Polyamine Transport and Ornithine Decarboxylase Activity in Hypoxic Pulmonary Artery Smooth Muscle Cells Cheryl A. Haven, Jack W. Olson, Santosh S. Arcot, and Mark N. Gillespie Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky A. B. Chandler Medical Center, Lexington, Kentucky
Hypoxia causes remodeling of the pulmonary circulation that is dependent on increases in lung polyamine contents. Mechanisms by which polyamines are regulated in hypoxic lung cells are unknown, but ornithine decarboxylase (ODC) activity, the initial enzyme in de novo biosynthesis, is depressed and polyamine transport is augmented in lungs from hypoxic rats (R.-T. Shiao et al. 1990. Am. J. Physiol. 259:L351L358). To determine if hypoxia directly influences polyamine regulatory mechanisms in pulmonary vascular cells, we examined [I4C]spermidine (SPD) transport and ODC activity in bovine main pulmonary artery smooth muscle cells (PASMCs) cultured under standard (culture medium Po2 : > 100 mm Hg), "normoxic" (culture medium Po2 : 50 to 70 mm Hg), or "hypoxic" (culture medium Po2 : 18 to 30 mm Hg) conditions. Uptake of [14C]SPD in cells cultured under standard conditions was temperature- and concentration-dependent, exhibited saturation kinetics, and was abolished by metabolic inhibition. Modeling of transport according to Michaelis-Menten kinetics revealed that [I4C]SPD uptake in cells cultured under standard conditions was characterized by K; and Vmax values of 0.78 J.tM and 4.5 pmol/min/tO" cells, respectively. In comparison to cells cultured under standard conditions, K; was unaffected by culture under normoxic or hypoxic conditions while Vmax was increased to 18 pmol/min/ 106 cells in normoxic cells and to 33 pmol/min/l'O' cells in preparations cultured under hypoxic conditions. Inhibition of ODC with o-difluoromethylomithine (DFMO) also induced SPD transport, as evidenced by an increase in the Vmax to 65 pmol/min/lf)" cells. Both hypoxia- and DFMO-induced increases in [I4C]SPD transport were suppressed by cycloheximide and actinomycin D, thus highlighting the importance of protein and RNA synthesis. ODC activity did not differ between cells cultured under standard and normoxic conditions (48 pmol/mg protein/60 min) but was reduced by 75% to 12 pmol/60 min/mg protein in hypoxic cells. The abundance of ODC mRNA also was reduced by 80 to 85 % in hypoxic cells relative to cells cultured under standard conditions. In contrast to the reduction in ODC activity, hypoxia failed to inhibit the activity of another important enzyme in polyamine synthesis, S-adenosylmethionine decarboxylase. These findings indicate that hypoxia induces polyamine transport in PASMCs, perhaps secondary to decreased ODC activity, and suggest that increases in lung polyamine contents necessary for hypoxic vascular remodeling may be ascribed in part to induction of transport in cells of the pulmonary circulation.
The polyamines, putrescine, spermine, and spermidine, are a family of low molecular weight organic cations that play central intracellular regulatory roles in cell growth and differentiation (1). In the lung, their importance is illustrated (Receivedin originalform November 1,1991 and in revisedform March 16, 1992) Addresscorrespondence to: Mark N. Gillespie, Ph. D., Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082. Abbreviations: S-adenosylmethionine decarboxylase, AdoMet-DC; o-difluoromethylornithine, DFMO; Dulbecco's modified Eagle's medium, DMEM; high performance liquid chromatography, HPLC; monocrotaline, MCT; ornithine decarboxylase, ODC; pulmonary artery smooth muscle cell, PASMC; phosphate-buffered saline, PBS; spermidine, SPD. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 286-292, 1992
by observations that elevated polyamine contents are required for normal lung development (2), for compensatory growth after pneumonectomy (3), for repair of lung injury evoked by hyperoxia (4), and for development of hypertensive pulmonary vascular disease induced by monocrotaline (MCT) (5, 6) or chronic hypoxic exposure (7). Because of the central role of polyamines in regulation of cell structure and function, mechanisms governing lung cell polyamine content are physiologically and pharmacologically significant. In this context, with the notable exception of chronic hypoxic pulmonary hypertension, the dominant regulatory mechanism' seems to involve induction of ornithine decarboxylase (ODC) , the initial rate-limiting enzyme in polyamine synthesis. ODC activity is markedly increased in lungs from neonatal rats (2) and in animals with
Haven, Olson, Arcot et al.: Polyamine Transport in Pulmonary Artery Smooth Muscle Cells
hyperoxic (4) or MCT-induced lung injury (5, 6). Inhibition of ODC with o-difluoromethylornithine (DFMO) prevents increases in lung polyamine contents and suppresses lung development in neonatal rats (2) and forestalls repair of hyperoxic lung injury (4). In MCT-treated rats, DFMO prevents development of pulmonary edema, medial thickening of pulmonary arteries, sustained pulmonary hypertension, and right ventricular hypertrophy (5, 6). In contrast to these model systems, hypoxic pulmonary hypertension is associated with profound decreases in lung ODC activity (8), despite the fact that lung polyamine contents are elevated (7, 8). Though the mechanisms underlying the hypoxia-induced increase in lung polyamines are not established, altered transmembrane transport is a likely possibility in light of our finding that perfused lungs from chronically hypoxic rats exhibit an increase in the Vmax for putrescine uptake and a prolonged half-life of putrescine efflux (8). Lung cell types in which hypoxia enhances putrescine transport are not known. Two lung cell populations, type II pneumocytes (9) and alveolar macrophages (0), exhibit polyamine transport systems in vitro. Cultured pulmonary vascular cells have yet to be examined for their polyamine transport capacities. Autoradiographic experiments in intact rats (11), isolated rat lungs (11), and rat lung slice preparations (2) indicate that transported polyamines localize principally in type II pneumocytes; localization in pulmonary vascular cells generally has not been appreciated. However, in a preliminary autoradiographic study, we found that while vascular spermidine (SPD) uptake was unimpressive in control rat lungs, the pulmonary vasculature in animals subjected to 4 and 7 days of hypoxia exhibited substantial localization of the radiolabel in intimal and medial regions (13). Based on these considerations, the present study determined if an SPD transport system is expressed by bovine main pulmonary artery smooth muscle cells (PASMCs) in culture and, more importantly, tested the hypothesis that polyamine transport and ODC activity in PASMCs are upregulated and downregulated, respectively, by culture under hypoxic conditions. Toverify the potential for induction of SPD transport in PASMCs, some cultures were treated with DFMO, which, along with inhibition of ODC, is a potent stimulus for polyamine transport in many cell culture systems (e.g., 14).
