Abscisic acid signaling through cyclic ADP-ribose in plants.

Abscisic acid (ABA) is the primary hormone that mediates plant responses to stresses such as cold, drought, and salinity. Single-cell microinjection experiments in tomato were used to identify possible intermediates involved in ABA signal transduction. Cyclic ADP-ribose (cADPR) was identified as a signaling molecule in the ABA response and was shown to exert its effects by way of calcium. Bioassay experiments showed that the amounts of cADPR in Arabidopsis thaliana plants increased in response to ABA treatment and before ABA-induced gene expression.

Subcellular localization of cyclic ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase activities in porcine airway smooth muscle.

Recent studies have provided evidence for a role of cyclic ADP-ribose (cADPR) in the regulation of intracellular calcium in smooth muscles of the intestine, blood vessels and airways. We investigated the presence and subcellular localization of ADP-ribosyl cyclase, the enzyme that catalyzes the conversion of beta-NAD(+) to cADPR, and cADPR hydrolase, the enzyme that degrades cADPR to ADPR, in tracheal smooth muscle (TSM). Sucrose density fractionation of TSM crude membranes provided evidence that ADP-ribosyl cyclase and cADPR hydrolase activities were associated with a fraction enriched in 5'-nucleotidase activity, a plasma membrane marker enzyme, but not in a fraction enriched in either sarcoplasmic endoplasmic reticulum calcium ATPase or ryanodine receptor channels, both sarcoplasmic reticulum markers. The ADP-ribosyl cyclase and cADPR hydrolase activities comigrated at a molecular weight of approximately 40 kDa on SDS-PAGE. This comigration was confirmed by gel filtration chromatography. Investigation of kinetics yielded K(m) values of 30.4+/-1.5 and 695. 3+/-171.2 microM and V(max) values of 330.4+/-90 and 102.8+/-17.1 nmol/mg/h for ADP-ribosyl cyclase and cADPR hydrolase, respectively. These results suggest a possible role for cADPR as an endogenous modulator of [Ca(2+)](i) in porcine TSM cells.

Cyclic ADP-ribose and its metabolic enzymes.

Cyclic ADP-ribose (cADPR) is a recently discovered cyclic nucleotide with Ca2+ signaling functions. There is a growing recognition that it is an endogenous modulator of the Ca(2+)-induced Ca2+ release mechanism in cells. The cyclic structure of cADPR has now been confirmed by x-ray crystallography. A series of analogs of cADPR has been synthesized, including antagonists and a novel analog, cyclic GDP-ribose. Considerable progress has been made in characterizing ADP-ribosyl cyclase, the synthetic enzyme, and cADPR hydrolase, the hydrolytic enzyme. A new class of bifunctional enzymes has been identified which catalyses both the synthesis and hydrolysis of cADPR. CD38, a lymphocyte differentiation antigen, is a member of this class. The understanding of the mechanisms of regulation of the metabolic enzymes and signaling by cADPR is likely to have important implications and several possibilities are discussed in this article.

Biologic priming of neutrophils in subcutaneous wounds.

Migration of neutrophils from blood into tissue is a complex response by circulating cells to chemotactic stimulation. Previous studies of the functional changes induced by this process have produced variable results. We compared neutrophils isolated from blood and from subcutaneous wounds in rabbits using established assays for adherence, chemotaxis, superoxide anion production, and hydrogen peroxide production. No differences in adherence to biologic surfaces or chemotaxis toward activated plasma were found. However, our results confirm the observation that wound neutrophils are "primed" for increased production of oxygen radicals. Primed responses were observed for both soluble (formyl methionyl leucylphenylalanine, phorbol myristate acetate) and particulate (opsonized zymosan) stimulants. Priming was also observed for peritoneal exudate neutrophils. The data suggest that the process of extravascular migration includes priming of the superoxide generating system.

Superoxide production by wound neutrophils. Evidence for increased activity of the NADPH oxidase.

