Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells

Walz, Marjorie A.; Holt, I. Lee; Howlett, A­llyn C.

doi:10.1002/jnr.490170314

Cited by 4 publications

(3 citation statements)

References 22 publications

(4 reference statements)

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“…Divalent cations have been shown to regulate the activation of G s by effecting the GDP/GTP exchange in the absence of a hormone receptor stimulation (20,21). Consistent with previously published data (20), Fig.…”

Section: No Inhibits Adenylyl Cyclase Activity Without Altering Thesupporting

confidence: 90%

Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

McVey¹,

Hill²,

Howlett

et al. 1999

Journal of Biological Chemistry

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Nitric oxide (NO) donors inhibit hormone-and forskolin-stimulated adenylyl cyclase activity in purified plasma membrane preparations from N18TG2 neuroblastoma cells. Northern blot analyses indicate that the predominant isoform of adenylyl cyclase in N18TG2 cells is the type VI. Our experiments eliminate all the known regulatory proteins for this isoform as possible targets of NO. NO decreases the V max of the enzyme without altering the K m for ATP. Occupancy of the substrate-binding site protects the enzyme from the inhibitory effects of NO, suggesting that the conformation of the enzyme determines its sensitivity. The inhibition is reversed by reducing agents, implicating a Cys residue(s) as the target for nitric oxide and an S-nitrosylation as the underlying modification. These findings implicate NO as a novel cellular regulator of the type VI isoform of adenylyl cyclase. Nitric oxide (NO)1 has been attributed roles in a variety of cellular activities throughout the body. For example, NO has been implicated as a regulator of vasodilation, synaptic plasticity, and immune defense. In each case, however, NO has potentially deleterious effects should its production be disturbed, as occurs in excitotoxicity and ischemia (reviewed in Refs. 1-3). To understand the means by which NO achieves its paradoxical effects, and eventually to control its actions, attempts have focused on characterizing the enzymes responsible for NO production (NOS), identifying target molecules that are altered by NO, and specifying the underlying mechanism(s) by which NO alters those targets.One target of NO is the soluble guanylyl cyclase. It is the subsequent increase in cGMP levels that is believed to mediate NO-induced vasodilation. Given that an activation of soluble guanylyl cyclase occurs almost universally in response to NO, changes in cGMP have received primary consideration as the mechanism by which NO acts. However, the multiplicity of NO actions are unlikely to be explained by a common mechanism and a number of laboratories have suggested alternative targets of NO. Generally, cellular components have been implicated as targets of NO based largely on the ability of NO or NO-releasing compounds to alter their activities in vitro. Thus, whether they are altered in intact cells in response to NO, or what the possible physiological consequences may be, remain speculative. Our approach to discerning how NO functions has been to examine its effects on intact cells. We previously demonstrated that NO inhibits the production of cAMP pulses in Dictyostelium discoideum and does so independently of any changes in guanylyl cyclase activity (4, 5). The sum of the data indicated that NO specifically alters either a regulatory domain of the adenylyl cyclase itself or a distinct regulatory moiety. We have also observed that the addition of NO gas or NO donor compounds to cultures of N18TG2 neuroblastoma cells inhibits G s -coupled and forskolin-stimulated cAMP accumulation (6). NO-mediated inhibition of forskolin-stimulated cAMP production is ...

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Section: No Inhibits Adenylyl Cyclase Activity Without Altering Thesupporting

confidence: 90%

Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

McVey¹,

Hill²,

Howlett

et al. 1999

Journal of Biological Chemistry

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“…3). Both agonists increased intracellular cyclic GMP within 5 min (to 20 min, data not shown) in the presence of cyclic nucleotide phosphodiesterase inhibitors IBMX and the phosphodiesterase (PDE) 4 inhibitor rolipram, targeting a prominent isoform in this cell line (Walz et al, 1987). The response to cannabinoid drugs was blocked by a concentration of ODQ that is expected to inhibit NOdependent GC (Garthwaite et al, 1995), indicating that the detected cyclic GMP was due to enzymatic synthesis by this form of GC.…”

Section: Effects Of Cannabinoid Drugs On No-sensitive Gc In N18tg2 Cellsmentioning

confidence: 87%

Cannabinoid receptor-mediated translocation of NO-sensitive guanylyl cyclase and production of cyclic GMP in neuronal cells

et al. 2008

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Cannabinoid agonists regulate NO and cyclic AMP production in N18TG2 neuroblastoma cells, leading to the hypothesis that neuronal cyclic GMP production could be regulated by CB(1) cannabinoid receptors. NO (nitric oxide)-sensitive guanylyl cyclase (GC) is a heterodimeric cytosolic protein that mediates the down-stream effects of NO. Genes of proteins in the cyclic GMP pathway (alpha(1), alpha(2), and beta(1) subunits of NO-sensitive GC and PKG1, but not PKG2) were expressed in N18TG2 cells, as was the CB(1) but not the CB(2) cannabinoid receptor. Stimulation of N18TG2 cells by cannabinoid agonists CP55940 and WIN55212-2 increased cyclic GMP levels in an ODQ-sensitive manner. GC-beta(1) in membrane fractions was increased after 5 or 20 min stimulation, and was significantly depleted in the cytosol by 1h. The cytosolic pool of GC-beta(1) was replenished after 48 h of continued cannabinoid drug treatment. Translocation of GC-beta(1) from the cytosol was blocked by the CB(1) antagonist rimonabant (SR141716) and by the Gi/o inactivator pertussis toxin, indicating that the CB(1) receptor and Gi/o proteins are required for translocation. Long-term treatment with rimonabant or pertussis toxin reduced the amount of GC-beta(1) in the cytosolic pool. We conclude that CB(1) receptors stimulate cyclic GMP production and that intracellular translocation of GC from cytosol to the membranes is intrinsic to the mechanism and may be a tonically active or endocannabinoid-regulated process.

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“…These findings suggest that CaM-PDE may undergo developmental regulation, and that the 75-kDa peptide may serve as a marker for differentiation in neuroblastoma. Other labs have studied the patterns of phosphodiesterase isozymes in C 1 300-derived murine neuroblastomas and found very little CaM-PDE activity (Walz et al, 1987); however, it is likely that considerable variation exists between human and murine neuroblastoma lines.…”

Section: Discussionmentioning

confidence: 99%

Expression of Calmodulin‐Dependent Phosphodiesterase, Calmodulin‐Dependent Protein Phosphatase, and Other Calmodulin‐Binding Proteins in Human SMS‐KCNR Neuroblastoma Cells

Pennypacker

Kincaid

Polli

et al. 1989

Journal of Neurochemistry

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Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.

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Cyclic nucleotide phosphodiesterase isozymes in neuroblastoma cells

Cited by 4 publications

References 22 publications

Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

Adenylyl Cyclase, a Coincidence Detector for Nitric Oxide

Cannabinoid receptor-mediated translocation of NO-sensitive guanylyl cyclase and production of cyclic GMP in neuronal cells

Expression of Calmodulin‐Dependent Phosphodiesterase, Calmodulin‐Dependent Protein Phosphatase, and Other Calmodulin‐Binding Proteins in Human SMS‐KCNR Neuroblastoma Cells

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