Mechanism and Tissue Specificity of Nicotine-mediated Lung S-Adenosylmethionine Reduction*

We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced.

Pneumocystis is an opportunistic fungus causing Pneumocystis pneumonia (PCP) 2 in people immunosuppressed because of AIDS, cancer chemotherapy, treatment to prevent transplant rejection, treatment for rheumatic diseases, severe malnutrition, etc. S-Adenosylmethionine (AdoMet) is a critical biochemical intermediate serving both as the methyl donor for a myriad of biochemical events and as the aminopropyl donor for polyamine synthesis. This pivotal intermediate is synthesized from ATP and methionine by all cells, the only known exceptions being Pneumocystis and some rickettsial bacteria that scavenge AdoMet from their hosts. The unprecedented requirement of a eukaryote for exogenous AdoMet was discovered in work initially directed toward improving Pneumocystis culture. We found that the addition of AdoMet to axenic Pneumocystis culture allows limited growth that is not possible without exogenous AdoMet and that Pneumocystis uses a highly specific transporter to acquire this essential metabolite (1). We also reported that AdoMet consumption by Pneumocystis affects host AdoMet; plasma AdoMet is depleted in a rat model of PCP (2) and in patients with PCP (3,4). This led to the prediction that deliberate reduction of host AdoMet will reduce susceptibility to PCP. A means for testing this was suggested by data reported 20 years ago: parenteral nicotine administration to guinea pigs causes selective reduction of lung AdoMet (5). Encouraging circumstantial support came from a large clinical study of factors that might relate to rates of PCP relapse in people with AIDS. Smoking was included as one of the parameters, and although no explanation was offered, a significant negative correlation was reported between smoking and PCP relapse (6). We tested whether nicotine would reduce rat lung AdoMet, as reported for guinea pigs (5) and found the effect to be similar (6). Infusion of the R-(ϩ)-nicotine isomer caused a 15-fold reduction in lung AdoMet with no change in plasma or liver AdoMet. When nicotine was evaluated in a rat model of PCP, it was strongly protective (6). Compared with controls infused with saline, rats infused with 475 g of R-(ϩ)-nicotine kg Ϫ1 h Ϫ1 starting at the time of inoculation had 99.9% fewer Pneumocystis organisms in their lungs 21 days post-5inoculation.
Understanding the shifts in metabolic pathways that drive nicotine-induced lung AdoMet depletion is interesting from a biochemical point of view and important for understanding how nicotine blocks development of PCP. Furthermore, it is possible that some part of nicotine toxicity relates to decreased lung AdoMet. We began our investigation by applying proteomic and biochemical techniques, and results led to the hypothesis that polyamine metabolism is involved (6). We found that nicotine causes no change in lung activity of the AdoMet-synthesizing enzyme methionine-adenosyl transfer-ase; thus we concluded that AdoMet synthesis is likely not affected. Because the major use of AdoMet is for methylation reactions, we looked for changes in response to nicotine. Shifts in methylation rates are reflected by changes in the "methylation index," the ratio of the byproduct of methyl donation, S-adenosyl homocysteine, to AdoMet (7). This index increases whenever there is a significant increase in the rate of AdoMetmediated methylation reactions for nucleic acid or protein methylation, for methionine regeneration, for lipid synthesis, or for any other methyl donation pathway or combination of pathways. We found nicotine treatment not to affect the lung methylation index, an observation confirmed by data in Table 2 of this publication; thus we concluded that AdoMet depletion was not driven by increased methylation activity. Using twodimensional gel separation of protein extracts from lungs of rats treated with nicotine and controls, densitometry of gel spots for quantitation of differentially expressed proteins, and peptide mass fingerprint for identification of these proteins, we found ornithine decarboxylase (ODC) to be up-regulated 14-fold. Subsequent enzymatic assay of lung homogenates showed a 20-fold increase. ODC is the regulating enzyme for polyamine synthesis, an AdoMet-consuming pathway. The first steps of polyamine synthesis are parallel decarboxylations of ornithine by ODC and AdoMet by AdoMetDC; the former produces putrescine, and the latter produces decarboxylated AdoMet (dcAdoMet). Spermidine is the result of an aminopropyl transfer from dcAdoMet onto one of the amino groups of putrescine. Spermine results from transfer of a second aminopropyl group onto the other amino group of the putrescine core of spermidine. This increase in ODC led us to consider up-regulation of