Activation of alveolar phospholipase A2 after hydrochloric acid aspiration in rats

Activation of Alveolar Phospholipase A2 After Hydrochloric Acid Aspiration in Rats YoshiakiTerao, Sumitaka Haseba, Harumasa Nakamura, Hiroaki Morooka, Osamu Shibata, and Koji Sumikawa Puroose: The present study was carried out to determine phospholipase A 2 (PLAz) activity in the bronchoalveolar lavage fluid (BALF) in rats subjected to HCI aspiration. Materials and Methods: Rats were allocated into one of five groups. Groups H-1 and H-3 received instillation of HCI into lungs. Groups S-1 and S-3 received saline instead of HCI. Group C received no instillation. BAL was performed according to the protocol, that is, 1 hour after the instillation in groups H-1 and S-1, 3 hours after the instillation in groups H-3 and S-3, and arbitrarily in group C. Obtain BALF was analyzed for the protein concentration, PLA2 activity, and the molecular mass of PLA2,

Results:The protein concentration in BALF showed an increase in groups H-1 and H-3. PLA2 activity decreased in group H-l, but increased in group H-3, compared with groups S-1 and S-3, respectively. PLAz in groups C and H-1 revealed a high molecular mass (HM), but that in group H-3 revealed a low molecular mass (LM). Conclusions:There is an increase in the alveolar LMPLA2 at inflammatory phase after Hci aspiration, suggesting the pathophysiologic role of LM-PLA2 in the acute lung injury. Copyright 9 2001 by W.B. Saunders Company

CID ASPIRATION-induced lung injury is one

role in the pathophysiology of A R D S , 6 but the role of PLA2 in the lung injury induced by acid aspiration remains unclear. This study was carried out to determine PLA2 activity in the BALF in rats subjected to HC1 aspiration.

of the causes of acute respiratory distress synA drome (ARDS). Acid aspiration causes direct damage to the alveolar-capillary membrane, leading to influx of protein rich edema fluid into the alveolar space I and neutrophil sequestration. 2 A biphasic injury phase was observed with the peaks 1 hour and 4 hours after aspiration. 3 Initial phase consists of an acute protein extravasation resulting from a direct chemical injury by hydrochloric acid (HC1). The second phase (the inflammatory phase) consists of inflammation resulting from the activation and accumulation of neutrophils. Acid aspiration lung injury is associated with lipid inflammatory mediator, arachidonic acid (AA) metabolites, including thromboxane A2 (TXA2) and leukotriene B4 (LTB4).4 Phospholipase A2 (PLA2) is a key enzyme releasing AA from the sn-2 position of phospholipids. Expressions of some PLA2 subtypes were cloned in human lungs. 5 PLA2 activity in bronchoalveolar lavage fluid (BALF) has been suggested to play an important

MATERIALS AND METHODS

Animal Preparation

From the Department of Anesthesiology, Nagasaki University School of Medicine; and the Intensive Care Unit, Nagasaki University Hospital, Nagasaki, Japan. Received April 20, 2000. Accepted November 1, 2000. Address reprint requests to Yoshiaki Terao, MD, PhD, Department of Anesthesiology, Nagasaki University School of Medicine, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Copyright 9 2001 by W.B. Saunders Company 0883-9441/01/1601-0007535. 00/0 doi: lO.1053/jcrc.2001.21796

