translation mechanism. Here, we assessed inosine RNA modifications in oocytes, eggs, and embryos from Cnot6l-/- and Btg4-/- mice, which display stabilization of mRNA and over-translation of the stabilized transcripts. If inosine modifications have a role in modulating RNA stability, we hypothesize that in these mutant backgrounds, we would observe changes or a disruption in inosine mRNA modifications. To test this, we used a computational approach to identify inosine RNA modifications in total and polysomal RNA-seq data during meiotic maturation (GV, MI, and MII stages). We observed pronounced depletion of inosine mRNA modifications in samples from Cnot6l-/-, but not in Btg4-/- mice. Additionally, analysis of ribosome-associated RNA revealed clearance of inosine modified mRNA. These observations suggest a novel mechanism of mRNA clearance during oocyte maturation, in which inosine-containing transcripts decay in an independent, but parallel mechanism to CCR4-NOT deadenylation.

Deoxyinosine triphosphate induces MLH1/PMS2- and p53-dependent cell growth arrest and DNA instability in mammalian cells.

Deoxyinosine (dI) occurs in DNA either by oxidative deamination of a previously incorporated deoxyadenosine residue or by misincorporation of deoxyinosine triphosphate (dITP) from the nucleotide pool during replication. To exclude dITP from the pool, mammals possess specific hydrolysing enzymes, such as inosine triphosphatase (ITPA). Previous studies have shown that deficiency in ITPA results in cell growth suppression and DNA instability. To explore the mechanisms of these phenotypes, we analysed ITPA-deficient human and mouse cells. We found that both growth suppression and accumulation of single-strand breaks in nuclear DNA of ITPA-deficient cells depended on MLH1/PMS2. The cell growth suppression of ITPA-deficient cells also depended on p53, but not on MPG, ENDOV or MSH2. ITPA deficiency significantly increased the levels of p53 protein and p21 mRNA/protein, a well-known target of p53, in an MLH1-dependent manner. Furthermore, MLH1 may also contribute to cell growth arrest by increasing the basal level of p53 activity.

Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.

[Effect of prenatal hypobaric hypoxia on activity of the rat brain phosphoinositide system].

Activity of the phosphoinositide system of the intracellular signalization was studied in offspring of rats exposed to severe hypobaric hypoxia at the 14-16th (group 1) or the 18-20th day (group 2) of prenatal development. At the age of 15 days, in animals of both experimental groups the basal level of triphosphoinositides in the brain cortex was shown to be elevated as compared with control. In the group 1 this parameters also remains elevated in adult animals. Application of glutamate produces a more pronounced increase of the inositephosphates in brain sections of the 15-day old rats of the group 1 than in sections of animals of the control group. In the 15-day old rats of the group 2, as compared with control, the phosphoinositide response to glutamate application was reduced. No changes in the inositephosphate levels were revealed after application of glutamate upon sections of adult (the 90-day old) control animals and of adult rats of the group 2. In sections of adult rats of the group 1, on the contrary, the glutamate application produced an increase of the inositephosphate content. The obtained data indicate essential changes of the phosphoinositide metabolism in the brain of rats exposed to action of hypoxia at the period of prenatal development. The character and the degree of these changes depend on the period of development when the action of hypoxia occurs.

NUDT16 is a (deoxy)inosine diphosphatase, and its deficiency induces accumulation of single-strand breaks in nuclear DNA and growth arrest.