polyamine metabolism as the mechanism underlying nicotine-induced lung AdoMet depletion (6). However, analysis of the polyamine content of lungs showed only an approximate 2-fold increase in putrescine, the product of ODC, and no change in the other major polyamines, spermidine and spermine, results confirmed by the data in Table 1. One interpretation is that, despite changes in ODC, polyamine synthesis does not increase significantly and is not responsible for AdoMet depletion. An alternative hypothesis is that polyamine synthesis does increase, but catabolism also increases, resulting in increased AdoMet consumption with little change in lung polyamine concentrations; we found evidence of increased polyamine catabolism. The key polyamine catabolic enzyme spermine/spermidine acetyl transferase (SSAT) is increased 4.5-fold in the lungs of nicotine-treated rats, and N-1-acetylspermidine, a product of that enzyme, is increased 11-fold. The 2-fold increase in putrescine without increases in spermidine and spermine is consistent with increased catabolism in that enhanced SSAT activity will accelerate spermidine/spermine degradation, whereas declining AdoMet concentration will cause slower spermidine/spermine synthesis, thus increasing the ratio of putrescine to spermidine/spermine. Although these data support the hypothesis that nicotine causes lung AdoMet depletion by stimulating polyamine metabolism, the evidence is indirect. Here we report data directly demonstrating that nicotine-induced rat lung AdoMet depletion treatment is driven by increased polyamine metabolism. We also report that AdoMet reduction in response to nicotine is not a general lung response but is specific for the alveolar region, the site of Pneumocystis infection.
Administration of Drugs-Nicotine and DFMO were delivered to animals by infusion pumps (Advanced Neuromodulation Systems, Plano, TX) implanted subcutaneously in the suprascapular region as previously described (8). The pumps had a chamber volume of 1.0 ml, a flow rate of 500 l day Ϫ1 , and a catheter to deliver drug to the peritoneum. Pump solutions were changed as follows. The needle of an "infusion set" was inserted percutaneously into the pump fill port, and the pump was drained into an attached syringe to measure the volume remaining; this allowed delivery rate calculation and confirmation of the manufacturer's reported delivery rate when implanted and filled with our drug solution. To flush and refill, a second infusion set was inserted, and 3 ml of fresh solution was slowly injected into the pump and drained through the first infusion set. The draining infusion set was then removed, and 1.0 ml of fresh solution was injected to refill the pump reservoir. The 1.0-ml reservoir required refilling every other day, but because AdoMet degrades rapidly, solutions containing AdoMet were replaced daily. At the time the pumps were implanted, the animals weighed 160 -170 g. Immunosuppression by dexamethasone without Pneumocystis carinii inoculation typically caused a weight drop to 130 -140 grams after 4 weeks. Nominal dose rates reported in the results were calculated at the beginning of the experiment based on a mean body weight of 160 g; however, because of weight loss resulting from dexamethasone treatment, the dose rate can increase by as much as 20% by the end of the treatment period.
Immunosuppression of Animals-Specific pathogen-free SD rats (Taconic Farms, Germantown, NY) were housed in a barrier colony and maintained on multiple antibiotics to avoid other opportunistic infections, as previously described (9). The rats were pretreated with a combination of trimethoprim and sulfamethoxazole for 21 days to reduce latent infections. After trimethoprim and sulfamethoxazole treatment, infusion pumps were implanted and delivered saline during the 7 days allowed for recovery before immunosuppression was begun by adding dexamethasone in the drinking water (1.5 mg l Ϫ1 ), and drug delivery was begun by changing pump solutions. This protocol was used because earlier nicotine lung AdoMet data were collected with immunosuppressed rats. To control for dexamethasone, we performed an experiment using four groups of four rats each that were treated by pump infusion of the following: saline only, saline plus dexamethasone, nicotine only, and nicotine plus dexamethasone. After 3 weeks treatment, the animals were sacrificed, and the lungs were analyzed for AdoMet. The respective mg of AdoMet (g lung) Ϫ1 were: 6.78 Ϯ 1.2, 7.22 Ϯ 0.55, 0.78 Ϯ 0.44, and 0.67 Ϯ 0.23. Thus nicotine treatment caused an 88.5% reduction in lung AdoMet in animals not treated with dexamethasone and a 91.7% reduction in animals treated with dexamethasone. Because these results are similar, we conclude that dexamethasone does not interfere with the action of nicotine on lung AdoMet, nor is it required.