Materials and Methods PASMC Cultures Bovine main pulmonary arteries were harvested from freshly slaughtered cattle (Dawson's Packing Co., Louisville, KY), immersed in cold (4° C) Pucks F6 solution supplemented with 300 U/rnl penicillin, 0.3 mg/ml streptomycin, and transported to the laboratory. The arteries were then opened longitudinally, and the endothelium was scraped away. Medial explants, approximately 2 X 2 mm, were dissected from the subintima and plated in culture flasks containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% defined fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/rnl streptomycin. Cells were then grown to confluence and propagated in culture. Cells from eight different adult cows were used in these experiments. Culture medium was'changed every 3 to 4 days. Cells were harvested using a 0.05% solution of trypsin. All experiments used cells
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from passages between 2 and 10. The smooth muscle phenotype of the cells was confirmed by the presence of smooth muscle-specific myosin, as evidenced by immunocytochemical analysis (data not shown). Cell counts were determined by hemocytometry. Hypoxic Exposure and DFMO Treatment To examine the effects of hypoxia and DFMO on SPD transport and ODC activity, PASMCs were seeded into 35-mm2 , six-well cluster plates (Costar) at a density of 75,000/well and cultured in DMEM supplemented with 10% serum for 24 h. Subsequently, the well plates were placed in specially constructed plastic chambers affixed with inflow and outflow ports that permitted the chambers to be exposed to selected gas mixtures. The chambers were then placed in standard tissue culture incubators after which they were exposed for an additional 24 h to one of three levels of hypoxia: "standard" conditions, wherein the cells were maintained in incubator chambers gassed with 95 % air-5 % CO 2 (Po 2 > 100 mm Hg); "normoxic" conditions, in which incubator chambers were gassed with a mixture of 12% O2 , 5% CO 2 and 83% N2 (Po 2 = 50 to 70 mm Hg), and "hypoxic" conditions, in which the incubators were purged with 3 % 02> 5 % C02> and 92 % N2 (Po 2 = 18 to 30 mm Hg). Aliquots of culture medium (1.0 ml) were withdrawn anaerobically at termination of hypoxic exposure, and Po, values were determined using an Instrument Laboratories model 213 blood gas analyzer. SPD Transport After the indicated incubation times, PASMCs were rinsed with fresh, serum-free DMEM after which I rnl of serumfree DMEM was added to each well and the cells were allowed to acclimate for a l-h period. Subsequently, [14C]SPD was added to each well in concentrations of 0.1 to 30 JLM, and cells were incubated for specific durations (up to 90 min) and at selected temperatures (4° and 37° C). At the appropriate time, media containing residual [14C]SPD was aspirated, and cells were placed on ice and rinsed with cold phosphate-buffered saline (PBS). The PASMCs were then digested for I h at room temperature in I N NaOH. Before determination of cell-associated radioactivity, 400 JLl of the cell digest was neutralized with 400 JLI of I N acetic acid. An additional 400 JLl of distilled H20 along with 2 rnl of scintillation cocktail was added to the neutralized digests, and radioactivity was determined using a Packard liquid scintillation counter. We elected to normalize [14C]SPD content and uptake rate in terms of cell number rather than protein or DNA content since culture under hypoxic conditions may alter these biochemical parameters (15, 16). Cell Viability Cell viability was assessed using trypan blue exclusion according to standard techniques. In brief, 500 JLl of cell suspension was mixed with the same volume of a 0.08% solution of trypan blue dye in PBS. After a 2- to 15-min incubation period, the numbers of stained and unstained cells were determined using a hemocytometer. Viability is expressed as a percentage of the total number of cells.
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Assessment of DOC and S-Adenosylmethionine Decarboxylase (AdoMet-OC) Activity and p·C]SPD Metabolism ODC and AdoMet-DC activities in PASMCs were determined as described previously (8). Enzyme activities were quantitated by determining the amount of l4C02 released from 0.5 /LCi of L-[1-14C]ornithine or 0.2 /LCi of S-adenosylL-[carboxyl-14C]methionine, respectively, during a 60-min incubation at 37° C. To determine if [14C]SPD was metabolized by PASMCs during the 30-min uptake period, cells were harvested and prepared for high performance liquid chro1l!atography (HPLC) analysis of polyamine contents as descnbed prevrously (8). The effluent from the HPLC column was collected in 2 min fractions, and the amount of radioactivity was determined in each fraction and related to the retention times of the specific polyamines. Northern Analysis of DOC mRNA Total RNA from cells cultured under standard conditions and cells cultured in a hypoxic environment was isolated and purified by the guanidinium isothiocyanate/CsCI method ~s described elsewhere (17). Briefly, PASMCs were washed m PBS, lysed in 5 vol of 4 M gu.anidinium isothiocyan~te solution, layered on a 1.5-ml cushion of 5.7 M CsCI solution, and centrifuged for 16 to 18 h in an SW50.1 rotor (Beckman) at 35,000 rpm. The purified RNA samples were analyzed by fractionating equal amounts (15 /Lg) of RNA on a 1.2 % agarose/2.2 M formaldehyde gel, after which they were transferred to a nylon membrane (Zetaprobe; Bio-Rad) which was baked in a vacuum at 80° C and hybridized with a 32P-labeled 707-bp fragment of mouse ODC cDNA (18). The intensity of hybridization was assessed by scanning densitometry of the autoradiograms. Statistical Analysis Values for K; and Vmax were determined from full concentration-rate plots of [14C]SPD uptake in cultured PASMCs using the PC Nonlin computer program. These values, along with ODC and AdoMet-DC activities, are presented as the mean ± SEM. Data were compared between experimental groups using a one-way ANOVA com.bined with Ne.umanKeuls test or an unpaired t test, dependmg on the specific experiment. In all cases, P values < 0.05 were considered to denote statistical significance.
Results Kinetics of SPD Uptake Preliminary experiments established that uptake by PASMCs of [14C]SPD in concentrations of 0.1 to 30 /LM was line~r over a 90-min period (data not shown). In subsequent expenments, full SPD concentration-rate plots were constructed using a 30-min incubation period. As illustrated in Figure 1, SPD uptake by PASMCs incubated at 37° C exhibited saturation kinetics as the concentration of substrate was increased from 0.1 to 30 /LM. Additionally, cell-associated radioactivity was proportional to SPD concentration when PASMCs were incubated at 4°C. Results identical to those obtained at reduced temperatures were obtained when the cells were incubated at 37° C in the presence of the metabolic inhibitors dinitrophenol (0.1 mM) and iodoacetic acid (1 mM) (data not shown). This component of uptake, which is assumed to reflect passive diffusion or adsorption, was subtracted from the total uptake at 37° C for subsequent kinetic analysis. Concentration-rate data modeled according to MichaelisMenten kinetics using PC Nonlin indicated that the K; and Vmax values describing [14C]SPD uptake by subconfluent PASMCs were 0.78 ± 0.03 /LM and 4.5 ± 0.3 pmol/min/ lQ6 cells, respectively. Impact of Hypoxia and DFMO on SPD Transport Kinetics Kinetics of ['4C]SPD uptake were assessed in PASMCs cultured under standard, normoxic, and hypoxic conditions and in PASMCs cultured under standard conditions and challenged with 1 mM DFMO. Values of s; and Vm~ des.cribing SPD transport in these four groups are shown in FIgure 2. Neither the severity of hypoxic culture nor DFMO altered Km • In contrast, Vmax increased with decreasing Po 2 of the culture media from 4.5 ± 0.3 pmol/min/ltf cells in cells cultured under standard conditions to 18 ± 1.2 pmol/min /1Q6 cells in cells cultured under normoxic conditions and
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Materials DMEM, trypsin-EDTA, and penicillin/streptomycin solution were purchased from GIBCO (Grand Island, NY). Defined bovine calf serum was provided by HyClone (Logan, UT). PBS, actinomycin D, and cyclohe~imide were p~r chased from Sigma Chemical Co. (St. LoUIS, MO). RadIOchemicals and related supplies used in assessment of SPD transport and ODC and AdoMet-DC. activities were p~r chased from Amersham (Arlington Heights, IL). All plasticware used for tissue culture was obtained from Costar (Cambridge, MA).
Concentration Spermidine (lJM)
Figure 1. Effect of substrate concentration on kinetics of spermidine (SPD) uptake in cultured bovine PASMCs. Cells were cultured under standard conditions (Po2 > 100 mm Hg) at 37° C (control) or at 4 ° C. Each point represents the mean ± SEM of eight observations. Results identical to those obtained at low temperature were obtained when cells were incubated at 37° C in the presence of the metabolic inhibitors dinitrophenol (0.1 mM) and iodoacetic acid (l mM) (data not shown).
