38

BBALIP 54249

ler an ichert Pulmonary Research Unit, Policlinic of the Department of internal Medicine, Philipps Uniniversi~ i%zrbui-g, Marburg (Germg~yj

(Received SO March 1993)

Key words: Type II pneumocyte; Nitrogen dioxide; Lsng surfactznt; Phospholipid synthesis; (Rat)

After exposure of rats to NO, (IO ppm, 72 h) HI pneumocytes were isolated and compared to cells from control animals in order to determine whether nitrogen dioxide lation affects surfactant phos~ho~i~~ synthesis. (I) &po& cells contain&

more DNA, protein and phospholipid than type II cells from controls. (2) Choline kinase, CTP : c~o~~~c~bos~ba~c cy~~dy~~~t~a~s~ ferase, and cholinephosphotransferase showed higher specific activities in the exposed cells. (3) In correspondence with this finding, the incorporation rates of choline into intermediate metabolic products were also higher in t N&-exposed cells. (4) The pool sizes of the intermediate metabolic products of the CDPcholine-pathway for the synthesis of ~§~~a~~dy~~~o~~~e \hiere

also higher in the cells isolated from exposed animals. This suggests that acute nitrogen dioxide exposure leads to an enhanced phospholipid synthesis that may be responsible for the higher amount of phospholipid detectable in lung 9avage.

Introduction

Lung surfactant is a highly surface-active material, composed of proteins and phospholipids, which lines

e alveolar surface and is essential for normal lung nction [1,2]. During breathing the lung is in direct

contact with the atmosphere and thus atmospheric air pollutants can reach the terminal lung tissue, d ing on size and solubility of the contaminants. ation of the lung lining material therefore is a conse- quence of the reactivity of the inhaled agents. all atmospheric pollutants nitrogen dioxide ( one of the principal oxidants present in the urban environment. It is known to generate free radicals that are sufficiently stable in ambient air (up to 4 ppmb and in cigarette smoke (up to 50 ppm) [3,4].

Experimental studies demonstrate, t dioxide causes an increase of protein in alveolar lavage material [5]. However, for the surfac- tant phospholipids both an increase [6-S] and a de- crease [9,10] is reported for this component after nitro- gen dioxide exposure. It is well documented that the type II pneumocytes synthesize and secrete the surfac- tant material [II]. Histological findings clearly demon- strate a degeneration of the type I pneumocytes and a

Correspondence to: B. Miiller, Pulmonary Research Uait, Policlinic

of the Department of Internal Medicine, Philipps University Mar-

burg, P.O. Box 2360, 35033 Marbnrg, Germany.

The basis for the increas

nitrogen dioxide i14,15]1. owever, for the CDPcho-

study was to analyse, if this pathway is also

and if this alteration may also contribute to the amount of ~bos~ho~ipids in the e~tracc~~~~ar surfactant mate- rial.

mCi/ mm011 was receiv many). Enzymes were obtained from Serva (Heidel- berg, Germa the other reagents were purchased from Sigma ( senhofen, Germany).

Animals and ,-e.xpomre FOP all experiments male istar rats :C%arles iver

Wiga, Sulzfeld Germany) were used. The

39

ranged between 200-230 g. The animals were kept in cages that were placed into gas tight boxes with water and food ad libitum. Breathing air or NO,-atmosphere were generated either by a constant flow of com- pressed air alone or a mixture of compressed air with NO, to a concentration of 10 ppm over a period of 72 h. The nitrogen dioxide atmosphere was controlled photometrically according to Salzman [16] after reac- tion with an absorption solution every 2 h.

Lung lavage and cell isolation After NO,-exposure the animals were anesthetized

with sodium pentobarbital solution (15 mg/kg body weight; NembutalR, Ceva, France) containing 500 IU heparin per rat by intraperitoneal administration. The following procedures were performed as described in detail by Dobbs et al. [17], all solutions and reagents used were identical to their method: after thoracotomy the uena cava was cut, the trachea cannulated and the lungs washed free of blood via the pulmonary artery and then excised. Lung lavage was performed to total lung capacity 8 times before elastase solution (9.25 mg/lung, Elastin Products, Owensville, MO) was in- stilled. The final cell suspension was transferred to rat IgG coated bacteriological plastic dishes to a density up to 30. lo6 cells and incubated for 1 h in a 10% CO, : air incubator. The unattached type II cells were removed, centrifuged and used for determination of specific activity of phospholipid synthesizing enzymes, choline incorporation rates, and pool sizes of the inter- mediate metabolic products of the CDPcholine-path- way.

