Lung Cellspecific Polyamine Transport Regulation

Given the substantial effect of hypoxia on lung vascular structure and the requirement for polyamines in cell proliferation and differentiation, we postulated that the hypoxia-induced increase in polyamine uptake would be prominent in PA cells. Previous autoradiographic studies on the cellular localization of polyamine uptake focused on the normoxic lung and identified the most prominent sites of uptake as alveolar type I and II cells (12). Uptake by cells of the normoxic pulmonary circulation had not been appreciated. Accordingly, we used rat lung and main PA explant preparations to examine the effects of culture in a hypoxic environment for 24 hours on [14C]-SPD localization in vascular cells (1). We found increases in the density of [UC]-SPD

labeling in both intimal and medial layers of conduit, muscularized, and partially-muscularized PAs. The extent of [14C]-SPD uptake in main PA explant preparations also was elevated in hypoxia and autoradiography revealed that the increase could be ascribed to augmented labeling of both intimal and medial arterial layers. The hypoxia-induced increase in [l4C]-SPD transport was most evident in smooth muscle cells of the media. Viewed collectively, these findings in lung and main PA explant preparations suggested that hypoxia increases SPD uptake in PAECs and PASMCs, most conspicuously in vascular smooth muscle.

To explore cell-cell interactions in the hypoxic pulmonary vasculature that could impact on polyamine transport properties, we examined the effect of hypoxia in rat denuded PA explants (1). Endothelial denudation reduced the baseline [14C]-SPD uptake and abolished the increase normally evoked by hypoxia. One explanation for this observation was that hypoxic PAECs elaborate afactor(s) that increases [l4C]-SPD uptake by the underlying smooth muscle. To address this possibility, a cross-over design was used in which media conditioned by normoxic or hypoxic PAECs was applied to denuded main PA explants. Normoxic PAEC-conditioned media failed to increase the [HC]-SPD uptake rate in either normoxic or hypoxic denuded explants, thus suggesting that induction of SPD transport in medial smooth muscle cells cannot be ascribed to the hypoxia-mediated activation of a factor(s) released constitutively by cultured, normoxic PAECs. However, while media conditioned by hypoxic PAECs also did not elevate the uptake rate in normoxic explants, it engendered substantial increases in the hypoxic explant preparation. This pattern of results was recapitulated using cultured PASMCs as the bioassay preparation and supports the idea that a factor(s) elaborated by hypoxic PAECs is permissive for the ability of hypoxia to increase [l4C]-SPD uptake rate in PA smooth muscle.

The identity of the postulated endothelium-derived factor(s) enabling hypoxic SPD uptake by underlying smooth muscle is unknown. There are many candidate mediators, such as endothelin, PDGF, and serotonin (8). Based on mounting evidence for involvement of proteases in vascular development and remodeling (26), including hypoxic pulmonary vascular remodeling (39), we examined the prospect that an endothelium-derived protease was important for hypoxic induction of polyamine transport in PASMCs. Media conditioned by hypoxic PAECs was treated with the non-selective protease inhibitor, aprotinin, or the serine protease inhibitor, 1-antitrypsin, and applied to denuded main PA explants cultured in either normoxic or hypoxic conditions. As shown in Figure 1, the ability of hypoxic PAEC-conditioned media to enable hypoxia-induced rises in the [14C]-SPD uptake rate in main PA explants was attenuated by both protease inhibitors. Identical results were obtained using cultured PASMCs as the bioassay preparation. Much work remains to be done, but the idea that an endothelium derived serine protease(s) is an activator of SPD transport in hypoxia by medial arterial smooth muscle is an attractive hypothesis.

Figure 1. Aprotinin (AP, lOpg/ml; A) and alpha-1-antitrypsin (AAT, 50|a.g/ml; B) attenuate hypoxic EC conditioned media (HECM)-induced hypoxia-dependent polyamine transport in rat main PA explants. Normoxic EC conditioned media (NECM) failed to increase polyamine transport in either hypoxic or normoxic PA explants. Mean± SE (n=4). * P<0.05 vs. NECM.