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Haven, Olson, Arcot et al.: Polyamine Transport in Pulmonary Artery Smooth Muscle Cells
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tinomycin D also suppressed, but did not abolish, the induction of [14C]SPD transport by DFMo. As illustrated in Figure 4, the protein synthesis inhibitor cycloheximide, in concentrations ranging from 0.001 to 10 J.tM, failed to influence [14C]SPD transport in cells cultured under standard conditions but was associated with concentrationdependent inhibition of the induction in transport caused by both hypoxia and DFMO. Neither actinomycin D nor cycloheximide reduced cell viability as determined by trypan blue exclusion (data not shown).
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Effect of Actinomycin D and Cycloheximide on SPD Transport The effects of RNA synthesis inhibition with actinomycin D on uptake of 3 j.tM (14C]SPD in PASMCs cultured under standard and hypoxic conditions and in PASMCs challenged with 1 mM DFMO are shown in Figure 3. Actinomycin D in concentrations ranging from 5 to 400 nM failed to alter [14C]SPD uptake in cells cultured under standard conditions. Hypoxia and DFMO, as expected, increased [14C]SPD uptake in the absence of actinomycin D, with DFMO being the more effective stimulus for induction. Actinomycin D caused concentration-dependent inhibition of hypoxiainduced [14C]SPD to the extent that there were no differences between cells cultured under standard and hypoxic conditions at actinomycin D concentrations> 50 nM. Ac-
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Figure 4. Concentration-dependent effects of cycloheximide on uptake of 3 JLM SPD by bovine PASMCs. PASMCs were cultured under standard (Po, > 100 mm Hg) or "hypoxic" conditions (Po2 = 18 to 30 mm Hg) or under standard conditions in the presence of 1 mM DFMO. Values for SPD uptake were normalized to 1 million (mil.) cells. Each point represents mean ± SEM of six observations. * Significantly different from uptake in cells cultured under standard conditions.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 71992
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Figure 5. Ornithine decarboxylase (ODC) activity in bovine PASMCs cultured under standardconditions (1'02 > 100 mm Hg), normoxic conditions (1'02 = 50 to 70 mm Hg), hypoxic conditions (1'02 = 18 to 30 mm Hg) or under standard conditions in whichthemediacontained1 mM DFMo. Eachpoint represents the mean ± SEM of at least six observations. * Significantly different from ODC activity in cells cultured under standard conditions.
Figure 7. Activity of S-adenosylmethionine decarboxylase (AdoMet-DC) in bovine PASMCs cultured under standard (1'02 > 100 mm Hg) or "hypoxic" conditions (1'02 = 18 to 30 mm Hg) in the absence or presence of 1 /LM putrescine (PUT). Each point represents the mean ± SEM of at least six observations. * Significantly different from AdoMet-DC activity in cells cultured under standard conditions.
ODC Activity and mRNA Abundance in PASMCs ODC activity in PASMCs cultured under standard, normoxie, and hypoxic conditions and in cells exposed to 1 mM DFMO is shown in Figure 5. ODC activity did not differ between cells cultured under standard and normoxic conditions but was significantly decreased in hypoxic cells and in normal cells exposed to the ODC inhibitor, DFMO. The degree of ODC inhibition associated with culture under hypoxic conditions did not differ from that caused by DFMo. Northern blots comparing the abundance of ODC mRNA in PASMCs cultured under standard and hypoxic conditions are shown in Figure 6. As determined by scanning densitometry, the abundance of ODC mRNA in hypoxic cells was approximately 15 % of that in cells cultured under standard conditions.
tween cells cultured under standard and hypoxic conditions. Because putrescine (and SPD) is a potent stimulus of AdoMet-DC activity in some systems (1), the impact of hypoxia on AdoMet-DC activity was reassessed in cells cultured in medium supplemented with 1 ~M of the diamine. As expected, putrescine increased AdoMet-DC activity in PASMCs cultured under standard conditions. The diamine also increased AdoMet-DC activity in hypoxic cells, but the extent of the increase did not differ from cells cultured under standard conditions. PASMCs cultured under standard and hypoxic conditions were harvested for HPLC and radiometric analysis of ['4C]SPD metabolism. All of the radioactivity was present in the 30- to 32-min fraction corresponding to the retention time of authentic SPD, with no accumulation at time points corresponding to the other polyamines (data not shown). There were no differences in the distribution of radioactivity between cells cultured under standard conditions and cells exposed to hypoxia. These findings suggest that transported ['4C]SPD is not metabolized during the 30-min uptake period in cells cultured under either standard or hypoxic conditions.
AdoMet-DC Activity and Metabolism of P4C]SP D in PASMCs The impact of hypoxia on AdoMet-DC activity in PASMCs is shown in Figure 7. AdoMet-DC activity failed to differ be-
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Figure 6. Northern analysis of steady-state levels of ODCmRNA abundance in bovine PASMCs cultured under standard conditions (lane 1: 1'02 > 100 mm Hg) and hypoxic conditions (lane 2: 1'02 = 18 to 30 mm Hg). The intensity of hybridization observed in hypoxic cells was 15 to 20 % of that in cells cultured under standardconditions, as determined by scanning densitometry. Note that the probe hybridizes nonspecificallyto 28SRNA and that the intensities of this signal are similar between experimental groups.
Discussion Lungs from chronically hypoxic rats exhibit polyaminedependent hypertensive pulmonary vascular remodeling accompanied by decreased ODC activity and increased polyamine transport and elevated AdoMet-DC activity (7, 8). Although polyamine transport properties of cultured pulmonary arterial cells have not been examined, preliminary autoradiographic studies in lungs from intact rats suggest that cells of the pulmonary vasculature may contribute to the augmented polyamine uptake observed in chronically hypoxic rat lungs (13). Against this background, the present study determined if bovine PASMCs in culture expressed an SPD transport system and whether SPD transport and ODC
Haven, Olson, Arcot et al.: Polyamine Transport in Pulmonary Artery Smooth Muscle Cells
and AdoMet-DC activities were modulated by exposure to hypoxic conditions in vitro. At the outset, it should be emphasized that the evidence for transport contributing to increased polyamine contents is derived from studies in intact rat lungs, whereas the present study used bovine PASMCs in culture. Species differences, along with the usual vagaries associated with experiments using cultured cells from large, conduit vessels, may thus complicate extrapolation of the present observations to the intact animal model. Bovine PASMCs exhibited time-, temperature-, and energy-dependent SPD uptake that could be characterized by Michaelis-Menten kinetics. Additionally, SPD transport is subject to adaptive regulation inasmuch as the Vmax was augmented by inhibition ofODC with DFMO or by exposure to hypoxia. In general, these characteristics are typical of polyamine transport systems in other cell types (14). Using lung cells as pertinent examples, the K; for SPD transport in PASMCs, 0.78 I-tM, is similar to rat type II epithelial cells (0.48 I-tM) (9) and rabbit alveolar macrophages (0.16 I-tM) (10). It is not possible to compare values for Vmax because of differences in the units used to express transport rate. Induction of SPD transport in PASMCs by DFMO and differences in the Vmax for SPD transport between growing and quiescent has been noted in other cell populations (14). Along with these similarities, there are interesting differences between bovine PASMCs, rattype II epithelial cells, and rabbit alveolar macrophages. Polyamine transport by bovine PASMCs and rat type II pneumocytes can be modeled by Michaelis-Menten kinetics; these equations do not apply to transport in rabbit alveolar macrophages. Neither