Cell fractionation For determination of enzyme activities only freshly

isolated type II pneumocytes were used. To obtain ‘cytosol fractions’ and ‘microsome fractions’ cell frac- tionation of the type II pneumocytes was performed in detail according to Burkhardt et al. [181.

Phosphatidylcholine synthesizing enzymes To test the results of nitrogen dioxide exposure on

phosphatidylcholine synthesis enzymes (i.e., choline ki- nase, CTP : cholinephosphate cytidylyltransferase, and cholinephosphotransferase) of the CDPcholine-path- way were analysed.

Choline kinase (EC 2.7.1.3.2) activity was measured according to Ulane et al. [19] with a modification of Burkhardt et al. 1181. In brief, enzyme activity was determined by conversion of [Me-i4C]choline to [ 14C]phosphocholine. The assay buffer contained 50 mM NaCI, 50 mM ATP, 67 mM Tris-HCI (pH 8.51, 100 mM MgCI,, 5 mM [Me-14Clcholine and 11-40 pg of cytosolic protein in a final test volume of 60 ~1. Reac- tion was allowed to proceed for 10 min at 37°C and was then stopped by boiling the reaction mixture. Choline

and phosphocholine were separated by paper chro- matography and the radioactivity in the phospho- choline was calculated by liquid-scintillation counting.

CTP : cholinephosphate cytidyltransferase (EC 2.7.7.15) was assayed by formation of [i4ClCDPcholine from [Me-14C]choline as reported by Weinhold et al. [20]. In type II cell cytosol the enzyme was measured with and without 1 mM phosphatidylglycerol as activa- tor; the final test volume was 100 ,ul and the protein content ranged from lo-37 pg for the ‘cytosol fraction’ and from 8-11 pug for the ‘microsome fraction’. The incubation (10 min at 37°C) was terminated by boiling. CDPcholine and phosphocholine were separated by thin-layer chromatography, and the radioactivity in the CDPcholine was determined as described by Post et al.

m. Cholinephosphotransferase (EC 2.7.8.2) was assayed

according to the procedure described by Haagsman et al. [22], using the microsomal fraction of the isolated type II pneumocytes (20 pug protein). As substrate 1,2-dioleyl-sn-glycerol (50 pg/ml), that was converted with [r4ClCDPcholine (end concentration 80 ,uM) to 1,2-dioleylphosphatidylcholine in a 0. 1 M Tris-HCl buffer, containing 0.03% Tween-20,4 mM gluthathione and 10 mM MgCI,, pH 7.4; reaction time was 30 min at 37°C. The reaction was terminated by addition of a mixture of chloroform/ methanol (1: 2), and phospho- lipids according to Bligh and Dyer [23] extracted. After separation of the extracted phospholipids via thin-layer chromatography on silica (solvent: chloroform/ methanol/water/ acetic acid, 65 : 35 : 4 : 11, the phos- phohpid bands were counted for radioactivity. The calculation of the activity of the choline phosphotrans- ferase was performed using the the synthesized phos- phatidylcholine and corrected for loss of radioactivity.

Rates of choline incorporation into choline-containing intermediate metabolic products

Freshly isolated type II cells were plated at a density of 5 . lo5 cells/plate in DME medium with 10% FCS, containing 1 &i [14C]choline, and cultured for 4 h. After washing the cells (4. 106) with Krebs-Ringer KRB) buffer they were scraped off from the plastic dishes in a mixture of water/ methanol/ chloroform (5 : 20 : 10; v/v). To achieve phase separation 1 ml of water/chloroform was added. The organic phase containing phosphatidylcholine was separated on silica G thin-layer chromatography plates (Merck, Darmstadt, Germany) with chloroform/ methanol/ water (65 : 35 : 4, v/v> as eluent and counted for ra- dioactivity. The aqueous phase, containing choline, phosphocholine and CDPcholine, was separated on silica H plates (Merck, Darmstadt, Germany) in a mixture of 0.15 M NaCl solution/methanol/NH, (50 : 50 : 5, v/v> and the amount of labelled precursors determined according to Post et al. 124,251. The rate of

esis was calculated as amount of incorporated me per hour and cell, taking into account the

changes in the pooi size of choline.

~~~~yses of the pool sizes of the ~~~~~~e-~o~ta~~~~~ inter- deviate metabolic ~r~~~~~~

Determination of the pool sizes of ~h’~spbat~~y~- choline and intermediate metabolic products were per- formed according the procedure of Post et al. [24]: isolated type II cold methanol/ phos~b~~ip~ds accordi

~~~Qrna~ogr~~~y t

fractions the enzymatic procedure described by al. E24] was used.