Nucleotides function in a variety of biological reactions; however, they can undergo various chemical modifications. Such modified nucleotides may be toxic to cells if not eliminated from the nucleotide pools. We performed a screen for modified-nucleotide binding proteins and identified human nucleoside diphosphate linked moiety X-type motif 16 (NUDT16) protein as an inosine triphosphate (ITP)/xanthosine triphosphate (XTP)/GTP-binding protein. Recombinant NUDT16 hydrolyzes purine nucleoside diphosphates to the corresponding nucleoside monophosphates. Among 29 nucleotides examined, the highest k(cat)/K(m) values were for inosine diphosphate (IDP) and deoxyinosine diphosphate (dIDP). Moreover, NUDT16 moderately hydrolyzes (deoxy)inosine triphosphate ([d]ITP). NUDT16 is mostly localized in the nucleus, and especially in the nucleolus. Knockdown of NUDT16 in HeLa MR cells caused cell cycle arrest in S-phase, reduced cell proliferation, increased accumulation of single-strand breaks in nuclear DNA as well as increased levels of inosine in RNA. We thus concluded that NUDT16 is a (deoxy)inosine diphosphatase that may function mainly in the nucleus to protect cells from deleterious effects of (d)ITP.

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa(-) mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa(-) embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa(-) primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa(-) MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa(-) embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa(-) embryos. However, immortalized Itpa(-) MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa(-) MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.

Structural basis for enzymatic evolution from a dedicated ADP-ribosyl cyclase to a multifunctional NAD hydrolase.

Cyclic ADP-ribose (cADPR) is a universal calcium messenger molecule that regulates many physiological processes. The production and degradation of cADPR are catalyzed by a family of related enzymes, including the ADP-ribosyl cyclase from Aplysia california (ADPRAC) and CD38 from human. Although ADPRC and CD38 share a common evolutionary ancestor, their enzymatic functions toward NAD and cADPR homeostasis have evolved divergently. Thus, ADPRC can only generate cADPR from NAD (cyclase), whereas CD38, in contrast, has multiple activities, i.e. in cADPR production and degradation, as well as NAD hydrolysis (NADase). In this study, we determined a number of ADPRC and CD38 structures bound with various nucleotides. From these complexes, we elucidated the structural features required for the cyclization (cyclase) reaction of ADPRC and the NADase reaction of CD38. Using the structural approach in combination with site-directed mutagenesis, we identified Phe-174 in ADPRC as a critical residue in directing the folding of the substrate during the cyclization reaction. Thus, a point mutation of Phe-174 to glycine can turn ADPRC from a cyclase toward an NADase. The equivalent residue in CD38, Thr-221, is shown to disfavor the cyclizing folding of the substrate, resulting in NADase being the dominant activity. The comprehensive structural comparison of CD38 and APDRC presented in this study thus provides insights into the structural determinants for the functional evolution from a cyclase to a hydrolase.

ITPase-deficient mice show growth retardation and die before weaning.

Inosine triphosphate pyrophosphatase (ITPase), the enzyme that hydrolyzes ITP and other deaminated purine nucleoside triphosphates to the corresponding purine nucleoside monophosphate and pyrophosphate, is encoded by the Itpa gene. In this study, we established Itpa knockout (KO) mice and used them to show that ITPase is required for the normal organization of sarcomeres in the heart. Itpa(-/-) mice died about 2 weeks after birth with features of growth retardation and cardiac myofiber disarray, similar to the phenotype of the cardiac alpha-actin KO mouse. Inosine nucleotides were found to accumulate in both the nucleotide pool and RNA of Itpa(-/-) mice. These data suggest that the role of ITPase in mice is to exclude ITP from the ATP pool, and the main target substrate of this enzyme is rITP. Our data also suggest that cardiomyopathy, which is mainly caused by mutations in sarcomeric protein-encoding genes, is also caused by a defect in maintaining the quality of the ATP pool, which is an essential requirement for sarcomere function.

Localization of the cyclic ADP-ribose-dependent calcium signaling pathway in bovine rod outer segments.