bovine PASMCs nor rat type II pneumocytes avidly metabolize transported polyamines, at least over 30- or 60-min periods, respectively, but polyamines transported into rabbit alveolar macrophages undergo substantial metabolism within only 15 min. These disparities in polyamine transport properties may be related to the cell type or species differences. Finally, transport in rabbit alveolar macrophages is nonselective for the three polyamines whereas rat type II pneumocytes seem to express discrete transporters for each of the polyamines with overlapping selectivities. The issue of whether bovine PASMCs express relatively discrete polyamine transporters versus a single, nonselective pathway is relevant to the induction of SPD transport by hypoxia. Although regulation of independent polyamine transporters has not been reported, it is conceivable that the induction of transport by hypoxia may be more prominent for putrescine than for SPD or spermine. This possibility is suggested by the fact that hypoxia depresses ODC activity, which might reduce the pool of putrescine, whereas AdoMet-DC activity is preserved or elevated (8, and see below). Putrescine transport may thus be disproportionately induced to provide substrate for AdoMetDC and conversion to higher order polyamines. As noted above, relative to cells cultured under standard incubator conditions where the Po, of the culture media exceeds 100 mm Hg, PASMCs exposed to Po2 values approximating the normal pulmonary arterial environment or a hypoxic environment exhibited increased values of Vmax for SPD transport. Activity of ODC, the initial rate-limiting enzyme in de novo polyamine biosynthesis, did not differ between cells 'cultured under standard or normoxic conditions but was reduced in hypoxic PASMCs. Whether these altera-
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tions in SPD transport and ODC activity evoked by hypoxia in bovine PASMCs are physiologically significant in terms of the development of hypoxic pulmonary hypertension cannot be discerned from the present studies. Nevertheless, inasmuch as lungs from chronically hypoxic rats exhibit increased putrescine transport and decreased ODC activity (8) and because preliminary autoradiographic experiments suggest enhanced spermidine uptake by pulmonary arterial cells (13), it is possible that induction of transport in cultured PASMCs can be used to examine selected aspects of polyamine regulatory mechanisms in the hypoxic lung. Neither cycloheximide nor actinomycin D, at the concentrations tested, affected SPD transport in PASMCs cultured under standard conditions. However, both agents abolished the induction of transport by hypoxia. This observation is similar to other cell populations in which blockade of RNA and protein synthesis with these agents prevents induction of polyamine transport. It thus appears that the induction of SPD transport by hypoxia also requires RNA and protein synthesis (14, 19). The effects of cycloheximide and actinomycin D on induction of SPD transport by DFMO were, in general, similar to their actions on the hypoxic response. However, it is interesting that RNA synthesis inhibition with actinomycin D suppressed, but did not abolish, the induction of transport by DFMO, in contrast to the complete blockade of hypoxia-induced increases in transport. This disparity may reflect the fact that DFMO was a more potent stimulus for transport than hypoxia or, as discussed subsequently, that DFMO and hypoxia induce SPD transport by different mechanisms. The stimulus for induction of SPD transport by hypoxia in cultured PASMCs is unknown. We considered that the mechanism might be similar to that ascribed to induction of transport by DFMO; namely, that the increase in transport occurs secondarily to decreased ODC activity in the face of a cellular demand for polyamines. In support of this contention, cells cultured under hypoxic conditions exhibited reduced ODC activity and an approximate lO-fold increase in the Vmax for SPD transport in comparison with PASMCs cultured under standard conditions. On the other hand, the Vmax for SPD transport in normoxic cells was increased nearly 4-fold in comparison with cells cultured under standard conditions despite the fact that ODC activity failed to differ between the two environments. In companion studies, we found that DFMO reduced ODC activity to a level similar to that of hypoxia, but that the Vmax for SPD transport increased nearly IS-fold. Although the present data do not permit firm conclusions to be drawn, the finding that ODC activity and induction of transport by hypoxia and DFMO are not closely linked suggests that the mechanism is more complex than initially postulated. As noted previously, hypoxia reduced ODC activity relative to PASMCs cultured under standard conditions. The decrease in ODC activity was accompanied by an approximate 80 % reduction in the abundance of ODC mRNA. The mechanism by which hypoxia suppresses ODC activity is not known. Inasmuch as AdoMet-DC activity was not affected by culture under hypoxic conditions, the depression in ODC activity cannot be ascribed to a generalized reduction in enzyme activities. Regulation of ODC activity is complex and involves incompletely understood transcriptional, post-
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transcriptional, translational, and post-translational mechanisms (20). The reduction in steady-state ODC mRNA content noted in hypoxic PASMCs suggests that transcriptional or post-transcriptional mechanisms are involved. Hypoxia also could promote elaboration of an endogenous inhibitor of ODC, perhaps similar to the ODC antizyme described by Heller and Canellakis (21). Activity of AdoMet-DC was detectable in normoxic and hypoxic PASMCs and, unlike ODC activity, was not affected by hypoxic exposure. Hypoxia also failed to increase AdoMet-DC activity in the presence of putrescine, which is known to stimulate AdoMet-DC activity in some systems (20). In intact, chronically hypoxic rat lungs, AdoMet-DC activity is profoundly increased (8), thus raising the question as to why its activity was unaffected by culture of PASMCs under hypoxic conditions. The distribution of AdoMet-DC activity among the various lung cells is not known, nor is it certain that hypoxia stimulates AdoMet-DC activity in all lung cells. Against this background, it is conceivable that hypoxia stimulates AdoMet-DC activity in lung cells other than PASMCs. Additional studies will be required to address this issue. Despite the fact that AdoMet-DC activity was detected in both PASMCs cultured under standard or hypoxic conditions, transported SPD was not metabolized in either cell population, at least during the 30-min period of exposure to the triamine. This observation, which is similar to that previously reported for rat type II epithelial cells (9), suggests that access of newly transported polyamines to metabolizing enzymes must be regulated. Very little is understood regarding the intracellular compartmentalization of polyamines, though binding to RNA, DNA, ribosomes, polyphosphates, and phospholipids is known to occur (22, 23). Additional studies will be required to delineate mechanisms governing interactions between transported polyamines and their biosynthetic and degradative enzymes. In summary, the present findings demonstrate that bovine PASMCs in culture express a saturable, energy- and temperature-dependent transport system for SPD. SPD transport can be upregulated by culture under hypoxic conditions or exposure to the ODC inhibitor, DFMo. The response of cultured PASMCs to hypoxia mimics the effect of hypoxic exposure on the intact rat lung in terms of decreased ODC activity and induction of polyamine transport, suggesting that this culture system may prove useful in delineating mechanisms by which lung cell polyamine contents are regulated during development of hypoxic pulmonary hypertension. Acknowledgments: This investigationwas supported in part bygrants (HL36404, HL38495, HL02055, and HL02174) from the National Institutes of Health.
References I. Heby, O. 1991. Role of polyamines in cell growth and differentiation. Differentiation 19:1-20.