Other methods hosphat~dylcho~ine was determined from the ii

extract by thin-layer chromatography on silica G plates erck, Mannheim, Germany) with chlorofor thanol/W,O (65 : 35 : 4, v/v) as eluent using

ethod of Post et al. [25]. Protein was determined according to D7i

using the Bio-Rad (Munich, Germany) r NA was calculated according to ichards [28] and phospho- lipid was calculated with the method o

Statistical analysis were done using e StatgraphicsR program on an I

esults

ronchoalueolar lavage AU rats used in the experiments bad about the same

body weight. Because the recovery of the extracorpo- rally performed bronchoalveolar lavage fluid was in the same range (about 95%) for all animals, the total washed out phospholipid material was calcul and compared to the experimental groups. Lung e of exposed animals contained 3.8 f 0.7 mg phospholipid when compared to controls (0.9 + 0.3 mg) (n = 18).

alyses of the individual phospholipid classes only showed a decrease for the phosphatidylcholine species (77.2 i 4,8% of total phospholipids in controls 61.2 i 4.7% for the NO,-exposed rats; y1 = 7). Concerning the cellular component of the bronchoalveolar lavage the treated animals showed a higher content of granulo- cytes and a decreased content of macrophages when compared to the controls.

(n) = number of cell isoiations; * * = P I 0.31 _

Ceiiular c~aracter~5t~~s After exposure to nitrogen dioxide (72 h, 10 ppmi

ease was found for t

The activities of &dine kinase, CTP : &dine- Iylhransferase and §~~~~~a~§~~~~§~ in fractions of fre isdated Type IE

the specific zc&ity 0f the kinase in the cytosolic fraction -was higher iil

e been isolated from XD,-exposed to cells from control animais.

ate ~y~~~~~~~tra~sf~rase is ~~rn~t~~g enzyme in

did not differ in brane fraction of the rats and controi ani fraction the activity of this enzyme was hi from N Is. Addition of phospbatidy’?..

cerol to the assa creased the ac:i.vity of b~sp~ocbol~~e cyti rase in the cytesol of

cells from control animals and of cells from exposed animals, but then the enzyme activitry in the cytosrsl from exposed animals was not ~~~~~~~~~~~~~ diffezrent

41

TABLE II TABLE IV

Injluence of in vivo exposure to NO, on activity of choline-kinase, CTP: cholinephosphate cytidylyltransferase, and cholinephosphotrans-

ferase in isolated type II cells

Pool sizes of choline-containing metabolites of the CDPcholine-pathway in isolated type II pneumocytes after in vivo exposure to NO, in

comparison to controls

After isolation of the type II pneumocytes the cells were incubated for 1 h in palmitate (0.2 mM bound to BSA in a molar ratio of 5.3 : 1) containing Krebs-Ringer buffer (pH 7.4). After homogenization of the cells the cytosol fractions were tested for choline kinase activity (nmol/min.mg protein) as described in the Materials and Methods section. For CTP : cholinephospate cytidylyltransferase in the pres- ence or absence of 1 mM phosphatidylglycerol (PG) in the test system, and for cholinephosphotransferase cells were not preincu- bated in palmitate. Each value is the mean+S.D. and * indicate values significantly different from those of control.

Metabolite concentrations were determined as described by Post et al. [25]. The numbers represent means+S.D. from four separate experiments and * indicate values significantly different from those of control.

(n = 7) Control NO,

Control NO,

pmol/106 cells

choline 606.7* 119.0 1349.9* 324.5 * phosphocholine 15829.8-+ 2795.5 54335.8+ 8604.9 * CDPcholine 3 891.2+ 1560.3 10244.0* 3000.3 * phosphatidylcholine 125 570.6+ 10836.5 298 633.9 + 49435.4 *

pmol/pg DNA * = P 5 0.01.

Choline kinase cytosol (n = 7) 3.6 +0.6 5.7 +0.9 *

CTP : cholinephosphate cytidylyltransferse (n = 4) cytosol - PG 0.52+0.4 0.97+0.4 * cytosol + PG 2.06 + 0.6 2.14kO.8 microsomes 2.30+0.9 2.07 + 0.8

Cholinephosphotransferase I(n = 6) microsomes 0.32 f 0.01 0.41+0.06 *

* = P ( 0.01.

from that of control animals. The choline phosphotransferase catalyses the final

reaction in the CDPcholine-pathway and the main activity was found in the membrane fraction. Determi- nation of its specific activity showed a higher activity in this fraction from NO,-exposed type II pneumocytes (Table II>.