Oxygen radical secretion by neutrophils is potentiated or "primed" by extravascular migration into wounds. To define this change in responsiveness more precisely we measured superoxide production by blood and wound neutrophils from rabbits using formylmethionyl-leucyl-phenylalanine and phorbol myristate acetate as agonists. In all experiments, the time- and dose-dependency of superoxide secretion were the same for blood and wound neutrophils. However, wound neutrophils produced significantly more superoxide. Furthermore, the cytochrome b component of the NADPH oxidase was found in greater quantities within wound neutrophils. We conclude that priming does little to alter the requirements for activating the NADPH oxidase but does significantly increase the velocity of superoxide generation. The data suggest that alterations in the assembly and function of the NADPH oxidase may contribute to enhanced superoxide secretion by wound neutrophils.

ADP-ribosyl cyclase and CD38. Multi-functional enzymes in Ca+2 signaling.

Mobilization of internal Ca+2 is an important signaling mechanism in cells. In addition to the inositol trisphosphate pathway, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide (NAADP) have been shown to mobilize Ca+2 via independent mechanisms. Although the structures of cADPR and NAADP are totally distinct, both nucleotides can be synthesized by ADP-ribosyl cyclase or CD38, a lymphocyte antigen. Both enzymes cyclize NAD to cADPR. In the presence of nicotinic acid the two enzymes catalyze a base exchange reaction resulting in the synthesis of NAADP from NADP. The switch between these two modes of catalysis is regulated by pH. Furthermore, both enzymes can also cyclize nicotinamide guanine dinucleotide (NGD) to produce a fluorescent product, cyclic GDP-ribose (cGDPR), which has a site of cyclization different from cADPR. A model is proposed to account for the multi-functionality of these enzymes. In order to be able to verify the model, a soluble ADP-ribosyl cyclase has been crystallized and X-ray diffraction shows that it is a dimer. Solution of the crystal structure of the cyclase should provide valuable insight into the structural features necessary for its multiple catalytic functions.

GDP-ribosyl cyclase activity as a measure of CD38 induction by retinoic acid in HL-60 cells.

Retinoic acid (RA) treatment of HL-60 cells induces surface expression of CD38. This lymphocytic antigen is also a novel bifunctional enzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR), a Ca2+ mobilizing metabolite of NAD+. The synthetic activity of CD38 is very difficult to detect because of the concurrent hydrolytic activity. In this study, a Ca2+ release assay capable of detecting submicromolar concentrations of cADPR was used to demonstrate the induction of ADP-ribosyl cyclase activity in HL-60 cells by RA. Concomitantly, cADPR hydrolase activity was also increased. The results were further substantiated by using a newly developed assay for GDP-ribosyl cyclase activity. This assay uses NGD+ as substrate instead of NAD+. The resulting fluorescent product, cyclic GDP-ribose, is resistant to hydrolysis and accumulates, making it a highly sensitive and convenient assay for CD38-like enzymes.

Structures and activities of cyclic ADP-ribose, NAADP and their metabolic enzymes.

ADP-ribosyl cyclase and CD38 are multi-functional enzymes involved in calcium signaling. Both can cyclize NAD and its guanine analog, NGD, at two different sites of the purine ring, N1 and N7, respectively, to produce cyclic ADP-ribose (cADPR) and cyclic GDP-ribose, a fluorescent but inactive analog. Both enzymes can also catalyze the exchange of the nicotinamide group of NADP with nicotinic acid, producing yet another potent activator of Ca2+ mobilization, nicotinic acid adenine dinucleotide phosphate (NAADP). The Ca2+ release mechanism activated by NAADP is totally independent of cADPR and inositol trisphosphate indicating it is a novel and hitherto unknown Ca2+ signaling pathway. This article summarizes the current results on the structures and activities of cADPR, NAADP and the enzymes that catalyze their syntheses. A comprehensive model accounting for the novel multi-functionality of ADP-ribosyl cyclase and CD38 is presented.

Oral filament proteins and their regulation in Tetrahymena pyriformis.

