1095(1991)223-229 £3 1091El~vier SciencePubtlshers O.V. AS rights reserved 411()74889/gl/S03 fit)
223
ni~himica et Biophysics Acts.
BBAMCR13053
Cytoehrome P-450-dependent metabolism in alveolar ~pe II epithelial cells: modulation by platelet-activating factor J e a n R a b o v s k y 2, W i l l i a m H . P a i l e s ~, D e l o r i s J. J u d y i a n d V i n c e n t C a s t r a n o v a ~ i National I ~ t i t o t e f o r Occopallonal Sa~el3, a,,d Health, Division o f Re~pir,,to~ Disease S,l.d#~. 'vforganto~n. Wit" I U S A l and 2 California State D~Tartment o f Heahh Sck'nce~. Sacramento, CA (U.S A )
(Received9 April 1991} (Revised rn~nuscript receivcd 18July 1991)
Platelet-activating factor (pAF), an ether lipid mediator released from activated pulmon~,ry phagocytes, was evaluated for its ability to affect cytochrome P-450.dependent activities in isolated tat alveolar type 11 cells. The data indicate that at non-toxic doses, PAF caused an increase in ~-naphtholla,~one (BNF) inducible/ ot-naphthoflavone (ANF) sensitive ethoxyphenoxazone deethylase (EtOPx'ase) activity. A. high concentrations of PAF, inhibition of both EtOPx'ese and metyrapone (MP) sensitive benzyloxyphenoxazone deb~azylase (BzOPx'ase) activities and aggregation of type II cells were observed. The PAI¢ analogs, lyso-PAF and enantio-PAF, exhibited actions similar to those observed with PAF. PAF-induced enhancement of EtOPx'ase activity required the presence of intact cells, whereas at high PAF concentrations decreased enzyme activities were observed in beth intact cell and sonieated cell preparations. The data thus suggest that xenobiotic metabolism in alveolar type Ii cells can be modified by an inflammatory mediator, such as PAF, produced by alveolar phagocytes.
Platelet-activating factor (PAF) (Fig. 1) is a unique naturally occurring phospholipid, in that it is an ether rather than the ester usually found in cell membranes. PAF is responsible for diverse biological responses [l] which may be divisible i'~to two major classes. One operates through highly specific receptors of high affinity and may represent normal tissue function. The other class, operating through nonspecific, low affinity but high capacity binding sites, appears to encompass responses to toxic conditions [2,3]. Among the biological responses effected by PAF are those directed towards pulmonary tissue. Exposure of the lung to PAF results in increased resistance, de-
c8~0-P-O-ell 2 ~ - q , bCH3
~o
o
Pkrl~et AclivuiMgFoctor p ~ / Fig. 1 structure of platelet-activatingfactor(PAF).
Abbreviations: pAF. platelet-activatirtgfactor. BNF, B-naphthoflavane; ANF, a-naphthoflavone; EtOPx'ase, ethoxYphenoxazone deethylase; BzOPx'a~, benzyloxyphen~on¢ deelhyla~: mP, iaelhyralmne: tlt, typeII; IFN, int¢fferon; E, enantio; MC, methylcholanthrone,
creased lung compliance, pulmonary hypertension and edema [4]. Along with increased airway and arterial pressures, increased syntheses of ~GE 2 and thromboxane A2 have been detected in PAF-treated guinea pig lungs [5]..~uperoxide anion release has also been detected in PAF-stimulated guinea-pig alveolar macrophages [6] but not in rat alveolar macrophages [7]. Despite the known effects of PAF on pulmonaw function and on the release of some biochemical substances, little is known regarding the effect of PAF on
Cortesoondence: V. Cagtran~a. rZhief, ni~hemisllT Section, Notional Institute tar Occupational Safety and Heaah. 944 Chestnut Ridge Road, MS 21I, Morgantown,WV 7/a505,U.S.A.