Male Wistar rats (n = 48) weighing 250 to 350 g were used in this study. The study protocol was approved by the Animal Care Committee of Nagasaki University School of Medicine. The care and handling of the rats were in accord with National Institutes of Health guidelines. Freely feeding rats were anesthetized with pentobarbital (50 mg/kg) administered intraperitoneally and fixed on the heated operating table. After induction of anesthesia, tracheotomy was performed and a catheter was inserted into the carotid artery for arterial pressure monitoring and arterial blood sampling. Anesthesia was maintained with pentobarbital (60 mg/kg/h) intraperitoneally and the rats were paralyzed with pancuronium bromide (0.5 mg/kg/h) intramuscularly. The lungs were mechanically ventilated with an infant ventilator (IV - 1 0 0 B , Sechrist, Anaheim, CA) under the following ventilator settings: ~ mode = pressure-control, Fit2 = 1.0, ventilation frequency = 30/rain, peak airway pressure (Ppe~a,) = 14 cm H20, positive end-expiratory pressure (PEEP) = 2 cm H20, and inspiratory/expiratory ratio = 1:1.9. After ventilation was maintained for 30 minutes to reach steady state, the rats were allocated into one of three groups, that is, groups C, H, and S. Group C (n = 9) underwent only bronchoalveolar lavage (BAL) with saline (37 ~ C; 30 mL/kg) for measurement of total protein concentration and PLA2 activity. Group H (n = 17) received instillation of a total of 3 rnL/kg HC1 (0.1 N; pH = 1.0) intratracheally. For this procedure, the rats received 1.5 mL/kg HCt followed by a bolus of air (30 mL/kg) while lying on their right side, and then received the same amount of HC1 and air while lying on their left side. Group S (n = 17) received instillation of a total of 3 mL/kg saline in-

42

Journal of Critical Care, Vol 16, No 1 (March), 2001: pp 42-46

PLA2 IN ASPIRATION PNEUMONIA

43

stead of HC1 by the same procedure as group H. After the instillation, Ppe~k was set at 26 cm H20 and PEEP at 6 cm H20 in all rats; these ventilator settings were maintained throughout the observation period. Group I~was subdivided to two groups, that is, groups H-1 and H-3. Group H-1 (n = 9) underwent BAL 1 hour after the instillation. Group H-3 (n = 8) underwent BAL twice 7 1 hour and 3 hours after the instillation, and only the second BAL fluid (BALF) was sampled for measurement. Group S was subdivided to two groups, that is, groups S-1 and S-3. Groups S-1 (n = 9) and S-3 (n = 8) underwent BAL in the same manner as groups H-1 and H-3, respectively. Arterial blood gas analysis was done for Pao2 and Paco2 immediately before BAL in all rats. BALF collected by aspiration was centrifuged for 10 minutes at 700g at 4 to remove alveolar ceils and debris, and then stored at - 8 0 until PLA2 assay. Protein concentration in BALF was measured using protein analysis kit (Bio-rad Laboratories, Richmond, CA).

PLAe Assay PLA2 activity in BALF was measured as previously described with some modifications.8'9 L-3-phosphatidylethanolamine, 1acyl-2-[l-14C]arachidonyl (PE) (Amersham, Buckinghamshire, UK) was used as the exogenous substrate, which was dried under N2 and resuspended in dimethylsulfoxide. The reaction was initiated by addition of the sample and 5 mmol/L CaC12 to the substrate (15 ixmol/L at a final concentration). After incubation for 30 minutes at 37 ~ C, the reaction was terminated by addition of ethanol containing 2% acetic acid and 100 ixg/ml free arachidonic acid (AA). The release of X4C-AA was analyzed using thin-layer chromatography with development fluid (ethylacetate/isooctane/HzO/acetic acid [55:75:100:8]. Lipids were visualized by I2 staining and the bands were scraped. Radioactivities were counted with a liquid scintillation counter (Aloka, Tokyo, Japan). The activity of PLA2 was expressed as picomol of AA released from PE per minute per milliliter of BALE

Gel Filtration Each of 2-mL BALF in group C, group H-l, or group H-3 was concentrated t o - 2 0 0 ixL using Centricon 10 (Amicon, Danvers, MA). The concentrated sample was injected into a Snperose 12 FPLC column (Pharmacia LKB Biotechnology, Pis-

cataway, NJ) preequilibrated with 50 mmol/L Tris/HC1 (pH 7.4)/1 mmol/L EDTA/1 mmol/L EGTA/0.15 mol/L NaC1. 6 Samples were eluted in this buffer at a flow rate of 0.5 mL/min. Fractions (1.0 mL) were collected and assayed for PLA2 activity. The column was calibrated using aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease (13.7 kDa) as standards.