Two proteins from the Triton X-100-insoluble fraction of Tetrahymena pyriformis have been isolated and shown by immunological methods to be major components of a pervasive system of filaments localized within the oral apparatus. These proteins, OF-1 and OF-2, have apparent molecular weights (MWapp) in polyacrylamide gels of 87,000 and 80,000 D, respectively. Peptide maps obtained and the absence of immunological cross-reactivity suggest that these proteins are not closely related to each other. Indirect immunofluorescence studies on dividing cells have shown that the oral filament system forms late in the cell cycle. The filaments appeared first after the basal bodies in the oral primordium had organized into groups and the fission furrow had begun to form. Dedifferentiation of the oral filament system in the anterior (old) oral apparatus was also observed at this point in the cell cycle. Following this, the oral filament systems in both old and new oral apparatuses completed development synchronously. Proteins showing antigenic similarity to OF-1 were found in a number of other cell types. Tests with heterologous antisera failed to demonstrate a relationship between vertebrate cytoskeletal proteins and the oral filament proteins of Tetrahymena.

Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin.

Cyclic ADP-ribose (cADPR) is emerging as an endogenous regulator of Ca2+-induced Ca2+ release (CICR), and we have recently demonstrated that its action is mediated by calmodulin (CaM) (Lee, H. C., Aarhus, R., Graeff, R., Gurnack, M. E., and Walseth, T. F. (1994) Nature 370, 307-309). In this study we show by immunoblot analyses that the protein factor in sea urchin eggs responsible for conferring cADPR sensitivity to egg microsomes was CaM. This was further supported by the fact that bovine CaM was equally effective as the egg factor. In contrast, plant CaM was only partially active even at 10-20-fold higher concentrations. This exquisite specificity was also shown by binding studies using 125I-labeled bovine CaM. The effectiveness of various CaMs (bovine > spinach > wheat germ) in competing for the binding sites was identical to their potency in conferring cADPR sensitivity to the microsomes. A comparison between bovine and wheat germ CaM in competing for the sites suggests only 10-14% of the total binding was crucial for the activity. Depending on the CaM concentration, the sensitivity of the microsomes to cADPR could be changed by several orders of magnitude. The requirement for CaM could be alleviated by raising the divalent cation concentration with Sr2+. Results showed that CaM, cADPR, and caffeine all act synergistically to increase the divalent cation sensitivity of the CICR mechanism. The combined action of any of the three agonists was sufficient to sensitize the mechanism so much that even the nanomolar concentration of ambient Ca2+ was enough to activate the release. Unlike the CICR mechanism, the microsomal inositol 1,4,5-trisphosphate-sensitive Ca2+ release showed no dependence on CaM. Using an antagonist of CaM, W7, it was demonstrated that the cADPR-but not the inositol 1,4,5-trisphosphate-dependent release mechanism could be blocked in live sea urchin eggs. These results indicate cADPR can function as a physiological modulator of CICR and, together with CaM, can alter the sensitivity of the release mechanism to divalent cation by several orders of magnitude.

Possible role of cyclic nucleotides in the mechanism of the protective effect of methylprednisolone on the hypoxic rat heart.

The isolated isovolumic rat heart was used as a model of cardiac hypoxia. Force of cardiac contraction and cardiac cyclic nucleotide levels (cyclic GMP and cyclic AMP) were monitored in hearts subjected to hypoxia for 5 min and allowed to recover by reoxygenation. Hearts were obtained from both control animals and animals pretreated with methylprednisolone at 18 hr and 1 hr prior to sacrifice. Myocardial levels of cyclic GMP which were significantly (p less than 0.05) elevated above control during all periods of hypoxia were found to be lower when hearts were pretreated with methylprednisolone prior to hypoxic exposure. Hearts of animals pretreated with methylprednisolone also demonstrated better recovery during reoxygenation than did control hearts. These studies suggest that methylprednisolone may be beneficial in the prevention of myocardial failure following hypoxia via a modulation in myocardial cyclid GMP content.

KGF and FGF-10 stimulate liquid secretion in human fetal lung.