Analysis for AdoMet, AdoMet Metabolites, and Polyamines-The AdoMet contents of lung, liver, and plasma samples were measured by HPLC analysis using Waters AccQ.Fluor derivitizing reagent as previously reported (1). For biological samples, the limit of detection is 0.5 nmol, and linearity extends to 5,000 nmol. All of the samples were analyzed in triplicate, and the coefficient of variation ranged from 5 to 17%, depending on the amount of AdoMet in the sample. The polyamines spermidine, spermine, and putrescine and AdoMet metabolites methylthioadenosine (MTA) and dcAdoMet were analyzed by HPLC using Waters' AccQ.Fluor derivitizing reagent as previously reported (10). Polyamine and AdoMet metabolite pools are expressed as nmol polyamine (g lung) Ϫ1 .
Analysis for Nicotine-Measurement of R-(ϩ)-nicotine in plasma was by our capillary zone electrophoresis method (6). A P/ACE MDQ system equipped with a photodiode array detector allowed electropherograms to be monitored at 257 and 205 nm. Sample preparation involved adding 20 l of 10% perchloric acid to 80 l of plasma to precipitate proteins that were removed by centrifugation at 5000 ϫ g for 10 min. The supernatants were stored for up to 7 days at Ϫ20°C before analysis. Just prior to analysis, the samples were diluted 1:1 with water. Separation utilized an amine capillary kit (Beckman, Inc.; 50-mm inner diameter ϫ 60-cm total length, 50 cm to the detector window) and the following protocol: 20 p.s.i. rinse with kit "amine regenerator solution" (2 min), 20 p.s.i. rinse with 50 mM Tris pH 8.0 buffer (2 min), sample injection, and separation using 30 kV in reverse polarity mode at 25°C (7 min). Specificity was assured by demonstrating that nicotine was resolved from all other peaks in plasma. Instrument precision was monitored by making triplicate injections from a single pooled standard.
Laser Capture Microdissection (LCM)-Sections (6 m) were cut from frozen lung tissue specimens embedded in OCT using a OTF/AS cryostat (Jencons, Brigeville, PA) and mounted on PEN membrane slides (Arcturus, Mountain View, CA). The sections were stained with hematoxylin to reveal lung architecture, and areas of interest were collected using a Veritas (Arcturus) LCM instrument equipped with Veritas image archiving software. The laser cutting mode was used to cut out and collect selected regions of sections. The cutting beam diameter was 3.0 M, and the laser intensity control was set at 4.46 mV. Typically, 10 -25 laser pulses were required to cut out the selected area, which was then captured on mounted films (Arcturus) according to manufacturer's directions. Portions of the films holding captured areas were placed in microcentrifuge tubes for further processing.
ODC Activity-The proteins were extracted from the pooled LCM material collected in microcentrifuge tubes by adding NKPD buffer (2.68 mM KCl, 1.47 mM KH 2 PO 4 , 51.1 mM Na 2 HPO 4 , 7.43 mM NaH 2 PO 4 , 62 mM NaCl, 1 mM EDTA, and 1.0 mM dithiothreitol), vortexing, sonicating for 20 min at 40 watts and 70% duty cycle (Heat System, Ultrasonics Inc., Plainview, NY), and clarifying by centrifugation at 30,000 ϫ g for 30 min. A 2-l aliquot was retained for protein assay (Nanodrop Technology, Wilmington, DC), and the balance was immediately used for ODC activity measurements as previously described in Ref. 11.
AdoMetDC Activity-A lung homogenate was prepared by freezing ϳ500 mg of tissue in liquid nitrogen and then grinding in a mortar and pestle. The resulting powder was immediately resuspended in 2 ml of buffer (150 mM KCl, 2 mM dithiothreitol, 25 mM HEPES, 5 mM MgSO 4 ), sonicated for about 2 min at 40 watts with a 70% duty cycle, and clarified by centrifugation at 10,000 ϫ g for 15 min. An aliquot of the collected supernatant was used for a protein assay (Bio-Rad Bradford assay), and the balance was for the AdoMetDC assay. Assay conditions were as described for ODC except for substitution of substrate. Detection of the dcAdoMet product was as described above.