PURPOSE: Calcium ions play a pivotal role in phototransduction. In this study, the presence and functional role of the adenosine diphosphoribosyl (ADPR)-cyclase-cyclic ADP-ribose (cADPR) system in bovine retinal rod outer segments (ROS) was investigated. METHODS: A Ca(2+) release from osmotically intact ROS discs elicited by cADPR was studied in the presence of the Ca(2+) tracer fluo-3. Endogenous cyclic guanosine diphosphate ribose (cGDPR) formation in discs was investigated by spectrophotometric detection of its synthesis from nicotinamide guanine dinucleotide (NGD(+)). ADPR-cyclase was also investigated at a structural level on mildly denaturing SDS-PAGE by production of cyclic inosine diphosphate ribose from nicotinamide hypoxantine dinucleotide (NHD(+)). Western immunoblot analysis with a specific antibody was conducted to verify the presence of ryanodine-sensitive Ca(2+) channels (RyRs) in ROS discs. RESULTS: cADPR-dependent Ca(2+) release was a linear function of extravesicular free Ca(2+) concentration, between 200 and 900 nM Ca(2+). When free Ca(2+) was 203 +/- 10 nM the mean Ca(2+) release was 23 +/- 3 pmol/mL per milligram protein. The average rate of cGDPR production was 13 +/- 2 nmol cGDPR/min per milligram protein, by a putative enzyme with an apparent molecular mass of 53 +/- 1 kDa. ROS ADPR-cyclase was localized in the membranous fraction. No nicotinamide adenine dinucleotide glycohydrolase (NADase) activity was detected. The presence of RyR channels in pure disc preparations was confirmed by confocal laser scanning microscopy. CONCLUSIONS: A cADPR metabolism may be present in retinal ROS discs, which may be Ca(2+) stores operated by cADPR. A model is proposed for the physiological role of cADPR-mediated Ca(2+) release in bovine ROS.

Plasma from patients exposed to ischemia reperfusion contains clastogenic factors and stimulates the chemiluminescence response of normal leukocytes.

Clastogenic factors (CFs) are released by cells exposed to superoxide radicals and are found in various situations of oxidative stress. Certain of their components stimulate further superoxide production by competent cells, as shown with cytochrome c assay in previous work. In the present study, we report CF formation after ischemia reperfusion in patients undergoing coronary bypass surgery. Plasma ultrafiltrates, collected 20 min after reperfusion, had clastogenic properties in contrast to those collected before ischemia. We also show that the luminol-enhanced chemiluminescence response of neutrophils from healthy persons is increased when these cells are exposed to CF-containing postreperfusion samples from patients. Light emission was reduced to control values in the presence of superoxide dismutase. The burst of oxyradicals upon reperfusion is probably the initiating event of CF formation, which in turn leads to further oxyradical generation. This amplification process may explain why detectable levels of CF need a delay of at least 10 min. The activated state of neutrophils in ischemia reperfusion is at once a consequence and a source of CFs. Individual variation in the persistence of this clastogenic and leukocyte-activating material was observed. Therefore, antioxidants for prevention of ischemia reperfusion injury should be continued during the postoperative course.

Glutamine 170 to tyrosine substitution in yeast mitochondrial F1 beta-subunit increases catalytic site interaction with GDP and IDP and produces negative cooperativity of GTP and ITP hydrolysis.

Glutamine 170 to tyrosine mutation in the beta-subunit from Schizosaccharomyces pombe mitochondrial F1 was found to increase both affinity for ADP, apparent negative cooperativity of ATPase activity, and sensitivity to azide inhibition (Falson, P., Di Pietro, A., Jault, J.-M., Gautheron, D.C., and Boutry, M. (1989) Biochim. Biophys. Acta 975, 119-126). The mutation is shown here to increase the affinity for GDP, IDP, and guanosine 5'-(beta,gamma-imidotriphosphate), which are competitive inhibitors of GTPase and ITPase activities. Various fluorescence approaches also reveal an increased affinity of the catalytic site in mutant as compared with wild-type enzyme for GDP, IDP, and 2'(3')-N-methylanthraniloyl GDP. The mutation alters the maximal rates and pH dependence of GTPase and ITPase activities, whereas wild-type F1 exhibits single optima at pH 7.5-8.0. The pH activity profiles of the mutant enzyme for these substrates are biphasic, with optima at pH 8.5-9.0 and below 6.5. The mutation increases the sensitivity of GTPase and ITPase activities to azide inhibition, which increases with decreasing pH. At pH 6.0-7.0, an apparent negative cooperativity is observed when mutant F1 hydrolyzes GTP or ITP, whereas the wild-type enzyme shows Michaelian kinetics. Addition of bicarbonate at pH 7.0 substantially stimulates GTP or ITP hydrolysis and abolishes the apparent negative cooperativity by the mutant enzyme; on the contrary, the anion produces a slight inhibition of these activities catalyzed by wild-type F1. The overall results suggest that apparent negative cooperativity can be observed with GTP or ITP hydrolysis provided that the release of the respective diphosphate is a rate-limiting step.