During fetal life, the pulmonary epithelium secretes liquid that distends the airways and is important for normal lung growth and development. The factors regulating human fetal lung liquid secretion are poorly understood; however, recent studies in murine models show that keratinocyte growth factor (KGF, FGF-7) and fibroblast growth factor 10 (FGF-10) stimulate liquid secretion. We asked whether KGF and FGF-10 stimulate liquid secretion in human fetal lung. First trimester fetal lung explants developed dose-dependent increases in intraluminal volume in response to KGF and FGF-10. Although there were no acute changes in explant transepithelial potential difference in response to KGF (0.1-1000 ng/mL), exposure to 5-50 ng/mL KGF over 60 h depolarized transepithelial potential difference compared with controls. We used ribonuclease protection assays to quantitate the ontogeny and regulation of mRNA expression for KGF and its receptor. Both mRNA were expressed in fetal and postnatal lung. Because the promoter region of the human KGF gene contains cAMP and IL-6 response elements, we asked whether cAMP or IL-6 stimulated expression of KGF or its receptor. We have previously shown that cAMP stimulates liquid secretion in this model. Both cAMP and IL-6 significantly increased expression of KGF but not KGF receptor during a 48-h experiment. Thus, stimulation of liquid secretion in explant models by cAMP may be mediated in part by induction of KGF expression. KGF and FGF-10 may be important paracrine factors regulating liquid secretion in human fetal lung.

The dynamics of cGMP metabolism in neuroblastoma N1E-115 cells determined by 18O labeling of guanine nucleotide alpha-phosphoryls.

The rates of phosphodiesterase-promoted hydrolysis of cGMP and cAMP have been measured in intact neuroblastoma N1E-115 cells by determining rates of 18O incorporation from 18O-water into the alpha-phosphoryls of guanine and adenine nucleotides. The basal rate of guanine nucleotide alpha-phosphoryl labeling ranged from 180 to 244 pmol X mg protein-1 X min-1. Sodium nitroprusside (SNP) caused a sustained 3.4-fold increase in this 18O-labeling rate in conjunction with 28- and 50-fold increases in cellular cGMP concentration at 3 and 6 min, respectively. This 18O-labeling rate (795 pmol X mg protein-1 X min-1) corresponded with the sum of the low (1.7 microM) and high (34 microM) Km phosphodiesterase activities assayable in cell lysates which exhibited a combined maximum velocity of 808 pmol X mg protein-1 X min-1 to which the high Km species contributed 84%. This information and the characteristics of the profile of 18O-labeled molecular species indicate that cGMP metabolism was restricted to a very discrete cellular compartment(s) of approximately 12% of the cell volume. Carbachol (1 mM) produced a transient increase (6-fold) in cellular cGMP concentration and a transient increase (90%) in the rate of 18O labeling of alpha-GTP during the first minute of treatment which translates into 30 additional cellular pools of cGMP hydrolyzed in this period. IBMX (1 mM) produced a relatively rapid increase in cellular cGMP (3- to 5-fold) and cAMP (2-fold) concentrations and a delayed inhibition of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls without further elevation of cyclic nucleotide levels. These results indicate that besides inhibiting cyclic nucleotide hydrolysis, IBMX also imparts a time-dependent inhibitory influence on the generation of cyclic nucleotides. The data obtained show that measurement of 18O labeling of guanine and adenine nucleotide alpha-phosphoryls combined with measurements of cyclic nucleotide steady state levels provides a means to assess the rates of cyclic nucleotide synthesis and hydrolysis within intact cells and to identify the site(s) of action of agents that alter cellular cyclic nucleotide metabolism.

Identification of the enzymatic active site of CD38 by site-directed mutagenesis.