specific lung biochemical pathways. As part of our program of studying biochemical processes in isolates cells of the rat alveolar region, we inquired into the
effect of PAF on alveolar type II (t n) cytochrome P-450-dependent metabolism. Cytoehreme P-450-dependent metabolism is the initial pathway responsible for the metabolism of foreign substances (xenobiotics) to less toxic or highly reactive species. In the alveolar region of the lung, the pathway exists at high levels in tll cells [8]. Exposure of the alveolar region, ill particular alveolar tll cells, to cellular mediators or to substances that activate the secretion of cellular mediators, could impact on the ability of this class of enzymes to properly metabolize xenobiotics. Changes in eytochrome P-450-dependent metabolism have been reported in pulmonary as well as nonpulmonary tissue after in vivo exposure to various purified cytokines. Results indicate that injection of interleukin-1, tumor necrosis factor, lymphotoxin or interferon (IFN) induce decreased P-450-dcpandent aetiv;ty in lung and liver 24 h after treatment [9-12]. The ability of cellular mediators to alter P-450-dependent activity was also tested in vitro. In cultured hepatocyles, cytoehrome P-450-dependent activity remained unchanged when tumor necrosis factor, lymphotoxin or interferon were added directly to the cells [12,13]. lnterleukin-l, however, caused a decrease in P-450-dependent activity under the same conditions [12,14]. These results suggest that only interleukin-I acted directly on hepatoeytes while the actions of the other cytokines were mediated through other substances. In the currenf study, we investigated the effect of another phagocyte-derived mediator, PAF, on P-450dependent metabolism in alveolar tll cells. Data indicate that in vitro treatment of type 11 cells with PAF caused a dose-dependent increase in one form of cytochrome P-450-activity.
Animals and isolation of type 1I cells. Specific pathogen.free male Sprague-Dawley rats (175-250 g) were injected, i.p., with one dose of/3-naphthoflavone (BNF) in corn oil at a final concentration of 80 mg per kg rat. 48 h later, animals were anesthetized with sodium pentobarbital (220 mg/kg body weight) and the lungs were excised, perfused with physiological saline and lavaged with ten 9 ml aliquots of physiological saline. Alveolar tll cells were isolated from the 0cr. fused and lavaged lungs of ~ix rats by centrifugal elutr;ation of the elastase-collagenase digested tissue [15]. tli cells were identified and counted as described previously using an electronic cell counter equipped with a cell sizer [15]. Data reported in this paper were obtained from 47 separate preparations of type I1 cells. The yield was 27.6 + 0.9 • 106 tll cells per g lung tissue with a purity of 96 5: 0.2%. The final t n preparation was suspended in 0.05 M Tris (pH 7.4)+ 0.25 M sucrose and used the same day. Sonieated preparations
of t a cells were obtained by pulse sonicating (0.33 s on, 0.67 s off) the whole cell preparations, kept in a testtube on ice, for 30 s at a power of 3 W. Enzyme assays. Two cytochrome P-450-dependent activities, ethoxyphenoxazone deethylase (EtOPx'ase) and benz-yloxyphenoxazone dehenzylase (BzOPx'ase) *, were assayed by the appearance of the fluorescent product resorufin [16]. Rat lung alveolar t . Et0Px'ese activity is associated with the BNF-inducihle, a-naphthoflavone (ANF)-sensitive form(s) of cytochrome ;'-450, whereas t u BzOPx'ase is associated with the ~v~-BNF-inducible (or constitutive), metyrapone tMP)-sensitive form(s) ** [8]. The enzyme activities of whole cell and sonicated cell preparations were assayed by a modified procedure of Pohl and Fouts [18]. The 1 ml reaction contained 0_5 mM NADPH (provided by a glucose 6-phosphate generating system [15]). 10 izM dicumarol, 0.1 M NaCI, and ceils ((1-7,5)- 106/ml) in 0.05 M Hepes (pH 7.8). The reaction was initiated with 2.5 ~ M substrat¢ (10 pA of 0.25 mM ROPx in DMSO). PAF (prepared in 0.05 M Hepes, pH 7.8) was added before the cells and the cells were in contact with the mediator for about 5 rain before the reaction was initiated. Dicumarol, at 10 ~M, was included to inhibit quinone reductese activity which interferes with the assay in cell preparations. EtOPx'asc and BzOPx'ase activities in mierosomal preparations were unaffected by the presence of 10 fzM diem'narol [8.15]. After a 10 rain incubation at 37 °, 2 ml of cold CH3OH were added and the tubes were kept on ice. Controls consisted of identical reactions, except the cells were added after the CH3OH. Reaction conditions were chosen to be linear with time and enzyme concentration and to be saturating with respect to substrate and co*factor concentrations. After a 10 rain centrifugation (205 × g) to remove the precipitate, the fluorescence of the supernatant liquid was incasureA at 585 nm (A~ = 530 nm), This procedure was chosen to circumvent interference problems that might occur in the presence of high concentrations of PAF (7]. When EtOPx'ase and BzOPx~ase activities were measured in the rat lung microsomal fraction, the direct kinetic assay outlined in Rabovsky and Judy [19] was used.