Free ADP-ribose in human erythrocytes: pathways of intra-erythrocytic conversion and non-enzymic binding to membrane proteins.

We have previously identified free ADP-ribose (ADPR) as a normal metabolite in mature human erythrocytes. In this study the metabolic transformations of ADPR were investigated in both supernatants from erythrocyte lysates and intact erythrocytes, loaded with ADPR by means of a procedure involving hypotonic haemolysis and isotonic resealing. In both experimental systems, the main pathway was a dinucleotide pyrophosphatase-catalysed hydrolysis to yield AMP, which was readily converted into the adenylic and inosinic nucleotide pools. To a lesser extent, ADPR underwent conversion into a compound that was identified as ADP-ribulose (ADPRu), on the basis of m.s., n.m.r. spectroscopy and enzymic analysis. ADPRu was also susceptible to degradation by the dinucleotide pyrophosphatase, which was partially purified from erythrocyte lysates and characterized with respect to its substrate specificity. Isomerization of ADPR to ADPRu was markedly enhanced by ATP. Incubation of unsealed haemoglobin-free erythrocyte membranes with labelled ADPR did not cause any transformation of this nucleotide and resulted in its trichloroacetic acid- and formic acid-resistant binding to a number of membrane cytoskeletal proteins. These proteins include spectrin, glyceraldehyde 3-phosphate dehydrogenase (Ga3PDH), three proteins of molecular masses 98, 79 and 72 kDa, which apparently comigrate with bands 3, 4.1 and 4.2 respectively, and two additional proteins of molecular masses 58 and 41 kDa. Acid-resistant binding of ADPR, as well as of NAD+, to Ga3PDH was confirmed for the enzyme purified from human erythrocytes.

Reduced alpha 1-adrenergic receptor-mediated inositide hydrolysis in cardiac atria of senescent rats.

We investigated the effect of age on epinephrine stimulation of phosphoinositide hydrolysis in atrial slices prepared from F-344 female rats. Three age groups were chosen for study: young adults (aged 6 months), mature adults (aged 15 months), and senescent animals (aged 25 months). Tissue slices were labeled with [3H]myoinositol and epinephrine-stimulated hydrolysis measured in the presence of LiCl. Epinephrine caused a dose-dependent increase in phosphoinositide hydrolysis in each age group. This increase was blocked by prazosin, suggesting that alpha 1-adrenergic receptors are involved. In animals aged 6 months, epinephrine caused a maximal increase in hydrolysis of 2.8-fold over basal. The maximal response was reduced at 15 months (2.52-fold increase, p less than 0.05) and at 25 months (2.02-fold increase, p less than 0.01). The potency for epinephrine stimulation of phosphoinositide hydrolysis was unchanged with age. The data indicate that alpha 1-mediated phosphoinositide hydrolysis in atria is reduced with age.

Influence of ATP turnover and metabolite changes on IMP formation and glycolysis in rat skeletal muscle.

