Immunohistochemical localization of 2′,3′-cyclic nucleotide 3′-phosphodiesterase in adult bovine cerebrum and cerebellum

We have previously shown that 2,3-cyclic nucleotide 3-phosphodiesterase (CNP; EC 3.1.4.37) in rat central nervous tissues can be immunohistochemically stained with anti-bovine CNP serum. However, the anti-bovine CNP serum prepared in our laboratory has only weak cross-reactivity with rat CNP. Sections of bovine nervous tissues were found to be stained effectively with the serum, and the localization of CNP has been revealed in greater detail. We describe here the immunohistochemical localization of CNP in adult bovine cerebrum and cerebellum. CNP stained was localized in myelin sheaths, oligodendrocytes, and the processes of oligodendrocytes; astrocytes and neurons were negative. All myelinated nerve fibers appeared to be stained with the anti-CNP serum. Perineuronal and perivascular oligodendrocytes, and oligodendrocytes extending their processes to isolated myelin fibers were stained. Interfascicular oligodendrocytes, however, did not react or reacted faintly to the anti-CNP serum; only their processes were reactive. Comparison with the stain for S-100 protein was helpful to distinguish oligodendrocytes from astrocytes particularly when both glial cells were situated together at the perineuronal and perivascular positions.Dedicated to Professor Yasuzo Tsukada.     

Topology of wolfgram proteins and 2′, 3′-cyclic nucleotide 3′-phosphodiesterase in CNS myelin: Studies with proteases

The topological disposition of Wolfgram proteins (WP) and their relationship with 2, 3-cyclic nucleotide 3-phosphodiesterase (CNPase) in human, rat, sheep, bovine, guinea pig and chicken CNS myelin was investigated. Controlled digestion of myelin with trypsin gave a 35KDa protein band (WP-t) when electrophoresed on dodecyl sulfate-polyacrylamide gel in all species. Western blot analysis showed that the WP-t was derived from WP. WP-t was also formed when rat myelin was treated with other proteases such as kallikrein, thermolysin and leucine aminopeptidase. Staining for CNPase activity on nitrocellulose blots showed that WP-t is enzymatically active. Much of the CNPase activity remained with the membrane fraction even after treatment with high concentrations of trypsin when WP were completely hydrolysed and no protein bands with M.W>14KDa were detected on the gels. Therefore protein fragments of WP with M.W<14KDa may contain CNPase activity. From these results, it is suggested that the topological disposition of all the various WP is such that a 35KDa fragment is embedded in the lipid bilayer and the remaining fragment exposed at the intraperiod line in the myelin structure which may play a role in the initiation of myelinogenesis.     

Distribution of lipid synthesizing enzymes, 2′,3′-cyclic nucleotide 3′-phosphodiesterase,and myelin proteins in rat forebrain subfractions during development

The distribution of UDP-galactose: ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2,3-cyclic nucleotide 3-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose: ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was ferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.     

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity in cultured nerve cell lines from central nervous system: Comparison of proliferating and resting growth states and cell cycle-dependent activity changes

1. The present communication is concerned with the expression and cell cycle-dependent regulation of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in cultured nerve cell lines derived from the rat central nervous system (CNS). 2. The enzyme activity was measured in relation to two reversible serum-controlled growth states (exponentially growing/quiescent) including a comparison of the enzyme activities in cell lines of neuronal and glial origin as well as in fibroblasts. CNPase is present in all cell types tested, but the enzyme activity is very sensitive to changes in the cellular growth state. Nerve cell lines in exponentially growing cultures express a 3 to 15 times higher specific CNPase activity than the nonneural cell types. In serum-starved quiescent cultures, the differences in specific enzyme activity between the nerve cell lines and the fibroblasts are enlarged even more up to a ratio of about 50 to 150, indicating a specific function of this enzyme within the central nervous system. 3. Neuron-like B104 cells could be stimulated to synchronized growth by serum readdition to quiescent cultures. A series of ordered activity changes of CNPase has been observed after the reinitiation of cell growth. The enzyme is stimulated at two particular stages during the cell cycle, leading to a biphasic activity profile. Maximum stimulation of CNPase correlates with the G1 phase. 4. Hydroxyurea-induced blockage of synchronized B104 cells to traverse the S phase also prevents the subsequent stimulation of CNPase activity. Therefore, we conclude that a correlation exists between the periodic activity changes of CNPase and particular phases of the B104 cell cycle.     