RESULTS
Nicotine and DFMO were delivered by implanted infusion pumps as previously reported (6,8) and summarized under "Experimental Procedures." After surgically implanting salinefilled infusion pumps in the suprascapular region of groups of five rats each, saline was delivered over a 7-day surgical recovery period. After recovery, the pump solution for Group 1 rats was replaced with one containing 10.5 mg of R-(ϩ)-nicotine tartrate ml Ϫ1 in saline to deliver a dosage of ϳ475 g nicotine kg Ϫ1 h Ϫ1 . For Group 2, the exchange solution contained 94.5 mg DFMO ml Ϫ1 to deliver a dosage of ϳ12 mg DFMO kg Ϫ1 h Ϫ1 . For Group 3, the exchange solution contained the nicotine content of Group 1 plus the DFMO content of Group 2. Group 4 rats were controls, and the exchange solution was fresh saline. After 21 days of treatment, the four groups were sacrificed, and the lungs were collected. Infusion of nicotine at this rate produced a blood concentration of 14.3 Ϯ 2.5 M at 21 days. Appropriate homogenates were prepared from portions of each lung with other portions immediately frozen in OTC embedding medium for subsequent sectioning. Aliquots of lung homogenate were used for analyses of polyamines, AdoMet, AdoMet metabolites, and AdoMetDC activity.
The data in Table 1 show the effects of nicotine, DFMO, and the combination of nicotine plus DFMO on rat lung polyamines. Compared with saline-treated controls (Group 4), lung polyamine concentrations of DFMO-treated animals (Group 2) show sharp declines: putrescine, ϳ9-fold; spermidine, ϳ14-fold; and spermine, ϳ3-fold. Nicotine-treated animals (Group 1) show a 1.8-fold increase in lung putrescine, but spermidine and spermine are essentially unchanged, results similar to those we previously reported (6). For animals given both nicotine and DFMO (Group 3), changes in lung polyamines are similar to those given DFMO alone: respective decreases of ϳ9-, ϳ19-, and ϳ3-fold. These data show that, with respect to polyamines, the effect of DFMO cancels the effect of nicotine. Data in Table 2 show the effects of nicotine, DFMO, and the combination of nicotine plus DFMO on rat lung AdoMet, AdoMetDC, and AdoMet metabolites. Compared with controls (Group 4), nicotine-treated animals (Group 1) show ϳ10fold decreases in AdoMet, ϳ9-fold increases in AdoMetDC, ϳ3-fold increases in dcAdoMet, and ϳ3-fold increases in MTA, all changes reflecting increased AdoMet flux through the polyamine synthesis pathway with attendant increased AdoMet consumption. Co-treatment with DFMO ablated the effect of nicotine.
LCM was used to collect samples from specific lung areas for measurement of ODC activity. Frozen sections from lungs of Group 1 nicotine-treated rats and Group 4 control rats were mounted, fixed, and stained with hematoxylin and eosin. An LCM instrument was used to identify, cut, and capture multiple samples of respiratory epithelium and alveolar regions. Fig. 1 shows lung sections before and after sample collection by LCM. Panel A1 is a section containing an airway, panel A2 shows the same section after the airway epithelium has been cut and removed, and panel A3 shows the captured sample. Panels B1, B2, and B3 show the equivalent images for capture of an alveolar region. Approximately 20,000 laser cuts/captures were performed for each area, for both nicotine-treated and control animals. The pooled extracts of captured airway epithelium areas of control group rats contained 1.4 g of protein and alveolar areas of 1.2 g. For nicotine-treated rats, the corresponding values were 1.4 and 0.99 g, respectively. Table 3 presents ODC specific activities of the extracts and also total lung homogenate. For the controls, ODC specific activities of airway epithelium, alveolar regions, and lung homogenate are roughly similar, and the increase in activity induced by nicotine is specific for alveolar regions. Fig. 2 shows the recovery of lung AdoMet after the cessation of nicotine treatment. Infusion pumps were implanted into a group of 15 rats, and nicotine was delivered at the same rate as above for 21 days, after which time the pump solutions were replaced with saline. At days 1, 3, 7, 14, and 17 post-cessation of nicotine treatment, three randomly chosen rats were sacrificed, and the lungs were collected for analysis of AdoMet content. Recovery of AdoMet after cessation of nicotine treatment FIGURE 1. Laser capture microdissection. These images illustrate marking, cutting, and capture of selected regions of lung sections. Panels A1 and A2, areas containing airway epithelium and alveoli, respectively, as marked by software for subsequent cut and capture. Panels B1 and B2, same sections after the marked areas were cut and removed. Panels C1 and C2, captured areas. The data for Group 2 were collected separately than for the other groups, but exactly the same protocols were used, and a separate group of saline-treated controls was included. The putrescine, spermidine, and spermine concentrations of Group 2 controls were 244 Ϯ 38, 828 Ϯ 66, and 1592 Ϯ 98 nmol (g wet tissue) Ϫ1 , respectively. Because these are essentially the same as the Group 4 controls in this table, and all of the comparative ratios are the essentially same regardless the set of controls used for calculation, we included Group 2 data in this table. However, the correct control data set was used to calculate changes induced by DFMO alone.