Figure 1. Aprotinin (AP, lOpg/ml; A) and alpha-1-antitrypsin (AAT, 50|a.g/ml; B) attenuate hypoxic EC conditioned media (HECM)-induced hypoxia-dependent polyamine transport in rat main PA explants. Normoxic EC conditioned media (NECM) failed to increase polyamine transport in either hypoxic or normoxic PA explants. Mean± SE (n=4). * P<0.05 vs. NECM.

We undertook additional studies in rat PAECs and PASMCs with the aims of determining the mechanism of the hypoxic effect on polyamine import and if there were multiple transporters regulated differentially by hypoxia (2, 28). Confluent cultures of both cell types were exposed to normoxia or hypoxia and uptake rates for [UC]-PUT, -SPD, and -SPM were determined as a function of polyamine concentration. Polyamine transport pathways in normoxic control populations of these lung vascular cells resembled other cells described in the literature; they exhibited time-, temperature-, and concentration-dependencies. Polyamine transport in lung vascular cells required ongoing RNA synthesis. Protein synthesis inhibition was associated with a transient increase in polyamine import, presumably as a result of relief from antizyme-mediated inhibition, while long term protein synthesis inhibition resulted in a reduction in polyamine import. Uptake of PUT, SPD, and SPM could be modeled according to Michaelis-Menten kinetics and values for K,,, and Vmax are shown in Table 1. In normoxic cells, the kinetic parameters are on the same order of magnitude.

Table 1. Values of K^ (top) and Vmas (bottom) for Polyamine Uptake in Rat PASMCs and ECs Cultured under Control (CON) and Hypoxic (HYP) conditions_

CON HYP

PASMCs ECs

PASMCs ECs 2.4S±0.66 2.5±0.45 2.41±0.73 2.8±0.65

PASMCs ECs 2.85 ± 0.92 5.6±U5 2.04 ± 0.77 11.3*3.0*

Vmas (pMoles/106 cells/min) PUT SPD

PASMCs ECs PASMCs ECs

CON 9.14 ± 2.33 5.60±0.30 21.23±1.92 4.60±0.25 HYP 29.3 ± 3.92* 10.9±0.06* 24.72 ± 2.53 8.60±0.65*

PASMCs ECs 17.44 ± 2.04 3.50±0.30 19.88 ±2.39 8.90±1.20*

Values are expressed as mean ± Standard error. * P<0.05 vs. control.

Figure 2, Competition between 0.1 [HC]-labeled polyamines with the two other unlabeled polyamines at the indicated concentrations for uptake in rat PAECs (A) and PASMCs (B). Mean±SE as percentage of control values (n=6).

In other key respects, however, the polyamine transport pathways operative in cultured rat main PAECs and PASMCs are different. For example, in PAECs there is relatively little cross-competition between the three polyamines for uptake, while in smooth muscle cells, SPD and SPM exhibit cross-competition and inhibit PUT uptake while PUT has minimal effect on the import of SPD and SPM (Fig. 2).

Regultory Control
Figure 3. Uptake of the three polyamines (0.1 |iM) from Na+-free media in cultured PAECs and PASMCs (% of uptake in control, normal Na+ conditions). MeaniSE (n=6).

The Na+-dependence of polyamine transport is also appears to differ between the cell types. Replacing Na+ with choline significantly (P<0.05) inhibits uptake of all three polyamines in ECs, while in PASMCs PUT import is more prominently inhibited by Na+ depletion than the other polyamines (Fig. 3). Finally, the most interesting difference pertains to the transport response to hypoxia. As shown in Table 1, PAECs responded to hypoxia with an increase in the Vm4x for transport for all three polyamines, while SMCs exhibited a selective increase in the Vmax for PUT uptake, with the values for SPD and SPM unchanged from controls.

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