Localization of 3′, 5′-cyclic nucleotide phosphodiesterase activity in isolated thyroid cells and intact thyroid tissue

Summary Cytochemical localization of 3,5-cyclic nucleotide phosphodiesterase (cPDEase) has been investigated by light and electron microscopy in dissociated bovine thyroid cells and in intact bovine thyroid tissue. By light microscopy in isolated thyroid cells reaction product deposition associated with cPDEase activity was localized at the level of the plasma membrane. In intact cryostat cut thyroid tissue, the activity was primarily observed in the cytoplasm and to a lesser extent at the level of the plasma membrane. By electron microscopy, cPDEase activity in isolated cells was found on the plasma membrane and was also encountered on the inner surface of membrane bound vacuoles, presumably pinocytic in origin. In intact tissue, cPDEase activity appeared mostly localized on the apical and lateral plasma membranes and was also present on the outer surface of the endoplasmic reticulum (ER).Even though cPDEase and 5-AMPase did share the same plasma membrane localization, the inhibitory response to theophylline and stimulation with Imidazole permitted the dissociation of their respective activities. 5-AMPase failed to respond to either theophylline or Imidazole suggesting absence of cross reactivity between 5-AMP and cyclic AMP. Thyrotropin (TSH) had no effect on cPDEase activity.We conclude that: (1) regardless of the nature of the material used, the cytomembranes of thyroid cells possess cPDEase activity; and that (2) the variability in distribution as well as in staining intensity recorded by light and electron microscopy between isolated thyroid cells and cryostat cut thyroid tissue is probably inherent to the methodology used.This paper was presented, in part, at the 60th Annual Meeting of the International Academy of Pathology, Montreal, Canada, March 1971 and was initiated in the Department of Pathology, Rhode Island Hospital, Providence, R.I., supported from a Grant-in-Aid of the American Cancer Society, Rhode Island Division, Inc. and the Brown-Hazen Fund.     

Density distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase and myelin proteins in particulate material from myelin deficient (mld) mutant and control brains

Total particulate material from control and myelin deficient (mld) brains was subjected to density centrifugation on a continuous sucrose gradient. Particles from control brains distributed in a bell-shaped mode with a peak density near 0.64 M-sucrose. In mld material only a slight elevation of optical density was observed near 0.8 M-sucrose. The highest specific activities of 2′,3′-cyclic nucleotide 3′-phosphodiesterase were observed at densities of 0.63 and 0.71 M-sucrose for mld and control brains, respectively. The peak of myelin basic protein in control fractions was near 0.60 M-sucrose. In mld fractions no peak was observed. Proteolipid and Wolfgram proteins had a maximum near 0.65 and 0.73 M-sucrose in control and mld fractions, respectively. The absence of myelin basic proteins in all the fractions makes it unlikely that, in mld mice, myelin basic proteins are synthesized but not incorporated into myelin.     

Interaction of myelin basic protein and 2′,3′-cyclic nucleotide phosphodiesterase with mitochondria

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and non-synaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca²⁺ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.     

Identification of phosphorylated form of 2′, 3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) as 46 kDa phosphoprotein in brain non-synaptic mitochondria overloaded by calcium

In our previous studies phosphorylation of several membrane-bound proteins in brain and liver mitochondria were found to be regulated by Ca²⁺ as a second messenger. One of the proteins, the 46 kDa phosphoprotein was found to be highly phosphorylated when Ca²⁺-induced permeability transition pore (mPTP) was opened in rat brain mitochondria (RBM). In the present study the 46 kDa phosphoprotein was identified as 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) after purification by 2D diagonal electrophoresis following mass spectrometric analysis and Western blot probed with anti-CNP antibody. CNPase was discovered in immunoprecipitates of mitochondria, phosphorylated under both conditions (control and with opened mPTP). Status phosphorylation of CNPase was found to be higher in the inmmunoprecipiates of calcium-overloaded RBM. The phospohoserine and phosphotyrosine residues were detected in phosphorylated 46 kDa band (CNPase) as well as in CNPase immunoprecipitates indicating possible participation of tyrosine and serine protein kinases in phosphorylation of CNPase in mitochondria. The levels of phospo-Ser and phospho-Tyr were increased in RBM with mPTP opened. It was found that CNPase substrate, 2′,3′-cAMP (5 μM) and, a non-competitive CNPase inhibitor, atractyloside (5 μM), were able to increase the level of CNPase phosphorylation in calcium-overloaded mitochondria, while CsA (mPTP blocker) was able to strong suppress the phosphorylation of the enzyme. Collectively, our results provide evidence that Ca²⁺-stimulated and mPTP-associated CNPase phosphorylation might be an important stage of mPTP regulation in mitochondria, revealing a new function of CNPase outside of myelin structure.     