Deamination of AMP to inosine monophosphate (IMP) and NH3 is thought to be regulated by the observed increases in ADP, AMP, and H+. We have examined this hypothesis by comparing the rate of IMP accumulation in contracting and noncontracting rat skeletal muscle. The rate of IMP formation was high during ischemic contraction, and consistent with previous studies, formation of IMP was associated with high levels of muscle lactate, depletion of phosphocreatine (PCr), and increased levels of ADP and AMP. When the contraction period was followed by 5-min anoxic recovery, the metabolic changes were maintained, but no further IMP or lactate was formed. During long-term (2-4 h) anoxia, the rate of IMP formation was less than 4% of that during contraction, despite similar changes in PCr, lactate, ADP, and AMP. It is concluded that the observed changes in the intracellular chemical environment are not sufficient to explain the high rate of IMP formation during contraction but that a combination of metabolic stress and a high ATP turnover rate is required. It is suggested that a high ATP turnover rate during conditions of metabolic stress results in transient increases in ADP and AMP at the site of ATP hydrolysis and that these activate AMP deaminase and glycolysis. An alternative hypothesis is that these processes are regulated by the increase in cytosolic Ca2+ in a contracting muscle.

Comparison of the self-association properties of the 5'-triphosphates of inosine (ITP), guanosine (GTP), and adenosine (ATP). Further evidence for ionic interactions in the highly stable dimeric [H2(ATP)]2(4-) stack.

The concentration dependence of the chemical shifts for the hydrogens H-2, H-8 and H-1' of ITP and for H-8 and H-1' of GTP has been measured in D2O at 25 degrees C under several degrees of protonation in the pD range 1.2-8.4. For reasons of comparison, inosine and guanosine have been included in the study The results are consistent with the isodesmic model of indefinite noncooperative stacking. The association constants for the nucleosides (Ns) inosine and guanosine decrease with increasing protonation: Ns greater than D(Ns)+/Ns in a 1:1 ratio greater than D(Ns)+. In contrast, a maximum is observed with ITP and GTP; the stacking tendency of GTP following the series: GTP4- less than or equal to D(GTP)3- (K approximately 0.7 M-1) less than D(GTP)3-/D2(GTP)2- in a 1:1 ratio (K approximately 2.9 M-1) greater than D2(GTP)2- greater than D3(GTP)- (K approximately 1.5 M-1). The order of the series with ITP corresponds to that with GTP, but the association constants are slightly smaller. At the maximum of the self-association tendency the triphosphate residue has only a minor influence; this follows from the fact that the association constants for the 1:1 ratios of Ino/D(Ino)+ and D(ITP)3-/D2(ITP)2- are identical within experimental error; this holds also for Guo/D(Guo)+ and D(GTP)3-/D2(GTP)2-; in all these pairs the K-7 site is 50% protonated. Comparison of the association constant for the deprotonated species shows that here charge effects, i.e. repulsion between the negatively charged triphosphate chains, are important: Ino (K approximately 3.3 M-1) greater than ITP4- (K approximately 0.4 M-1) and Guo (K approximately 8 M-1) greater than GTP4- (K approximately 0.8 M-1). In addition the series holds: Ado (K approximately 15 M-1) greater than Guo greater than Ino. However, most important is the comparison of the ITP and GTP series with previous data for ATP: ATP4- (K approximately 1.3 M-1) less than D(ATP)3- (2.1 M-1) less than 1:1 ratio of D(ATP)3-/D2(ATP)2- (6 M-1) much less than D2(ATP)2- (approximately 200 M-1) much greater than D3(ATP)- (K less than or equal to 17 M-1).(ABSTRACT TRUNCATED AT 400 WORDS)

[Enzymes of the inosinic crossing point in human lymphocytes]. / Enzimi del punto d'incrocio inosinico in linfociti umani.