S-Adenosylmethionine, Nicotine, and Pneumocystis
occurred over 3 weeks, the same period required for depletion upon initiation of nicotine treatment.

DISCUSSION
A critical test of the hypothesis that nicotine causes lung AdoMet depletion by stimulating polyamine metabolism was to determine whether blocking polyamine synthesis would block nicotine-induced AdoMet depletion. One approach would be to inhibit the AdoMet-consuming step of polyamine metabolism, decarboxylation of AdoMet by AdoMetDC. Although many inhibitors of AdoMetDC are known, and one is available in adequate amounts for in vivo experiments, all have metabolic effects beyond inhibition of AdoMetDC, and these off target effects would necessarily limit confidence in conclusions. An alternative approach was to block AdoMet consumption indirectly by inhibiting the other initial step of de novo polyamine synthesis, decarboxylation of ornithine by ODC to produce putrescine; limiting putrescine limits aminopropyl acceptors, thereby restricting dcAdoMet use and thus consumption of AdoMet to produce dcAdoMet. We chose to block putrescine production because it is only one step downstream from direct inhibition of AdoMetDC and because an ideal tool was available: DFMO. In contrast to inhibitors of AdoMetDC, DFMO is extraordinarily specific and has no other metabolic effects. This specificity is due to DFMO being an enzyme-activated suicide inhibitor that is metabolically inert until the carboxyl group is cleaved by ODC. Cleavage activates the ␣-difluoromethyl group, which alkylates a residue within the active site, cysteine 360, causing irreversible inhibition. A possible complication was that DFMO treatment has been found to cause a Ͼ10-fold up-regulation in AdoMetDC and a Ͼ500-fold increase in dcAdoMet (12), effects that might have stimulated AdoMet consumption independent of nicotine, thereby masking any reduction in nicotine-induced AdoMet consumption. However, not all cells respond similarly (13), and Table 2 data show that DFMO causes only moderate up-regulation of rat lung AdoMetDC and accumulation of dcAdoMet: 2.0-fold and 21-fold increases in AdoMetDC and dcAdoMet, respectively. Nicotine treatment produces a greater increase in AdoMetDC, 8.5-fold, but a smaller increase in dcAdoMet, 2.6-fold, a pattern consistent with our hypothesis. Table 1 data show that DFMO-treated animals have severe reductions in all three polyamines compared with controls, thereby demonstrating dosage suitability. The declines in putrescine and spermidine are greater than spermine, suggesting that despite blockage of de novo polyamine synthesis, some AdoMet consumption continues for spermine synthesis from pre-existing putrescine and spermidine, lower polyamines catabolically produced from spermine, and polyamines obtained from the diet. This is supported by Table 2 data that show the continuing presence of MTA in lung homogenates of DFMO-treated animals. In stark contrast to the 10-fold reduction in lung AdoMet of nicotine-treated animals compared with controls, the lung AdoMet content of animals treated with both nicotine and DFMO is essentially the same as animals treated with DFMO alone; i.e. DFMO reverses the effect of nicotine on lung AdoMet as predicted. Similarly, Table 1 shows that the effects on polyamines are essentially the same for animals given DFMO treatment alone and for animals given both nicotine and DFMO. Table 2 shows that DFMO alone, or in combination with nicotine, causes a large increase in dcAdoMet as expected because consumption of this intermediate is blocked by inhibition of putrescine production. Thus our hypothesis passed the critical test.