2. Thet, L. A., and S. C. Parra. 1986. Role of ornithine decarboxylase and polyamines in early postnatal lung growth. J. Appl. Physiol. 61:16611666. 3. Tatar-Kiss, S., S. Bardocz, and P. Kertai. 1984. Changes in L-ornithine decarboxylase activity in regenerating lung lobes. FEBS Lett. 175:131134. 4. Thet, L. A., S. C. Parra, and J. D. Shelburne. 1984. Repair of oxygeninduced lung injury. The role of ornithine decarboxylase and polyamines. Am. Rev. Respir. Dis. 129:174-181. 5. Olson, J. W., A. D. Hacker, R. J. Altiere, and M. N. Gillespie. 1984. Polyamines and the development of monocrotaline-induced pulmonary hypertension. Am. J. Physiol. 247:H682-H685. 6. Olson, J. W., J. E. Atkinson, A. D. Hacker, R. J. Altiere, and M. N. Gillespie. 1985. Suppression of polyamine synthesis prevents monocrotaline-induced pulmonary edema and arterial medial thickening. Toxicol. Appl. Pharmacol. 81:91-99. 7. Atkinson, J. E., J. W. Olson, R. J. Altiere, and M. N. Gillespie. 1987. Evidence that hypoxic pulmonary vascular remodeling is polyaminedependent. J. Appl. Physiol. 62:1562-1568. 8. Shiao, R.-T., H. B. Kostenbauder, J. W. Olson, and M. N. Gillespie. 1990. Mechanisms of lung polyamine accumulation in chronic hypoxic pulmonary hypertension. Am. J. Physiol. 259:L351-L358. 9. Kameji, P., S. R. Rannels, A. E. Pegg, and D. E. Rannels. 1989. Spermidine uptake by type II pulmonary epithelial cells in primary culture. Am. J. Physiol. 256:CI60-CI67. 10. Saunders, N. A., K. F. lIett, andR. F. Minchin. 1989. Pulmonary alveolar macrophages express a polyamine transport system. J. Cell. Physiol. 139:624-631. 11. Wyatt, I., A. R. Soames, M. F. Clay, and L. L. Smith. 1988. The accumulation and localisation of putrescine, spermidine, spermine, and paraquat in the rat lung. In vitro and in vivo studies. Biochem. Pharmacol. 37: 1909-1918. 12. Nemery, B., L. L. Smith, and W.. N. Aldridge. 1987. Putrescine and 5-hydroxytryptamine accumulation in rat lung slices: cellular localization and response to cell-specific lung injury. Toxicol. Appl. Pharmacol. 91: 107-112. 13. Marple, S. L., C. Haven-Doll, B. F. Sisken, J. W. Olson, and M. N. Gillespie. 1990. Autoradiographic localization of spermidine uptake in lungs from chronically hypoxic rats. FASEB J. 4:A577. (Abstr.) 14. Seiler, N., and F. Dezeure. 1990. Polyamine transport in mammaliancells. Int. J. Biochem. 22:211-218. 15. King, S. J., F. M. Booyse, P.-H. Lin, M. Traylor, A. J. Narkates, and S. Oparil. 1989. Hypoxia stimulates endothelial cell angiotensin-converting enzyme antigen synthesis. Am. J. Physiol. 256:CI231-CI238. 16. Dempsey, E. C., I. F. McMurtry, and R. F. O'Brien. 1991. Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia. Am. J. Physiol. 260:L136-L145. 17. Claycomb, W. C., and N. A. Lanson, Jr. 1987. Proto-oncogene expression in proliferating and differentiating cardiac and skeletal muscle. Biochem. J. 247:701-706. 18. Berger, F., D. Loose, H. Meisner, and G. Watson. 1986. Androgen induction of messenger RNA concentrations in mouse kidney is posttranscriptional. Biochemistry 25:1170-1175. 19. Martin, R. L., K. F. lIett, and R. F. Minchin. 1990. Characterization of putrescine uptake by cultured adult mouse hepatocytes. Biochim. Biophys. Acta 1051:25-29. 20. Pegg, A. E. 1988. Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy. Cancer Res. 48:759-774. 21. Heller, J. S., and E. S. Canellakis. 1981. Cellular control of ornithine decarboxylase activity by its antizyme. J. Cell. Physiol. 107:209-217. 22. Davies, R. H., andJ. L. Ristow. 1988. Polyarnine transport in Neurospora crassa. Arch. Biochem. Biophys. 267:479-489. 23. Davies, R. H., andJ. L. Ristow. 1989. Uptake, intracellular binding, and excretion of polyamines during growth of Neurospora crassa. Arch. Biochem. Biophys. 271:315-322.
Hypoxia causes remodeling of the pulmonary circulation that is dependent on increases in lung polyamine contents. Mechanisms by which polyamines are regulated in hypoxic lung cells are unknown, but ornithine decarboxylase (ODC) activity, the initial enzyme in de novo biosynthesis, is depressed and polyamine transport is augmented in lungs from hypoxic rats (R.-T. Shiao et al. 1990. Am. J. Physiol. 259:L351L358). To determine if hypoxia directly influences polyamine regulatory mechanisms in pulmonary vascular cells, we examined [I4C]spermidine (SPD) transport and ODC activity in bovine main pulmonary artery smooth muscle cells (PASMCs) cultured under standard (culture medium Po2 : > 100 mm Hg), "normoxic" (culture medium Po2 : 50 to 70 mm Hg), or "hypoxic" (culture medium Po2 : 18 to 30 mm Hg) conditions. Uptake of [14C]SPD in cells cultured under standard conditions was temperature- and concentration-dependent, exhibited saturation kinetics, and was abolished by metabolic inhibition. Modeling of transport according to Michaelis-Menten kinetics revealed that [I4C]SPD uptake in cells cultured under standard conditions was characterized by K; and Vmax values of 0.78 J.tM and 4.5 pmol/min/tO" cells, respectively. In comparison to cells cultured under standard conditions, K; was unaffected by culture under normoxic or hypoxic conditions while Vmax was increased to 18 pmol/min/ 106 cells in normoxic cells and to 33 pmol/min/l'O' cells in preparations cultured under hypoxic conditions. Inhibition of ODC with o-difluoromethylomithine (DFMO) also induced SPD transport, as evidenced by an increase in the Vmax to 65 pmol/min/lf)" cells. Both hypoxia- and DFMO-induced increases in [I4C]SPD transport were suppressed by cycloheximide and actinomycin D, thus highlighting the importance of protein and RNA synthesis. ODC activity did not differ between cells cultured under standard and normoxic conditions (48 pmol/mg protein/60 min) but was reduced by 75% to 12 pmol/60 min/mg protein in hypoxic cells. The abundance of ODC mRNA also was reduced by 80 to 85 % in hypoxic cells relative to cells cultured under standard conditions. In contrast to the reduction in ODC activity, hypoxia failed to inhibit the activity of another important enzyme in polyamine synthesis, S-adenosylmethionine decarboxylase. These findings indicate that hypoxia induces polyamine transport in PASMCs, perhaps secondary to decreased ODC activity, and suggest that increases in lung polyamine contents necessary for hypoxic vascular remodeling may be ascribed in part to induction of transport in cells of the pulmonary circulation.