At the "inosinic branch point", inosinic acid (IMP) can be channelled either to guanylic acid (GMP) or to adenylic acid (AMP). The 4 enzymes involved in these processes are IMP-dehydrogenase (IMP-DH) and GMP synthetase for the formation of GMP and adenylosuccinate (AMP-S) synthetase and lyase for the formation of AMP. The Authors study the behavior of these enzymes in peripheral blood lymphocytes from normal and leukemic patients. The cells were isolated as previously reported. GMP synthetase was assayed with radiochemical method, IMP-DH and AMP-S synthetase with a radiochemical method coupled to HPLC, while AMP-S lyase was determined following the formation of AMP separated by AMP-S by HPLC, without using labelled precursors. Except for GMP synthetase, which was very low, no activity was detectable in normal lymphocytes; while AMP-S was absent also in leukemic cells, the remaining three activities were well evident. The results open the possibility of using the inosinic branch point enzymes as tumor markers.

The cholinesterase inhibitor soman increases inositol trisphosphate in rat brain.

Studies were conducted to determine the effect of the cholinesterase inhibitor soman on the amount of inositol trisphosphate in the neocortex, striatum, cerebellum, and medulla-pons regions of rat brain in vivo. The studies indicate that treatment with soman increases inositol trisphosphate in the neocortex and striatum, but not in the cerebellum or medulla-pons region. In the neocortex the most pronounced increases were observed in animals with severe poisoning symptoms, however inositol trisphosphate was also found to be elevated in animals with only mild poisoning symptoms.

DNA-synthesis regulation and correlation with inositol trisphosphate levels in cultured bovine retinal capillary pericytes.

Inositol phosphate (IP), inositol bisphosphate (IP2) and inositol trisphosphate (IP3) in cultured bovine retinal capillary pericytes (BRCP) were quantitated by an ion-pair reverse-phase HPLC. BRCP were grown in media with standard (5 mM) or high (30 mM) glucose, and were either labeled with myo-[2-3H]inositol (20 microCi ml-1) for 60 hr or with dual isotopes (20 microCi ml-1 myo-[2-3H]inositol and 2 microCi ml-1 [14C]glycerol) for 8 hr. In parallel, BRCP in different glucose-media were incubated with 1 microCi ml-1 [3H]thymidine for 4 hr. High glucose significantly suppressed the accumulation of [3H]label in IP, IP2 and IP3, and specifically reduced the incorporation of [14C]glycerol into inositol phospholipids, but not that of neutral lipids and other types of phospholipids. The reduced IP3 level correlated with the decrease in the incorporation of [3H]thymidine into DNA. Both the reduced IP3 formation and DNA synthesis which were induced by high glucose were significantly reversed by adding either myo-inositol or AL1576, an aldose reductase inhibitor (ARI). However, the addition of neither myo-inositol nor ARI stimulated IP3 formation and/or DNA synthesis when BRCP were grown in the standard medium (5 mM glucose). These findings indicate that myo-inositol metabolism and the polyol pathway affect inositol phospholipid-mediated pericyte division in vitro only under the high-glucose condition. These data are compatible with the hypothesis that altered inositol phospholipid metabolism accounts for the loss of pericytes in diabetic retinopathy.

The effect of M&B 22948 on methacholine- and histamine-induced contraction and inositol 1,4,5-trisphosphate levels in guinea-pig tracheal tissue.

The effect of a cyclic GMP phosphodiesterase inhibitor, M&B 22948, on methacholine- and histamine-induced contraction and inositol 1,4,5-trisphosphate (IP3) elevation was studied in guinea-pig tracheal rings. After addition of methacholine or histamine the rise in IP3 content was rapid and transient reaching a maximum after 5-15 s, which coincided with the maximum rate of tension development. Cyclic GMP levels of the tissue were elevated by M&B 22948 before agonist stimulation and further elevated by addition of methacholine or histamine. Cyclic AMP levels were not altered by any of these agents. M&B 22948 abolished IP3 generation induced by methacholine or histamine, but did not alter the rate or magnitude of tension development. Thus, IP3 generation does not appear to be responsible for the contractions induced by methacholine or histamine in this tissue.