Other data in Table 2 provide additional support. Not only is lung AdoMet reduced by nicotine treatment, but also AdoMet catabolites dcAdoMet and MTA are increased 2.6-and 2.7fold, respectively. Because such catabolites can only be the result of AdoMet utilization for polyamine synthesis, these increases indicate increased flux in the polyamine pathway, as predicted. This shift is especially notable because it occurs despite severe AdoMet depletion and therefore indicates a strong diversion of AdoMet to polyamine metabolism. Although the 0.65 methylation index of Group 1 is similar to the 0.70 index of Group 4 controls, the absolute concentration of lung S-adenosyl homocysteine declines 10-fold in nicotinetreated animals compared with controls; this result also points away from increased activity in methylation pathways being involved in lung AdoMet depletion. Although it might seem that additional supporting data could be obtained by using DFMO to reverse the ability of nicotine to suppress PCP, this is not possible because DFMO itself is highly active against PCP both in the rat model (14) and clinically (15)(16)(17)(18)(19). Because of the very rapid response of this pathogenic fungus to manipulation of polyamine metabolism, DFMO is particularly effective when steadily infused (11,20).
There is precedence for increased polyamine flux causing cellular AdoMet reduction. When cells of the LNCaP prostate tumor line were genetically manipulated to increase SSAT mRNA 10-fold leading to a 20-fold increase in SSAT activity, the cells compensated for increased polyamine catabolism by increasing polyamine synthesis, and the polyamine content of the cells did not decline (21). ODC activity increased 10-fold, and AdoMetDC activity increased 8-fold. This increased polyamine flux required increased consumption of AdoMet, and cell AdoMet content declined. With respect to the increase in putrescine and lack of change in spermidine and spermine con- tent, the effects of nicotine on rat lungs and SSAT overexpression in LNCaP prostate tumor line cells are similar. Other effects, however, are dissimilar. The 20-fold SSAT activity increase in the cell line is greater than the 4.4-fold increase in lungs of nicotine-treated rats, and the Ͼ1000-fold increase in the SSAT product N-1-acetylspermidine in the cell line is far greater than the 11-fold increase in the lungs. These data suggest a greater polyamine flux increase in the cell line than in the lung, yet AdoMet declined only 2-fold in the cell line overexpressing SSAT, far less than in lungs of rats treated with nicotine. It is possible that, despite the greater shift in enzyme activities, polyamine flux changes less in the cell line than in the lungs, but a more plausible explanation is that the cell line is better able to compensate for increased AdoMet consumption by increasing AdoMet production. This possibility brings up an interesting point. Increases in ODC and SSAT are common responses to stress, including exposure to nicotine (22,23), but a nicotine-induced decline in AdoMet has never been described except for lungs. Therefore, the selective effect of nicotine on lung AdoMet may not depend so much on a selective increase in lung polyamine flux, but on an inability of lung cells to increase the AdoMet supply to compensate for increased consumption. Perhaps methionine needed for AdoMet synthesis is limited in lungs because of competing needs for 1-carbon intermediates for lipid synthesis, or perhaps lungs are unable to increase AdoMet synthesis for another reason. These questions cannot be answered with available data, but experimental approaches are feasible.
Even considering the remarkable degree of lung AdoMet depletion, the ability of nicotine to prevent PCP development in a rat model is extraordinary. We considered the possibility that AdoMet depletion may be especially marked in the cells Pneumocystis uses for attachment and nourishment: Type I pneumocytes of the lung parenchyma. To explore this, we used LCM to collect samples of lung parenchyma from sections of lungs taken from nicotine-treated and control rats; samples of airway epithelium were collected for the control (see Fig. 2 for examples). Nicotine caused an 18-fold increase in alveolar region ODC activity, but only a 10% increase in airway epithelium activity ( Table 3). The increases in lung parenchyma and total lung homogenate are similar, reflecting the relative composition of the lung. Although these data show that lung AdoMet depletion is not general, LCM resolution does not allow discrimination among various cell types present in the alveolar region: Type I pneumocytes, Type II pneumocytes, macrophages, fibroblasts, capillary endothelia, and blood cells within the capillaries. One could speculate that Type I pneumocytes are the target because they are the most common cell type of the parenchyma and Pneumocystis adheres to them. However, because nicotine causes a 10-fold AdoMet reduction overall (6,14), and the parenchyma is not 90% Type I cells, this would mean that the base-line AdoMet in all other lung parenchyma cells would have to be very low if depletion of Type I cells results in 10-fold reduction of lung AdoMet. Although data for AdoMet concentrations within lung regions is unavailable, broadening the speculation to include both Type I and II pneumocytes reduces this problem because these cells do comprise the bulk of the parenchyma. Although Type I and II cells have different morphologies, compositions, and functions, one develops directly from the other, so it is not unreasonable to speculate that they respond similarly to nicotine. This is another interesting question requiring further work for resolution.