The polyamines, putrescine, spermine, and spermidine, are a family of low molecular weight organic cations that play central intracellular regulatory roles in cell growth and differentiation (1). In the lung, their importance is illustrated (Receivedin originalform November 1,1991 and in revisedform March 16, 1992) Addresscorrespondence to: Mark N. Gillespie, Ph. D., Division of Pharmacology and Experimental Therapeutics, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082. Abbreviations: S-adenosylmethionine decarboxylase, AdoMet-DC; o-difluoromethylornithine, DFMO; Dulbecco's modified Eagle's medium, DMEM; high performance liquid chromatography, HPLC; monocrotaline, MCT; ornithine decarboxylase, ODC; pulmonary artery smooth muscle cell, PASMC; phosphate-buffered saline, PBS; spermidine, SPD. Am. J. Respir. Cell Mol. BioI. Vol. 7. pp. 286-292, 1992
by observations that elevated polyamine contents are required for normal lung development (2), for compensatory growth after pneumonectomy (3), for repair of lung injury evoked by hyperoxia (4), and for development of hypertensive pulmonary vascular disease induced by monocrotaline (MCT) (5, 6) or chronic hypoxic exposure (7). Because of the central role of polyamines in regulation of cell structure and function, mechanisms governing lung cell polyamine content are physiologically and pharmacologically significant. In this context, with the notable exception of chronic hypoxic pulmonary hypertension, the dominant regulatory mechanism' seems to involve induction of ornithine decarboxylase (ODC) , the initial rate-limiting enzyme in polyamine synthesis. ODC activity is markedly increased in lungs from neonatal rats (2) and in animals with
Haven, Olson, Arcot et al.: Polyamine Transport in Pulmonary Artery Smooth Muscle Cells
hyperoxic (4) or MCT-induced lung injury (5, 6). Inhibition of ODC with o-difluoromethylornithine (DFMO) prevents increases in lung polyamine contents and suppresses lung development in neonatal rats (2) and forestalls repair of hyperoxic lung injury (4). In MCT-treated rats, DFMO prevents development of pulmonary edema, medial thickening of pulmonary arteries, sustained pulmonary hypertension, and right ventricular hypertrophy (5, 6). In contrast to these model systems, hypoxic pulmonary hypertension is associated with profound decreases in lung ODC activity (8), despite the fact that lung polyamine contents are elevated (7, 8). Though the mechanisms underlying the hypoxia-induced increase in lung polyamines are not established, altered transmembrane transport is a likely possibility in light of our finding that perfused lungs from chronically hypoxic rats exhibit an increase in the Vmax for putrescine uptake and a prolonged half-life of putrescine efflux (8). Lung cell types in which hypoxia enhances putrescine transport are not known. Two lung cell populations, type II pneumocytes (9) and alveolar macrophages (0), exhibit polyamine transport systems in vitro. Cultured pulmonary vascular cells have yet to be examined for their polyamine transport capacities. Autoradiographic experiments in intact rats (11), isolated rat lungs (11), and rat lung slice preparations (2) indicate that transported polyamines localize principally in type II pneumocytes; localization in pulmonary vascular cells generally has not been appreciated. However, in a preliminary autoradiographic study, we found that while vascular spermidine (SPD) uptake was unimpressive in control rat lungs, the pulmonary vasculature in animals subjected to 4 and 7 days of hypoxia exhibited substantial localization of the radiolabel in intimal and medial regions (13). Based on these considerations, the present study determined if an SPD transport system is expressed by bovine main pulmonary artery smooth muscle cells (PASMCs) in culture and, more importantly, tested the hypothesis that polyamine transport and ODC activity in PASMCs are upregulated and downregulated, respectively, by culture under hypoxic conditions. Toverify the potential for induction of SPD transport in PASMCs, some cultures were treated with DFMO, which, along with inhibition of ODC, is a potent stimulus for polyamine transport in many cell culture systems (e.g., 14).
Materials and Methods PASMC Cultures Bovine main pulmonary arteries were harvested from freshly slaughtered cattle (Dawson's Packing Co., Louisville, KY), immersed in cold (4° C) Pucks F6 solution supplemented with 300 U/rnl penicillin, 0.3 mg/ml streptomycin, and transported to the laboratory. The arteries were then opened longitudinally, and the endothelium was scraped away. Medial explants, approximately 2 X 2 mm, were dissected from the subintima and plated in culture flasks containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% defined fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/rnl streptomycin. Cells were then grown to confluence and propagated in culture. Cells from eight different adult cows were used in these experiments. Culture medium was'changed every 3 to 4 days. Cells were harvested using a 0.05% solution of trypsin. All experiments used cells
287
from passages between 2 and 10. The smooth muscle phenotype of the cells was confirmed by the presence of smooth muscle-specific myosin, as evidenced by immunocytochemical analysis (data not shown). Cell counts were determined by hemocytometry. Hypoxic Exposure and DFMO Treatment To examine the effects of hypoxia and DFMO on SPD transport and ODC activity, PASMCs were seeded into 35-mm2 , six-well cluster plates (Costar) at a density of 75,000/well and cultured in DMEM supplemented with 10% serum for 24 h. Subsequently, the well plates were placed in specially constructed plastic chambers affixed with inflow and outflow ports that permitted the chambers to be exposed to selected gas mixtures. The chambers were then placed in standard tissue culture incubators after which they were exposed for an additional 24 h to one of three levels of hypoxia: "standard" conditions, wherein the cells were maintained in incubator chambers gassed with 95 % air-5 % CO 2 (Po 2 > 100 mm Hg); "normoxic" conditions, in which incubator chambers were gassed with a mixture of 12% O2 , 5% CO 2 and 83% N2 (Po 2 = 50 to 70 mm Hg), and "hypoxic" conditions, in which the incubators were purged with 3 % 02> 5 % C02> and 92 % N2 (Po 2 = 18 to 30 mm Hg). Aliquots of culture medium (1.0 ml) were withdrawn anaerobically at termination of hypoxic exposure, and Po, values were determined using an Instrument Laboratories model 213 blood gas analyzer. SPD Transport After the indicated incubation times, PASMCs were rinsed with fresh, serum-free DMEM after which I rnl of serumfree DMEM was added to each well and the cells were allowed to acclimate for a l-h period. Subsequently, [14C]SPD was added to each well in concentrations of 0.1 to 30 JLM, and cells were incubated for specific durations (up to 90 min) and at selected temperatures (4° and 37° C). At the appropriate time, media containing residual [14C]SPD was aspirated, and cells were placed on ice and rinsed with cold phosphate-buffered saline (PBS). The PASMCs were then digested for I h at room temperature in I N NaOH. Before determination of cell-associated radioactivity, 400 JLl of the cell digest was neutralized with 400 JLI of I N acetic acid. An additional 400 JLl of distilled H20 along with 2 rnl of scintillation cocktail was added to the neutralized digests, and radioactivity was determined using a Packard liquid scintillation counter. We elected to normalize [14C]SPD content and uptake rate in terms of cell number rather than protein or DNA content since culture under hypoxic conditions may alter these biochemical parameters (15, 16). Cell Viability Cell viability was assessed using trypan blue exclusion according to standard techniques. In brief, 500 JLl of cell suspension was mixed with the same volume of a 0.08% solution of trypan blue dye in PBS. After a 2- to 15-min incubation period, the numbers of stained and unstained cells were determined using a hemocytometer. Viability is expressed as a percentage of the total number of cells.
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Assessment of DOC and S-Adenosylmethionine Decarboxylase (AdoMet-OC) Activity and p·C]SPD Metabolism ODC and AdoMet-DC activities in PASMCs were determined as described previously (8). Enzyme activities were quantitated by determining the amount of l4C02 released from 0.5 /LCi of L-[1-14C]ornithine or 0.2 /LCi of S-adenosylL-[carboxyl-14C]methionine, respectively, during a 60-min incubation at 37° C. To determine if [14C]SPD was metabolized by PASMCs during the 30-min uptake period, cells were harvested and prepared for high performance liquid chro1l!atography (HPLC) analysis of polyamine contents as descnbed prevrously (8). The effluent from the HPLC column was collected in 2 min fractions, and the amount of radioactivity was determined in each fraction and related to the retention times of the specific polyamines. Northern Analysis of DOC mRNA Total RNA from cells cultured under standard conditions and cells cultured in a hypoxic environment was isolated and purified by the guanidinium isothiocyanate/CsCI method ~s described elsewhere (17). Briefly, PASMCs were washed m PBS, lysed in 5 vol of 4 M gu.anidinium isothiocyan~te solution, layered on a 1.5-ml cushion of 5.7 M CsCI solution, and centrifuged for 16 to 18 h in an SW50.1 rotor (Beckman) at 35,000 rpm. The purified RNA samples were analyzed by fractionating equal amounts (15 /Lg) of RNA on a 1.2 % agarose/2.2 M formaldehyde gel, after which they were transferred to a nylon membrane (Zetaprobe; Bio-Rad) which was baked in a vacuum at 80° C and hybridized with a 32P-labeled 707-bp fragment of mouse ODC cDNA (18). The intensity of hybridization was assessed by scanning densitometry of the autoradiograms. Statistical Analysis Values for K; and Vmax were determined from full concentration-rate plots of [14C]SPD uptake in cultured PASMCs using the PC Nonlin computer program. These values, along with ODC and AdoMet-DC activities, are presented as the mean ± SEM. Data were compared between experimental groups using a one-way ANOVA com.bined with Ne.umanKeuls test or an unpaired t test, dependmg on the specific experiment. In all cases, P values < 0.05 were considered to denote statistical significance.