Statistics Values were presented as mean • SE. The Man-Whitney U test was used for statistical analysis with P < .05 regarded as significant.

RESULTS

There were no significant differences in Pao2 and Paco2 values before intratracheal instillation among the five groups (Table 1). Intratracheal instillation of HC1 caused an increase in Paco2 and a decrease in Pao2 in group H-1 and group H-3. Because ventilator setting was altered after intratracheal instillation, Paco2 values in group S-1 and S-3 were significantly lower than that in group C. There were no significant differences in systolic blood pressure (SBP) before intratracheal instillation among five groups. SBP before BAL significantly decreased in group H-3 compared with group C or group S-3 (Table 2). Recovery percentage of BALF was significantly greater in group H-3 compared with group C or group S-3. The concentrations of protein in BALF in group H-1 and H-3 were higher compared with group S-1 and group S-3, respectively. PLA2 activity significantly decreased in group H-l, but increased in group H-3 compared with groups S-1 and S-3, respectively (Table 3). To characterize PLA2 in BALF, samples in groups C, H-l, and H-3 were fractionated with gel filtration chromatography. As shown in Figure 1,

Table 1, Blood-Gas Analysis Before and After Instillation of HCI (n) Control S-1 H-1 S-3 H-3

(9) (9) (9) (8) (8)

pre-Paco2 (mm Hg) 52.5 44.5 46.6 54.9 47.7

• • • + +

3.8 2.1 1.4 3.5 3.1

BAL- Paco2 (mm Hg) 52.5 34.3 50.2 30.8 87.1

• • • • •

3.8 2.9* 3.9t 1.1" 11.5"1"

pre-Pao2 (mm Hg) 403 439 416 427 449

• 16 -+ 17 + 9 _+ 22 • 21

BAL- Paco2 (mm Hg) 403 443 289 473 159

• • • • •

16 15 43"t 29

40"1"

NOTE. Values are mean • SE. Abbreviations: Control, without intratracheal instillation; S, intratracheel saline; H, intratracheal HCI; 1 or 3, time after intratracheal instillation; pre-, before intratracheal instillation; BAL, bronchoalveolar lavage; BAL-, before BAL; n, number. *P < .05 vs. control group. t P < .05 for group S-1 vs. group H-1 or group S-3 vs. group H-3.

44

TERAO ET AL

Table 2. SBP Before and After Instillation pre-SBP (mm Hg)

(n)

BAL-SBP (turn Hg)

Control

(9)

140 -+ 7

140 -+ 7

S-1

(9)

124 -+ 6

121 • 13

H-1

(9)

' 134-+7

S-3 H-3

(8) (8)

134 -+ 5 128 -+ 5

~-

134+7 129 • 7 94 -+ 11"1"

NOTE. Values are mean • SE. Abbreviations: Control, w i t h o u t intratracheal instillation; S, intratracheal saline; H, intratracheal HCI; 1 or 3, time after intratracheal instillation; SBP, systolic blood pressure; pre-, before intratracheal instillation; BAL, bronchoalveolar l a v a g e ; BAL, before BAL; n, number. * P < .05 vs. control group. 1.P< .05 for group S-1 vs, group H-1 or group S-3 vs. group H-3.

PLA2 activities in groups C and H-1 migrated to a peak as the fraction number 8 (estimated molecular mass 67 to 158 kDa), but PLA2 activity in group H-3 migrated to a peak as the fraction number 17 (approximately molecular mass 14 kDa). DISCUSSION

The results show that intratracheal instillation of HC1 causes gradual decreases in pulmonary and cardiovascular functions associated with influx of protein-rich fluid into the alveolar space, indicating that lung injury develops in 3 hours after acid aspiration. The results of PLA2 assay show that there is a biphasic change of PLA2 activity in BALF after HC1 aspiration, that is, a decrease in 1 hour, and then an increase in 3 hours and that a highmolecular mass PLA2 (HM-PLA2) seen in normal rats is depressed after HC1 instillation, whereas a low-molecular mass PLA2 (LM-PLA2) is activated 3 hours after the instillation.