A recent paper reported the effects of smoking and nicotine on related aspects of lung metabolism (24). But because the focus of that work was on asthma, the concentration was on airway epithelium, not alveolar regions. The authors found airway epithelium to show increases in expression of both ODC and arginase I, the enzyme that produces the ornithine substrate for ODC. The results were based on immunochemistry to detect protein and in situ hybridization to reveal corresponding mRNA. Quantification was by visual inspection of coded slides. They also studied the response of cultured cells to nicotine stimulation using quantitative PCR. These authors report a 2-fold increase in airway epithelium ODC protein and mRNA in sections and a 1.8-fold increase in mRNA in cultured cells. In contrast, we found only a 10% increase in ODC enzyme activity in airway epithelium. We cannot explain this difference, but note the different techniques used and the fact that their samples were from asthmatic patients, smokers and nonsmokers, and cultured cells, whereas our samples were from otherwise healthy laboratory animals, some treated with nicotine and some not. Besides those differences, they measured ODC protein and mRNA, and we measured activity in these experiments. Because control of ODC activity involves many posttranslational mechanisms in addition to control of expression, it is possible that both sets of results are correct and would be the same even if the samples were the same. This is a very new area of investigation, and better explanations will likely arise with further work.
We previously showed that rat lung AdoMet declines slowly upon initiation of nicotine treatment (6), and the data here (Fig.  2) show that recovery upon withdrawal is also very slow. The curves are mirror images with each requiring about 3 weeks for completion. Because pneumocytes are replaced over a similar time period (25), the notion arises that the presence of nicotine may cause a change in the phenotype of pneumocytes as they are produced in a manner reminiscent of enterocyte iron transport phenotype being dependent on iron stores at the time they are produced (26). However, other data show a rapid increase in lung ODC upon exposure to nicotine and cigarette smoke and therefore argue against this notion (24,27,28). It may be that nicotine causes relatively rapid changes in enzyme activities, but these changes cause only a slightly greater rate of AdoMet consumption than production; thus lung AdoMet declines slowly and recovers slowly upon removal of nicotine. This remains an open question.
The possibility of nicotine being used to treat PCP or to prophylax against this disease is another issue, one beyond the scope of this report. However, we do note that the 9.6 mg kg Ϫ1 day Ϫ1 dose of nicotine used to collect most of the data presented here produces a steady state plasma concentration of 2.32 g ml Ϫ1 , near a toxic level. But 1% of this dose, or 96 g kg Ϫ1 day Ϫ1 , produces a steady state plasma concentration of 300 nM or 4.9 ng ml Ϫ1 and, when given to immunosuppressed rats starting at the time of inoculation with P. carinii, reduces S-Adenosylmethionine, Nicotine, and Pneumocystis MARCH 21, 2008 • VOLUME 283 • NUMBER 12 JOURNAL OF BIOLOGICAL CHEMISTRY 7695 the lung burden at 3 weeks by 85% compared with salinetreated controls (6). Further insight is offered by the results of a human study using well tolerated nicotine-containing gum or lozenges that are ordinarily used as aids for those trying to cease smoking or chewing tobacco. Lozenges and gum containing 4 mg of nicotine produced plasma nicotine concentrations rising over the first hour to ϳ10 ng ml Ϫ1 , dropping to about 5 ng ml Ϫ1 at 3 h and dropping further to about 3 ng ml Ϫ1 at 6 h. Because these plasma concentrations exceed those that suppressed PCP by 85% in rats, it is possible that a nontoxic nicotine dose could be clinically active against PCP. Furthermore, once the initial interaction between nicotine and lung cells that initiates increased polyamine cycling is better understood, it might be possible to identify analogues that retain this activity but are less toxic than nicotine.
In summary, data here demonstrate that nicotine-induced lung AdoMet depletion is tied to increased polyamine metabolism. This effect on AdoMet is not general but is restricted to alveolar regions of the lung. We know that AdoMet depletion is a slow process and recovery is as well, but we do not know whether this is due to a nicotine-mediated phenotype change as pneumocytes are produced or to a small but persistent change in the net difference between AdoMet production and consumption. As far as is known, the effect of nicotine on cell AdoMet content is specific for lungs, but it is unknown whether this is because lungs respond to nicotine by increasing polyamine metabolism more strongly than other organs or whether this is general cell response to nicotine that has a greater effect on lungs because they are less able to compensate by increasing AdoMet production.