Results Kinetics of SPD Uptake Preliminary experiments established that uptake by PASMCs of [14C]SPD in concentrations of 0.1 to 30 /LM was line~r over a 90-min period (data not shown). In subsequent expenments, full SPD concentration-rate plots were constructed using a 30-min incubation period. As illustrated in Figure 1, SPD uptake by PASMCs incubated at 37° C exhibited saturation kinetics as the concentration of substrate was increased from 0.1 to 30 /LM. Additionally, cell-associated radioactivity was proportional to SPD concentration when PASMCs were incubated at 4°C. Results identical to those obtained at reduced temperatures were obtained when the cells were incubated at 37° C in the presence of the metabolic inhibitors dinitrophenol (0.1 mM) and iodoacetic acid (1 mM) (data not shown). This component of uptake, which is assumed to reflect passive diffusion or adsorption, was subtracted from the total uptake at 37° C for subsequent kinetic analysis. Concentration-rate data modeled according to MichaelisMenten kinetics using PC Nonlin indicated that the K; and Vmax values describing [14C]SPD uptake by subconfluent PASMCs were 0.78 ± 0.03 /LM and 4.5 ± 0.3 pmol/min/ lQ6 cells, respectively. Impact of Hypoxia and DFMO on SPD Transport Kinetics Kinetics of ['4C]SPD uptake were assessed in PASMCs cultured under standard, normoxic, and hypoxic conditions and in PASMCs cultured under standard conditions and challenged with 1 mM DFMO. Values of s; and Vm~ des.cribing SPD transport in these four groups are shown in FIgure 2. Neither the severity of hypoxic culture nor DFMO altered Km • In contrast, Vmax increased with decreasing Po 2 of the culture media from 4.5 ± 0.3 pmol/min/ltf cells in cells cultured under standard conditions to 18 ± 1.2 pmol/min /1Q6 cells in cells cultured under normoxic conditions and
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Concentration Spermidine (lJM)
Figure 1. Effect of substrate concentration on kinetics of spermidine (SPD) uptake in cultured bovine PASMCs. Cells were cultured under standard conditions (Po2 > 100 mm Hg) at 37° C (control) or at 4 ° C. Each point represents the mean ± SEM of eight observations. Results identical to those obtained at low temperature were obtained when cells were incubated at 37° C in the presence of the metabolic inhibitors dinitrophenol (0.1 mM) and iodoacetic acid (l mM) (data not shown).
289
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tinomycin D also suppressed, but did not abolish, the induction of [14C]SPD transport by DFMo. As illustrated in Figure 4, the protein synthesis inhibitor cycloheximide, in concentrations ranging from 0.001 to 10 J.tM, failed to influence [14C]SPD transport in cells cultured under standard conditions but was associated with concentrationdependent inhibition of the induction in transport caused by both hypoxia and DFMO. Neither actinomycin D nor cycloheximide reduced cell viability as determined by trypan blue exclusion (data not shown).
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 71992
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Figure 5. Ornithine decarboxylase (ODC) activity in bovine PASMCs cultured under standardconditions (1'02 > 100 mm Hg), normoxic conditions (1'02 = 50 to 70 mm Hg), hypoxic conditions (1'02 = 18 to 30 mm Hg) or under standard conditions in whichthemediacontained1 mM DFMo. Eachpoint represents the mean ± SEM of at least six observations. * Significantly different from ODC activity in cells cultured under standard conditions.
Figure 7. Activity of S-adenosylmethionine decarboxylase (AdoMet-DC) in bovine PASMCs cultured under standard (1'02 > 100 mm Hg) or "hypoxic" conditions (1'02 = 18 to 30 mm Hg) in the absence or presence of 1 /LM putrescine (PUT). Each point represents the mean ± SEM of at least six observations. * Significantly different from AdoMet-DC activity in cells cultured under standard conditions.
ODC Activity and mRNA Abundance in PASMCs ODC activity in PASMCs cultured under standard, normoxie, and hypoxic conditions and in cells exposed to 1 mM DFMO is shown in Figure 5. ODC activity did not differ between cells cultured under standard and normoxic conditions but was significantly decreased in hypoxic cells and in normal cells exposed to the ODC inhibitor, DFMO. The degree of ODC inhibition associated with culture under hypoxic conditions did not differ from that caused by DFMo. Northern blots comparing the abundance of ODC mRNA in PASMCs cultured under standard and hypoxic conditions are shown in Figure 6. As determined by scanning densitometry, the abundance of ODC mRNA in hypoxic cells was approximately 15 % of that in cells cultured under standard conditions.
tween cells cultured under standard and hypoxic conditions. Because putrescine (and SPD) is a potent stimulus of AdoMet-DC activity in some systems (1), the impact of hypoxia on AdoMet-DC activity was reassessed in cells cultured in medium supplemented with 1 ~M of the diamine. As expected, putrescine increased AdoMet-DC activity in PASMCs cultured under standard conditions. The diamine also increased AdoMet-DC activity in hypoxic cells, but the extent of the increase did not differ from cells cultured under standard conditions. PASMCs cultured under standard and hypoxic conditions were harvested for HPLC and radiometric analysis of ['4C]SPD metabolism. All of the radioactivity was present in the 30- to 32-min fraction corresponding to the retention time of authentic SPD, with no accumulation at time points corresponding to the other polyamines (data not shown). There were no differences in the distribution of radioactivity between cells cultured under standard conditions and cells exposed to hypoxia. These findings suggest that transported ['4C]SPD is not metabolized during the 30-min uptake period in cells cultured under either standard or hypoxic conditions.
AdoMet-DC Activity and Metabolism of P4C]SP D in PASMCs The impact of hypoxia on AdoMet-DC activity in PASMCs is shown in Figure 7. AdoMet-DC activity failed to differ be-
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Figure 6. Northern analysis of steady-state levels of ODCmRNA abundance in bovine PASMCs cultured under standard conditions (lane 1: 1'02 > 100 mm Hg) and hypoxic conditions (lane 2: 1'02 = 18 to 30 mm Hg). The intensity of hybridization observed in hypoxic cells was 15 to 20 % of that in cells cultured under standardconditions, as determined by scanning densitometry. Note that the probe hybridizes nonspecificallyto 28SRNA and that the intensities of this signal are similar between experimental groups.