* Eto]'x'ase an~ nzoFx'ase are ssnonym~for ed',ox~-and be.z~lore- ,csomfln o
223
ni~himica et Biophysics Acts.
BBAMCR13053
Cytoehrome P-450-dependent metabolism in alveolar ~pe II epithelial cells: modulation by platelet-activating factor J e a n R a b o v s k y 2, W i l l i a m H . P a i l e s ~, D e l o r i s J. J u d y i a n d V i n c e n t C a s t r a n o v a ~ i National I ~ t i t o t e f o r Occopallonal Sa~el3, a,,d Health, Division o f Re~pir,,to~ Disease S,l.d#~. 'vforganto~n. Wit" I U S A l and 2 California State D~Tartment o f Heahh Sck'nce~. Sacramento, CA (U.S A )
(Received9 April 1991} (Revised rn~nuscript receivcd 18July 1991)
Platelet-activating factor (pAF), an ether lipid mediator released from activated pulmon~,ry phagocytes, was evaluated for its ability to affect cytochrome P-450.dependent activities in isolated tat alveolar type 11 cells. The data indicate that at non-toxic doses, PAF caused an increase in ~-naphtholla,~one (BNF) inducible/ ot-naphthoflavone (ANF) sensitive ethoxyphenoxazone deethylase (EtOPx'ase) activity. A. high concentrations of PAF, inhibition of both EtOPx'ese and metyrapone (MP) sensitive benzyloxyphenoxazone deb~azylase (BzOPx'ase) activities and aggregation of type II cells were observed. The PAI¢ analogs, lyso-PAF and enantio-PAF, exhibited actions similar to those observed with PAF. PAF-induced enhancement of EtOPx'ase activity required the presence of intact cells, whereas at high PAF concentrations decreased enzyme activities were observed in beth intact cell and sonieated cell preparations. The data thus suggest that xenobiotic metabolism in alveolar type Ii cells can be modified by an inflammatory mediator, such as PAF, produced by alveolar phagocytes.
Platelet-activating factor (PAF) (Fig. 1) is a unique naturally occurring phospholipid, in that it is an ether rather than the ester usually found in cell membranes. PAF is responsible for diverse biological responses [l] which may be divisible i'~to two major classes. One operates through highly specific receptors of high affinity and may represent normal tissue function. The other class, operating through nonspecific, low affinity but high capacity binding sites, appears to encompass responses to toxic conditions [2,3]. Among the biological responses effected by PAF are those directed towards pulmonary tissue. Exposure of the lung to PAF results in increased resistance, de-
c8~0-P-O-ell 2 ~ - q , bCH3
~o
o
Pkrl~et AclivuiMgFoctor p ~ / Fig. 1 structure of platelet-activatingfactor(PAF).
Abbreviations: pAF. platelet-activatirtgfactor. BNF, B-naphthoflavane; ANF, a-naphthoflavone; EtOPx'ase, ethoxYphenoxazone deethylase; BzOPx'a~, benzyloxyphen~on¢ deelhyla~: mP, iaelhyralmne: tlt, typeII; IFN, int¢fferon; E, enantio; MC, methylcholanthrone,
creased lung compliance, pulmonary hypertension and edema [4]. Along with increased airway and arterial pressures, increased syntheses of ~GE 2 and thromboxane A2 have been detected in PAF-treated guinea pig lungs [5]..~uperoxide anion release has also been detected in PAF-stimulated guinea-pig alveolar macrophages [6] but not in rat alveolar macrophages [7]. Despite the known effects of PAF on pulmonaw function and on the release of some biochemical substances, little is known regarding the effect of PAF on
Cortesoondence: V. Cagtran~a. rZhief, ni~hemisllT Section, Notional Institute tar Occupational Safety and Heaah. 944 Chestnut Ridge Road, MS 21I, Morgantown,WV 7/a505,U.S.A.