Insulin: Its binding to specific receptors and its stimulation of DNA synthesis and 2′,3′-cyclic nucleotide phosphohydrolase activity in cerebral cells cultured from embryonic mouse brain

The presence and specificity of insulin receptors was investigated in cultured cells obtained from 15–16 days old embryonic mouse cerebra. Developmental studies suggested that the maximum insulin binding occurred at about 11 days in vitro (DIV). Scatchard analysis of binding data revealed two types of binding sites. One type of receptor was the high affinity type (K _d=7.77×10^–9 M; number of receptor sites,B _max=350 fmol/mg protein) while the other type was of low affinity type (K _d=5.75×10^–8 M;B _max=1150 fmol/mg protein). The specificity of receptors for insulin was also confirmed by showing that [¹²⁵I]insulin was displaced by non-radioactive insulin but not by glucagon or growth hormone. Insulin displayed a clear dose-dependent stimulation of thymidine incorporation. It also stimulated the activity of the enzyme 2,3-cyclic nucleotide phosphohydrolase (CNPase), which is specifically associated with myelin produced from oligodendroglia. Thus insulin has a positive influence on the proliferation and differentiation of brain cells.     

Generation of a monoclonal antibody against the myelin protein CNP (2′,3′-cyclic nucleotide 3′-phosphodiesterase) suitable for biochemical and for immunohistochemical investigations of CNP

The functional role of CNP (2,3-cyclic nucleotide 3-phosphodiesterase), a minor component of central and peripheral myelin is still unclear. Here we describe preparation of a monoclonal antibody directed against CNP. The antibody, of the immunoglobulin IgG₁ type, raised with a basic 46 kDa membrane-associated protein solubilized from pig cerebellar membranes, can be used to detect immunoreactivity in solubilized brain homogenates from pig, mouse, rat, sheep, cow and man, in cerebrum and cerebellum, but not in other tissues such as liver, skeletal and heart muscle. The antibody recognizes the CNP doublet band and shows no cross-reactivity with any of the other brain proteins solubilized. In tissue sections from paraformaldehyde-fixed rat brain the antigen was localized in oligodendrocytes. In cultured glial cells from newborn mice the antibody stained cells which were identified as oligodendrocytes by co-localization of myelin basic protein. Even cells from a C6 rat glioma cell line, which contain very little of CNP, were labeled by the monoclonal antibody. Thus the monoclonal antibody recognizing CNP from several species is suitable for immunocytochemical investigations and also for biochemical studies of CNP, since the antibody has been employed for immunoprecipitation and immunopurification of CNP in crude brain homogenates.     

Select 3′,5′-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain

3′,5′-cyclic nucleotide phosphodiesterases (PDEs) are the only known enzymes to compartmentalize cAMP and cGMP, yet little is known about how PDEs are dynamically regulated across the lifespan. We mapped mRNA expression of all 21 PDE isoforms in the adult rat and mouse central nervous system (CNS) using quantitative polymerase chain reaction (qPCR) and in situ hybridization to assess conservation across species. We also compared PDE mRNA and protein in the brains of old (26 months) versus young (5 months) Sprague–Dawley rats, with select experiments replicated in old (9 months) versus young (2 months) BALB/cJ mice. We show that each PDE isoform exhibits a unique expression pattern across the brain that is highly conserved between rats, mice, and humans. PDE1B, PDE1C, PDE2A, PDE4A, PDE4D, PDE5A, PDE7A, PDE8A, PDE8B, PDE10A, and PDE11A showed an age-related increase or decrease in mRNA expression in at least 1 of the 4 brain regions examined (hippocampus, cortex, striatum, and cerebellum). In contrast, mRNA expression of PDE1A, PDE3A, PDE3B, PDE4B, PDE7A, PDE7B, and PDE9A did not change with age. Age-related increases in PDE11A4, PDE8A3, PDE8A4/5, and PDE1C1 protein expression were confirmed in hippocampus of old versus young rodents, as were age-related increases in PDE8A3 protein expression in the striatum. Age-related changes in PDE expression appear to have functional consequences as, relative to young rats, the hippocampi of old rats demonstrated strikingly decreased phosphorylation of GluR1, CaMKIIα, and CaMKIIβ, decreased expression of the transmembrane AMPA regulatory proteins γ2 (a.k.a. stargazin) and γ8, and increased trimethylation of H3K27. Interestingly, expression of PDE11A4, PDE8A4/5, PDE8A3, and PDE1C1 correlate with these functional endpoints in young but not old rats, suggesting that aging is not only associated with a change in PDE expression but also a change in PDE compartmentalization.     