Discussion Lungs from chronically hypoxic rats exhibit polyaminedependent hypertensive pulmonary vascular remodeling accompanied by decreased ODC activity and increased polyamine transport and elevated AdoMet-DC activity (7, 8). Although polyamine transport properties of cultured pulmonary arterial cells have not been examined, preliminary autoradiographic studies in lungs from intact rats suggest that cells of the pulmonary vasculature may contribute to the augmented polyamine uptake observed in chronically hypoxic rat lungs (13). Against this background, the present study determined if bovine PASMCs in culture expressed an SPD transport system and whether SPD transport and ODC
Haven, Olson, Arcot et al.: Polyamine Transport in Pulmonary Artery Smooth Muscle Cells
and AdoMet-DC activities were modulated by exposure to hypoxic conditions in vitro. At the outset, it should be emphasized that the evidence for transport contributing to increased polyamine contents is derived from studies in intact rat lungs, whereas the present study used bovine PASMCs in culture. Species differences, along with the usual vagaries associated with experiments using cultured cells from large, conduit vessels, may thus complicate extrapolation of the present observations to the intact animal model. Bovine PASMCs exhibited time-, temperature-, and energy-dependent SPD uptake that could be characterized by Michaelis-Menten kinetics. Additionally, SPD transport is subject to adaptive regulation inasmuch as the Vmax was augmented by inhibition ofODC with DFMO or by exposure to hypoxia. In general, these characteristics are typical of polyamine transport systems in other cell types (14). Using lung cells as pertinent examples, the K; for SPD transport in PASMCs, 0.78 I-tM, is similar to rat type II epithelial cells (0.48 I-tM) (9) and rabbit alveolar macrophages (0.16 I-tM) (10). It is not possible to compare values for Vmax because of differences in the units used to express transport rate. Induction of SPD transport in PASMCs by DFMO and differences in the Vmax for SPD transport between growing and quiescent has been noted in other cell populations (14). Along with these similarities, there are interesting differences between bovine PASMCs, rattype II epithelial cells, and rabbit alveolar macrophages. Polyamine transport by bovine PASMCs and rat type II pneumocytes can be modeled by Michaelis-Menten kinetics; these equations do not apply to transport in rabbit alveolar macrophages. Neither bovine PASMCs nor rat type II pneumocytes avidly metabolize transported polyamines, at least over 30- or 60-min periods, respectively, but polyamines transported into rabbit alveolar macrophages undergo substantial metabolism within only 15 min. These disparities in polyamine transport properties may be related to the cell type or species differences. Finally, transport in rabbit alveolar macrophages is nonselective for the three polyamines whereas rat type II pneumocytes seem to express discrete transporters for each of the polyamines with overlapping selectivities. The issue of whether bovine PASMCs express relatively discrete polyamine transporters versus a single, nonselective pathway is relevant to the induction of SPD transport by hypoxia. Although regulation of independent polyamine transporters has not been reported, it is conceivable that the induction of transport by hypoxia may be more prominent for putrescine than for SPD or spermine. This possibility is suggested by the fact that hypoxia depresses ODC activity, which might reduce the pool of putrescine, whereas AdoMet-DC activity is preserved or elevated (8, and see below). Putrescine transport may thus be disproportionately induced to provide substrate for AdoMetDC and conversion to higher order polyamines. As noted above, relative to cells cultured under standard incubator conditions where the Po, of the culture media exceeds 100 mm Hg, PASMCs exposed to Po2 values approximating the normal pulmonary arterial environment or a hypoxic environment exhibited increased values of Vmax for SPD transport. Activity of ODC, the initial rate-limiting enzyme in de novo polyamine biosynthesis, did not differ between cells 'cultured under standard or normoxic conditions but was reduced in hypoxic PASMCs. Whether these altera-
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tions in SPD transport and ODC activity evoked by hypoxia in bovine PASMCs are physiologically significant in terms of the development of hypoxic pulmonary hypertension cannot be discerned from the present studies. Nevertheless, inasmuch as lungs from chronically hypoxic rats exhibit increased putrescine transport and decreased ODC activity (8) and because preliminary autoradiographic experiments suggest enhanced spermidine uptake by pulmonary arterial cells (13), it is possible that induction of transport in cultured PASMCs can be used to examine selected aspects of polyamine regulatory mechanisms in the hypoxic lung. Neither cycloheximide nor actinomycin D, at the concentrations tested, affected SPD transport in PASMCs cultured under standard conditions. However, both agents abolished the induction of transport by hypoxia. This observation is similar to other cell populations in which blockade of RNA and protein synthesis with these agents prevents induction of polyamine transport. It thus appears that the induction of SPD transport by hypoxia also requires RNA and protein synthesis (14, 19). The effects of cycloheximide and actinomycin D on induction of SPD transport by DFMO were, in general, similar to their actions on the hypoxic response. However, it is interesting that RNA synthesis inhibition with actinomycin D suppressed, but did not abolish, the induction of transport by DFMO, in contrast to the complete blockade of hypoxia-induced increases in transport. This disparity may reflect the fact that DFMO was a more potent stimulus for transport than hypoxia or, as discussed subsequently, that DFMO and hypoxia induce SPD transport by different mechanisms. The stimulus for induction of SPD transport by hypoxia in cultured PASMCs is unknown. We considered that the mechanism might be similar to that ascribed to induction of transport by DFMO; namely, that the increase in transport occurs secondarily to decreased ODC activity in the face of a cellular demand for polyamines. In support of this contention, cells cultured under hypoxic conditions exhibited reduced ODC activity and an approximate lO-fold increase in the Vmax for SPD transport in comparison with PASMCs cultured under standard conditions. On the other hand, the Vmax for SPD transport in normoxic cells was increased nearly 4-fold in comparison with cells cultured under standard conditions despite the fact that ODC activity failed to differ between the two environments. In companion studies, we found that DFMO reduced ODC activity to a level similar to that of hypoxia, but that the Vmax for SPD transport increased nearly IS-fold. Although the present data do not permit firm conclusions to be drawn, the finding that ODC activity and induction of transport by hypoxia and DFMO are not closely linked suggests that the mechanism is more complex than initially postulated. As noted previously, hypoxia reduced ODC activity relative to PASMCs cultured under standard conditions. The decrease in ODC activity was accompanied by an approximate 80 % reduction in the abundance of ODC mRNA. The mechanism by which hypoxia suppresses ODC activity is not known. Inasmuch as AdoMet-DC activity was not affected by culture under hypoxic conditions, the depression in ODC activity cannot be ascribed to a generalized reduction in enzyme activities. Regulation of ODC activity is complex and involves incompletely understood transcriptional, post-
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transcriptional, translational, and post-translational mechanisms (20). The reduction in steady-state ODC mRNA content noted in hypoxic PASMCs suggests that transcriptional or post-transcriptional mechanisms are involved. Hypoxia also could promote elaboration of an endogenous inhibitor of ODC, perhaps similar to the ODC antizyme described by Heller and Canellakis (21). Activity of AdoMet-DC was detectable in normoxic and hypoxic PASMCs and, unlike ODC activity, was not affected by hypoxic exposure. Hypoxia also failed to increase AdoMet-DC activity in the presence of putrescine, which is known to stimulate AdoMet-DC activity in some systems (20). In intact, chronically hypoxic rat lungs, AdoMet-DC activity is profoundly increased (8), thus raising the question as to why its activity was unaffected by culture of PASMCs under hypoxic conditions. The distribution of AdoMet-DC activity among the various lung cells is not known, nor is it certain that hypoxia stimulates AdoMet-DC activity in all lung cells. Against this background, it is conceivable that hypoxia stimulates AdoMet-DC activity in lung cells other than PASMCs. Additional studies will be required to address this issue. Despite the fact that AdoMet-DC activity was detected in both PASMCs cultured under standard or hypoxic conditions, transported SPD was not metabolized in either cell population, at least during the 30-min period of exposure to the triamine. This observation, which is similar to that previously reported for rat type II epithelial cells (9), suggests that access of newly transported polyamines to metabolizing enzymes must be regulated. Very little is understood regarding the intracellular compartmentalization of polyamines, though binding to RNA, DNA, ribosomes, polyphosphates, and phospholipids is known to occur (22, 23). Additional studies will be required to delineate mechanisms governing interactions between transported polyamines and their biosynthetic and degradative enzymes. In summary, the present findings demonstrate that bovine PASMCs in culture express a saturable, energy- and temperature-dependent transport system for SPD. SPD transport can be upregulated by culture under hypoxic conditions or exposure to the ODC inhibitor, DFMo. The response of cultured PASMCs to hypoxia mimics the effect of hypoxic exposure on the intact rat lung in terms of decreased ODC activity and induction of polyamine transport, suggesting that this culture system may prove useful in delineating mechanisms by which lung cell polyamine contents are regulated during development of hypoxic pulmonary hypertension. Acknowledgments: This investigationwas supported in part bygrants (HL36404, HL38495, HL02055, and HL02174) from the National Institutes of Health.
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