Kennedy et al 3 published a model of acute acid aspiration-induced lung injury in the rat undergoing intratracheal instillation with HC1. In their model, the injury was quantitated by permeability measurements of the leakage of radiolabeled protein from the circulation into the lung. A biphasic injury pattern was observed with injury peaks 1 hour and 4 hours after aspiration. Histologic studies at 4 hours revealed significant increases in neutrophil in the alveolar space. Eijking et al 1 instilled HC1 into the trachea of the rats and showed that HC1 caused direct damage to the alveolar-capillary membrane, leading to influx of protein-rich edema fluid into the alveolar space. These plasma-derived proteins contained inhibitors of pulmonary surfactant function. Martling and Lundberg 1~instilled either HC1 or gastric juice into the trachea of the rats and showed that the protein extravasation was reduced by about 70% after capsaicin treatment. They concluded that acute protein extravasation after aspiration was mainly dependent on capsaicinsensitive sensory nerves. These findings indicate that the biphasic injury pattern in acid aspiration lung injury might be caused by diverse pathways. The initial phase would be produced by a direct damage to the alveolar-capillary membrane, and the second phase (the inflammatory phase) would be produced by the inflammation associated with polymorphonuclear neutrophil (PMN) sequestration. The present results show that PLA2 in groups C and H-1 have a high molecular mass (HM), but that in group H-3 has a low molecular mass (LM). Kim et al 6 reported two subtypes of PLA2 in BALF in patients with ARDS, and that lung injury score correlated positively with PLA2 activity in BALF but not with the protein concentration in BALE The two subtypes described by them had low and high

Table 3. BALF Assay

Control S-1 H-1 S-3 H-3

(n)

Recovery (%)

(9) (9) (9) (8) (8)

85 82 85 86 97

• 1 _+ 2 + 2 -+ 1 -+ l * f

Protein Concentration (mg/mL) 0.2 0.1 3.7 0.1 4.5

_+ 0.0 • 0.4 • 0.4"1-+ 0.3 • 0.1"1"

PLA2 Activity (pmol/rnin/mL) 6.5 6.2 4.5 2,6 3.7

• -+ + -+ •

0.5 0.6 0.2"1" 0.2* 0.2"1.

NOTE. Values are mean -+ SE. Abbreviations: Control, Without intratracheal instillation; S, intratracheal saline; H, intratracheal HCI; 1 or 3, time after intratracheal instillation; BALF, bronchoalveolar lavage fluid; PLA2, phospholipase A2; n, number. *P < .05 vs. control group. I P < .05 for group S-1 vs. group H-1 or group S-3 vs. group H-3.

PLA2 IN ASPIRATION PNEUMONIA

45

A; Control 158

67

43

13.7

4~

.>_.

3-

~ 2 -,I a.

1 0

I

I

10

B; H-1

158

67

158

I 10 67

I

15 43

20 13.7

"F, E"

0 Fig 1. Fractionation of BALF PLA2 activity in group C (A: the upper), in group H-1 (B: the middle), and in group H-3 (C: the lower). Fractionation was performed by gel-filtration, and assayed for PLA2 activity. There was one peak (fraction number 8) of high molecular mass PLA2 (estimated molecular mass 67158 kDa) in group C and group H-l, but there was one peak (fraction number 17) of low molecular mass PLA2 (approximately molecular mass 14 kDa) in group H-3. The column was calibrated using aldolase (MW = 158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and ribonuclease (13.7 kDa).

C; H-3 4

I 15 43

i 20 13.7

13.

molecular mass, that is, 14 kDa type IIA and 80 to 90 kDa. It is possible that the two PLA2 subtypes in this study could be the same as the two PLA2 subtypes reported by Kim et al. In this study, there was a decrease of HM-PLA2 activity after instillation of HC1. HM-PLA2 activity might be inhibited by the plasma-derived proteins in bronchoalveolar space. Sixteen-kDa Clara

10

15

20

Fraction n u m b e r cell protein, an inhibitor of phospholipase A2 activity, was detectable in BALF in patients with acute lung injury.11 This protein might be secreted into alveolar space after instillation of HC1. Although the mechanism involved in normal catabolism of pulmonary surfactant is yet unknown, 12 the HM-PLA 2 may play a role in the catabolism of pulmonary surfactant in steady state.