Incorporation rates and pool sizes of metabolic interme- diate products of the CDPcholine-pathway

When type II cells were cultured in primary culture in the presence of labelled choline for 4 h, the cells isolated from exposed animals showed a higher amount

TABLE III

Choline incorporation into metabolites of the CDPcholine-pathway in isolated type II pneumocytes after in vivo exposure to NO,

Incorporation rates of [‘4C]choline were determined after 3 h of primary culture in the presence of 25 PM palmitate. The numbers represent means f S.D. and * indicate values significantly different from those of controls.

Control NO,

[i4C]choline incorporation rate pmol/h lo6 cells

choline uptake 4 098.6 * 528.2 8584.2k1046.2 * phosphocholine 629.8 + 132.9 1341.6+ 191.4 * CDPcholine 198.3 f 78.2 474.6c 196.5 * phosphatidylcholine 2 238.7 f 684.0 4898.4 + 1023.4 *

* P 0.01. = 5

of incorporated choline than those from control ani- mals. All incorporations were linear over the periods studied (data not shown). The incorporation of label into phosphocholine was also higher in the NO,-ex- posed cells, as it was the incorporation into CDP- choline and into newly synthesized phosphatidylcholine (Table III>.

The pool sizes of choline and its derivates in the isolated cells were higher in the exposed cells when compared to the controls. Hereby it is remarkable that the phosphocholine pool is much more larger than the choline and the CDPcholine pools for both cell popula- tions (Table IV).

Discussion

Nitrogen dioxide is probably the key substance in the photochemical chain leading to oxidant smog [29]. Due to its low solubility in aqueous solutions it will enter the lower respiratory tract and thus may affect the surfactant system, including the type II pneumo- cytes. In the used exposure model intact animals were exposed and then type II pneumocytes were isolated. During this in vivo exposure we believe that also indi- rect effects of the NO,-exposure could influence these cells thus representing the in vivo situation. From preliminary studies we know that lower NO,-con- centration or shorter exposure periods did not cause significant alterations in surfactant phosphatidylcholine synthesis (unpublished data). Although the NO,-con- centration used in these experiments (10 ppm) appear to be high in relation to environmental conditions, there are circumstances (e.g., in smokers) in which these concentrations are considerably higher and reach values up to 250 ppm [3,4]. In addition to short term NO,-exposure studies (5 h) in high concentrations (40 ppm) [14] the results of this study provide further biochemical characterization of the response of the type II cell to long term exposure (72 h). Since the type

IT ~~e~~~Q~~es were isdated directly after the expo-

sure procedure, the ~~ssibi~i~ of recovery within the

we was fixnd nst to be sure since viability and purity

of the cells were identical ts the sontrds. Maawever the yieBd of tine s was doub%ed from ani s that were

2. Similar results were scribed fm isslated celEs by Wr t et al. [I51 and by Ev

tologicaliy the pro mure to nitrogen

the cells from exposed ative state of these ceils

those were consideaably hi r than reported in the literature [31,32]. For su it was found that the

expQsure in viva In the the specific activity of the

-pathway bar the phsspko-

activity of susfactant zymes also represent re NO,-exposure.

wn that aheolas

nthesis is also enhanced in in order to regulate the pha

ystem the enzyme a difference was

assay was car-

e translocation

known to be enhanced [31,42,43]. Interestingly, effects of ozone to type II cells in vitro and in vivo showed mainly decreased enzyme activities and incorporation rates [22,44]. From these observations it might be con- cluded that different phases of injury in response to an oxidant exist. Additionally during in vivo exposure an- tioxidative mechanisms may be developed, due to the oxidative potential of the gas that prevent type II pneumocytes from direct effects of the oxidant [45].

We conclude that the phase of acute alveolar ep- ithelial damage is followed in vivo by an adaptive or recovery stage in which type II cells proliferate. At this stage the capacity to produce surfactant phospholipids may be higher due to the increase in type II cells [30] and/or the reported increase in specific activity of phospholipid synthesizing enzymes. Furthermore, en- hanced surfactant phospholipd synthesis after acute nitrogen dioxide exposure may be responsible for the higher amount of phospholipid that could be detected in lung lavage.

Acknowledgements

The excellent technical work of Christiane Skurk and Annette Piichner is gratefully acknowledged. For discussion of the manuscript the authors thank Dr. Henk P. Haagsman, Lab. Vet. Biochem., University of Utrecht, Netherlands. The study was supported by Grants PEF 9O/OOl/lC and PUG 91/OOl/lC.

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