Ontogeny of CLCN3 chloride channel gene expression in human pulmonary epithelium.

Human fetal bronchopulmonary epithelia secrete liquid, and this chloride (Cl)-dependent process is important for normal lung growth. At the time of birth there is a maturational transition from a secretory to an absorptive phenotype. The pathways for Cl exit from the apical membrane which are required for fetal lung liquid secretion are unknown but are thought to be independent of the cystic fibrosis transmembrane conductance regulator. We determined the ontogeny of expression of the CLCN family of voltage-dependent Cl channel genes (CLCN2 through 6, K(a) and K(b)) in the human lung to identify potential pathways for pulmonary liquid secretion. Only CLCN3 and CLCN6 messenger RNA were detected by Northern analysis of fetal whole lung tissue. Ribonuclease protection assays confirmed the expression of CLCN3 and also revealed expression of CLCN2. The ontogeny of expression of these two channels was similar, peaking in midgestation and declining postnatally. In situ hybridization localized the CLCN2 and CLCN3 messages to airway and distal pulmonary epithelia and to pulmonary blood vessels. We conclude that CLCN3 is expressed in human airway epithelia and expression is developmentally regulated. The contribution of these channels to pulmonary epithelial liquid transport and lung development remains to be determined.

A single residue at the active site of CD38 determines its NAD cyclizing and hydrolyzing activities.

CD38 is a multifunctional enzyme involved in metabolizing two Ca(2+) messengers, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). When incubated with NAD, CD38 predominantly hydrolyzes it to ADP-ribose (NAD glycohydrolase), but a trace amount of cADPR is also produced through cyclization of the substrate. Site-directed mutagenesis was used to investigate the amino acid important for controlling the hydrolysis and cyclization reactions. CD38 and its mutants were produced in yeast, purified, and characterized by immunoblot. Glu-146 is a conserved residue present in the active site of CD38. Its replacement with Phe greatly enhanced the cyclization activity to a level similar to that of the NAD hydrolysis activity. A series of additional replacements was made at the Glu-146 position including Ala, Asn, Gly, Asp, and Leu. All the mutants exhibited enhanced cyclase activity to various degrees, whereas the hydrolysis activity was inhibited greatly. E146A showed the highest cyclase activity, which was more than 3-fold higher than its hydrolysis activity. All mutants also cyclized nicotinamide guanine dinucleotide to produce cyclic GDP. This activity was enhanced likewise, with E146A showing more than 9-fold higher activity than the wild type. In addition to NAD, CD38 also hydrolyzed cADPR effectively, and this activity was correspondingly depressed in the mutants. When all the mutants were considered, the two cyclase activities and the two hydrolase activities were correlated linearly. The Glu-146 replacements, however, only minimally affected the base-exchange activity that is responsible for synthesizing NAADP. Homology modeling was used to assess possible structural changes at the active site of E146A. These results are consistent with Glu-146 being crucial in controlling specifically and selectively the cyclase and hydrolase activities of CD38.

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.

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.

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.

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.

Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 microM cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mM and 10 mM, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.

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.

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.

Keratinocyte growth factor stimulates CFTR-independent fluid secretion in the fetal lung in vitro.

Keratinocyte growth factor (KGF) caused cystic dilation of mouse fetal lung explants in vitro, markedly increasing the luminal volume of lung buds and disrupting branching morphogenesis. Effects of KGF were dose dependent, were detected within 4 h of treatment, and were blocked by cycloheximide but not by actinomycin D, indicating that de novo protein synthesis mediated the response. Effects of KGF were inhibited by bumetanide, an inhibitor of the Na(+)-K(+)-Cl- cotransporter, and ouabain, an inhibitor of the Na(+)-K(+)-ATPase. KGF stimulated fluid secretion equally in lung buds from cystic fibrosis transmembrane conductance regulators (CFTR) -/- and wild-type embryos, indicating that the effects were mediated by CFTR-independent Cl- transport. Microelectrode studies demonstrated that, whereas KGF did not acutely alter the transepithelial potential difference (PD) across the respiratory epithelium, the PD decreased while luminal volume increased during chronic exposure. KGF inhibited expression of alpha-subunit of epithelial Na+ channel (alpha-ENaC) mRNA, suggesting that KGF may inhibit Na+ absorption, which may contribute to KGF-induced fluid accumulation. KGF-induced fluid accumulation is driven by CFTR-independent Cl- transport and associated with decreased expression of alpha-ENaC.

Activation and inactivation of Ca2+ release by NAADP+.

Nicotinic acid adenine dinucleotide phosphate (NAADP+) is a recently identified metabolite of NADP+ that is as potent as inositol trisphosphate (IP3) and cyclic ADP-ribose (cADPR) in mobilizing intracellular Ca2+ in sea urchin eggs and microsomes (Clapper, D. L., Walseth, T. F., Dargie, P. J., and Lee, H. C. (1987) J. Biol. Chem. 262, 9561-9568; Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). The mechanism of Ca2+ release activated by NAADP+ and the Ca2+ stores it acts on are different from those of IP3 and cADPR. In this study we show that photolyzing caged NAADP+ in intact sea urchin eggs elicits long term Ca2+ oscillations. On the other hand, uncaging threshold amounts of NAADP+ produces desensitization. In microsomes, this self-inactivation mechanism exhibits concentration and time dependence. Binding studies show that the NAADP+ receptor is distinct from that of cADPR, and at subthreshold concentrations, NAADP+ can fully inactivate subsequent binding to the receptor in a time-dependent manner. Thus, the NAADP+-sensitive Ca2+ release process has novel regulatory characteristics, which are distinguishable from Ca2+ release mediated by either IP3 or cADPR. This battery of release mechanisms may provide the necessary versatility for cells to respond to diverse signals that lead to Ca2+ mobilization.

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.

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.

Functional expression of soluble forms of human CD38 in Escherichia coli and Pichia pastoris.

Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively. The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast. We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38. For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities.

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.

Magnesium ions but not ATP inhibit cyclic ADP-ribose-induced calcium release.

The pharmacology of the cyclic ADP-ribose (cADPR)-dependent Ca2+ release mechanism is very similar to that of the ryanodine receptor (RyR). Here we showed that MgCl2, a known inhibitor of RyR, blocked cADPR-induced Ca+2 release in sea urchin egg homogenates with a half maximal concentration of about 2.5 mM. The effect was specific since up to 10 mM Mg+2 had no effect on the Ca+2 release induced by inositol trisphosphate. K2ATP, another known modulator of RyR, at up to 10 mM did not affect the half-maximal concentration of cADPR, which remained at about 96 nM. These results indicate cADPR is a specific Ca+2 release activator and not merely an adenine nucleotide acting on the ATP-site. The inhibitory effects of Mg+2 further demonstrate the similarity between RyR and the cADPR-dependent Ca+2 release system.

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.