specific lung biochemical pathways. As part of our program of studying biochemical processes in isolates cells of the rat alveolar region, we inquired into the
effect of PAF on alveolar type II (t n) cytochrome P-450-dependent metabolism. Cytoehreme P-450-dependent metabolism is the initial pathway responsible for the metabolism of foreign substances (xenobiotics) to less toxic or highly reactive species. In the alveolar region of the lung, the pathway exists at high levels in tll cells [8]. Exposure of the alveolar region, ill particular alveolar tll cells, to cellular mediators or to substances that activate the secretion of cellular mediators, could impact on the ability of this class of enzymes to properly metabolize xenobiotics. Changes in eytochrome P-450-dependent metabolism have been reported in pulmonary as well as nonpulmonary tissue after in vivo exposure to various purified cytokines. Results indicate that injection of interleukin-1, tumor necrosis factor, lymphotoxin or interferon (IFN) induce decreased P-450-dcpandent aetiv;ty in lung and liver 24 h after treatment [9-12]. The ability of cellular mediators to alter P-450-dependent activity was also tested in vitro. In cultured hepatocyles, cytoehrome P-450-dependent activity remained unchanged when tumor necrosis factor, lymphotoxin or interferon were added directly to the cells [12,13]. lnterleukin-l, however, caused a decrease in P-450-dependent activity under the same conditions [12,14]. These results suggest that only interleukin-I acted directly on hepatoeytes while the actions of the other cytokines were mediated through other substances. In the currenf study, we investigated the effect of another phagocyte-derived mediator, PAF, on P-450dependent metabolism in alveolar tll cells. Data indicate that in vitro treatment of type 11 cells with PAF caused a dose-dependent increase in one form of cytochrome P-450-activity.
Animals and isolation of type 1I cells. Specific pathogen.free male Sprague-Dawley rats (175-250 g) were injected, i.p., with one dose of/3-naphthoflavone (BNF) in corn oil at a final concentration of 80 mg per kg rat. 48 h later, animals were anesthetized with sodium pentobarbital (220 mg/kg body weight) and the lungs were excised, perfused with physiological saline and lavaged with ten 9 ml aliquots of physiological saline. Alveolar tll cells were isolated from the 0cr. fused and lavaged lungs of ~ix rats by centrifugal elutr;ation of the elastase-collagenase digested tissue [15]. tli cells were identified and counted as described previously using an electronic cell counter equipped with a cell sizer [15]. Data reported in this paper were obtained from 47 separate preparations of type I1 cells. The yield was 27.6 + 0.9 • 106 tll cells per g lung tissue with a purity of 96 5: 0.2%. The final t n preparation was suspended in 0.05 M Tris (pH 7.4)+ 0.25 M sucrose and used the same day. Sonieated preparations
of t a cells were obtained by pulse sonicating (0.33 s on, 0.67 s off) the whole cell preparations, kept in a testtube on ice, for 30 s at a power of 3 W. Enzyme assays. Two cytochrome P-450-dependent activities, ethoxyphenoxazone deethylase (EtOPx'ase) and benz-yloxyphenoxazone dehenzylase (BzOPx'ase) *, were assayed by the appearance of the fluorescent product resorufin [16]. Rat lung alveolar t . Et0Px'ese activity is associated with the BNF-inducihle, a-naphthoflavone (ANF)-sensitive form(s) of cytochrome ;'-450, whereas t u BzOPx'ase is associated with the ~v~-BNF-inducible (or constitutive), metyrapone tMP)-sensitive form(s) ** [8]. The enzyme activities of whole cell and sonicated cell preparations were assayed by a modified procedure of Pohl and Fouts [18]. The 1 ml reaction contained 0_5 mM NADPH (provided by a glucose 6-phosphate generating system [15]). 10 izM dicumarol, 0.1 M NaCI, and ceils ((1-7,5)- 106/ml) in 0.05 M Hepes (pH 7.8). The reaction was initiated with 2.5 ~ M substrat¢ (10 pA of 0.25 mM ROPx in DMSO). PAF (prepared in 0.05 M Hepes, pH 7.8) was added before the cells and the cells were in contact with the mediator for about 5 rain before the reaction was initiated. Dicumarol, at 10 ~M, was included to inhibit quinone reductese activity which interferes with the assay in cell preparations. EtOPx'asc and BzOPx'ase activities in mierosomal preparations were unaffected by the presence of 10 fzM diem'narol [8.15]. After a 10 rain incubation at 37 °, 2 ml of cold CH3OH were added and the tubes were kept on ice. Controls consisted of identical reactions, except the cells were added after the CH3OH. Reaction conditions were chosen to be linear with time and enzyme concentration and to be saturating with respect to substrate and co*factor concentrations. After a 10 rain centrifugation (205 × g) to remove the precipitate, the fluorescence of the supernatant liquid was incasureA at 585 nm (A~ = 530 nm), This procedure was chosen to circumvent interference problems that might occur in the presence of high concentrations of PAF (7]. When EtOPx'ase and BzOPx~ase activities were measured in the rat lung microsomal fraction, the direct kinetic assay outlined in Rabovsky and Judy [19] was used.
* Eto]'x'ase an~ nzoFx'ase are ssnonym~for ed',ox~-and be.z~lore- ,csomfln o