CD38 is a ubiquitous protein originally identified as a lymphocyte antigen and recently also found to be a multifunctional enzyme participating in the synthesis and metabolism of two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate. It is homologous to Aplysia ADP-ribosyl cyclase, where the crystal structure has been determined. Residues of CD38 corresponding to those at the active site of the Aplysia cyclase were mutagenized. Changing Glu-226, which corresponded to the catalytic residue of the cyclase, to Asp, Asn, Gln, Leu, or Gly eliminated essentially all enzymatic activities of CD38, indicating it is most likely the catalytic residue. Photoaffinity labeling showed that E226G, nevertheless, retained substantial NAD binding activity. The secondary structures of these inactive mutants as measured by circular dichroism were essentially unperturbed as compared with the wild type. Other nearby residues were also investigated. The mutants D147V and E146L showed 7- and 19-fold reduction in NADase activity, respectively. The cADPR hydrolase activity of the two mutants was similarly reduced. Asp-155, on the other hand, was crucial for the GDP-ribosyl cyclase activity since its substitution with either Glu, Asn, or Gln stimulated the activity 3-15-fold, whereas other activities remained essentially unchanged. In addition to these acidic residues, two tryptophans were also important, since all enzyme activities of W125F, W125Y, W189G and W189Y were substantially reduced. This is consistent with the two tryptophans serving a substrate positioning function. A good correlation was observed when the NADase activity of all the mutants was plotted against the cADPR hydrolase activity. Homology modeling revealed all these critical residues are clustered in a pocket near the center of the CD38 molecule. The results indicate a strong structural homology between the active sites of CD38 and the Aplysia cyclase.

ADP-ribosyl cyclase and CD38 catalyze the synthesis of a calcium-mobilizing metabolite from NADP.

ADP-ribosyl cyclase catalyzes the cyclization of NAD+ to produce cyclic ADP-ribose (cADPR), which is emerging as an endogenous regulator of the Ca(2+)-induced Ca2+ release mechanism in cells. CD38 is a lymphocyte differentiation antigen which has recently been shown to be a bifunctional enzyme that can synthesize cADPR from NAD+ as well as hydrolyze cADPR to ADP-ribose. In this study, we show that both the cyclase and CD38 can also catalyze the exchange of the nicotinamide group of NADP+ with nicotine acid (NA). The product is nicotinic acid adenine dinucleotide phosphate (NAADP+), a metabolite we have previously shown to be potent in Ca2+ mobilization (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The switch of the catalysis to the exchange reaction requires acidic pH and NA. The half-maximal effective concentration of NA is about 5 mM for both the cyclase and CD38. In the absence of NA or at neutral pH, the cyclase converts NADP+ to another metabolite, which is identified as cyclic ADP-ribose 2'-phosphate. Under the same conditions, CD38 converts NADP+ to ADP-ribose 2'-phosphate instead, which is the hydrolysis product of cyclic ADP-ribose 2'-phosphate. That two different products of ADP-ribosyl cyclase and CD38, cADPR and NAADP+, are both involved in Ca2+ mobilization suggests a crucial role of these enzymes in Ca2+ signaling.

Fluorescent analogs of cyclic ADP-ribose: synthesis, spectral characterization, and use.

Cyclic ADP-ribose (cADPR) is a Ca(2+)-mobilizing cyclic nucleotide derived from NAD+. Accumulating evidence indicates that it is an endogenous modulator of the Ca(2+)-induced Ca2+ release mechanism in cells. In this study, we show that ADP-ribosyl cyclase catalyzes the cyclization of not only NAD+ but also several of its analogs with various purine bases (guanine, hypoxanthine, or xanthine) substituting for adenine. Unlike cADPR, the resulting cyclic products are fluorescent. Comparisons with various model compounds indicate that only 7-methyl substituted purine nucleosides and nucleotides are fluorescent, and the pH-dependence of their UV spectra is most similar to that of the fluorescent cADPR analogs, indicating that the site of cyclization of these analogs is at the N7-position of the purine ring. This finding is novel since the site of cyclization is at the N1-position for cADPR as determined by X-ray crystallography. That a single enzyme can cyclize a variety of substrates at two different sites has important implications mechanistically, and a model is proposed to account for these novel catalytic properties. Among the analogs synthesized, cyclic GDP-ribose is highly resistant to hydrolysis, while cyclic IDP-ribose can be readily hydrolyzed by CD38, a bifunctional enzyme involved in the metabolism of cADPR. These unique properties of the analogs can be used to develop fluorimetric assays for monitoring separately the cyclization and hydrolytic reactions catalyzed by the metabolic enzymes of cADPR. The convenience of the method in measuring kinetic parameters, pH-dependence, and modulator activity of the metabolic enzymes of cADPR is illustrated.