Induction of 2′,3′-cyclic nucleotide 3′-phosphohydrolase and morphological alterations in C6 glioma cells by dexamethasone, (3-butoxy-4-methoxybenzyl)-2-imidazolinone and prostaglandin E1

Summary Dexamethasone, R020-1724 and prostaglandin E₁ all induced morphological alterations and increased the glial specific enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) in rat C6 glioma cells in culture. Morphological alterations consisted mainly in the development of astrocytelike changes. Increases in dexamethasone-induced CNP activity was time dependent. Dexamethasone reduced cell growth rate, depending on the concentration employed. This paper is supported in part by N.I.M.H. Research Grant AA02372. Dr. S. Kamath participated initially in this study.     

Simultaneous inhibition of guinea pig brain 2′, 3′-cyclic nucleotide 3′-phosphohydrolase and myelin protein synthesis by 2′-adenosine monophosphate

Although the function of 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) in myelin is unknown, the enzyme has been implicated in the metabolism of myelin proteins. Using 2′-AMP to inhibit CNPase, we examined the effect of reduced enzyme activity on the $\text{in vitro}$ incorporation of ¹⁴C-leucine into brain proteins. The results of this study revealed that (1) guinea pig brain homogenates incorporate leucine into protein from a sucrose medium in a linear fashion, (2) all brain fractions (cytosol, myelin, and microsomes) are labelled within 1 hr, (3) 2′-AMP inhibition of CNPase by 50% results in a similar inhibition of brain protein synthesis, and (4) the reduced protein synthesis is accompanied by a shift in label from myelin proteins to those found in the microsomes. These results are consistent with a role for CNPase in myelin protein synthesis.     

Identification of cytidine 2′,3′-cyclic monophosphate and uridine 2′,3′-cyclic monophosphate in Pseudomonas fluorescens pfo-1 culture

Cytidine 2′,3′-cyclic monophosphate (2′,3′-cCMP) and uridine 2′,3′-cyclic monophosphate (2′,3′-cUMP) were isolated from Pseudomonas fluorescens pfo-1 cell extracts by semi-preparative reverse phase HPLC. The structures of the two compounds were confirmed by NMR and mass spectroscopy against commercially available authentic samples. Concentrations of both intracellular and extracellular 2′,3′-cCMP and 2′,3′-cUMP were determined. Addition of 2′,3′-cCMP and 2′,3′-cUMP to P. fluorescens pfo-1 culture did not significantly affect the level of biofilm formation in static liquid cultures.     

Stress-induced formation of echinatin and a metabolite, 5′-prenyl-licodione,in cultured Glycyrrhiza echinata cells

The production of a retrochalcone, echinatin, by isoflavone-rich Glycyrrhiza echinata (M-2) cultured cells was stimulated by the addition of yeast extract or calcium alginate beads to the culture medium. Combined addition of yeast extract and cycloheximide suppressed the formation of retrochalcone, suggesting de novo synthesis. A new metabolite was isolated from the induced cells and its structure was determined to be 1-[2,4-dihydroxy-5-(3-methyl-2-butenyl)phenyl]-3-(4-hydroxyphenyl)-1,3-propanedione (5′-prenyl-licodione).     

Differential expression of FCRLA in naïve and activated mouse B cells

FCRLA is an intracellular B cell protein that belongs to the FcR-like family. Using newly generated FCRLA-specific antibodies, we studied the constitutive expression pattern of mouse FCRLA and monitored changes during an immune response and following in vitro B cell activation. All B cell subpopulations examined expressed FCRLA. However, the level of FCRLA expression is determined by the stage of B cell differentiation. Low expression of FCRLA is characteristic of naïve follicular and marginal zone B cells. High expression was detected in a small fraction of activated B cells scattered along migratory pathways in the lymphoid tissues. FCRLA-bright cells could be subdivided into two subpopulations, with high and low/undetectable level of intracellular immunoglobulins, which phenotypically resemble either plasma or memory B cells. High expression of FCRLA in subset(s) of terminally differentiated B-cells suggests that, being an ER protein, FCRLA may participate in the regulation of immunoglobulin assembly and secretion.