46

TERAO ET AL

Four subfamilies of PLA2, (ie, secretory PLA2 [sPLA2], cytosolic PLA2 [cPLA2], Ca2+-independent PLA2, and platelet activating factor [PAF] acety, lhydrase [PAF-AH]), have been detected and isolated from a variety of mammalian tissues and cells. 13 Cytosolic PLA2 is detected in the cytosolic fraction in the cell and its role is well unknown. The role of Ca2+-independent PLA2 is considered to remodel membranous phospholipids. PAF-AH hydrolyzes PAF and oxidized phospholipid preferentially. Subfamily of sPLA2 is classified into five subtypes, that is, IB, IIA, IIC, V, and X. Type IB is pancreatic type in the pancreatic juice 9 Because types IIC, V, and X have been recently identified since 1994, their properties remain unclear. It has been reported that the activity of type IIA PLA2 increases in the serum of septic patients, 14"15 and in the serum 16 and BALF 6 in patients with ARDS, and that type IIA PLA2 would be activated in the inflammatory tissue9 In this study, there was a significant increase in the activity of LM-PLA2 3 hours after instillation of HC1. This time course of pathological change after HC1 is consistent with the inflammatory phase9 Thus, it is likely that LM9

PLA2, with a molecular mass 14 kDa, which increased in the inflammatory phase in this study could be type IIA PLA2. In this study, pulmonary function worsened 1 hour and 3 hours after HC1 aspiration, when the systolic blood pressure also decreased significantly. These changes in pulmonary and cardiac functions are in accordance with the findings of previous investigators. Nesti et all7 instilled gastric aspirate titrated to pH 1.0 into the trachea of the piglets and showed that the oxygenation went from bad to worse in the inflammatory phase9 Schertel et al is instilled HC1 into the lung of the dog and also showed that acid aspiration resulted in significant decreases in mean arterial pressure and cardiac output. In conclusion, we found a significant increase in alveolar LM-PLA2 after HC1 aspiration in rats, suggesting a possible pathophysiologic role of LMPLA 2 in the development of acute lung injury.

ACKNOWLEDGMENT

We acknowledgeDaido Hokusan (TokyoJapan) for lending us mechanicalventilator(Sechrist IV-100B).

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10. MartlingCR, LundbergJM: Capsaicinsensitiveafferents contributeto acute airway edema followingtracheal instillation of hydrochloric acid or gastric juice in the rat. Anesthesiology 68:350-356, 1988 11. Jorens PG, SibilleY, GouldingNJ, et al: Potential role of Clara cell protein,an endogenousphospholipaseA2 inhibitor, in acute lung injury.Eur Respir J 8:1647-1653, 1995 12. Lewis JF, Jobe AH: Surfactantand the adult respiratory distress syndrome.Am Rev Respir Dis 147:218-233, 1993 13. DennisEA: The growingphospholipaseA2 snperfamilyof signal transdnctionenzymes.Trends BiochemSci 22:1-2, 1997 14. TeraoY, Hashimoto S, Nakamura H, et al: Is circulatory phospholipase A2 removed by continuoushemodiafiltrationin septic acute renal failure?J Jpn Soc IntensiveCare Med 5:385388, 1998 15. S6rensenJ, Kald B, TagessonC, et al: Platelet-activating factor and phospholipaseA2 in patients with septic shock and trauma. IntensiveCare Med 20:555-561, 1994 16. RomaschinAD, Demajo WC, WintonT, et al: Systemic phospholipase A2 and cathectinlevels in adult respiratory distress syndromeand multiple-organfailure. Clin Biochem25:5560, 1992. 17. Nesti FD, FuhrmanBR SteiuhornDM, et al: Perfluorocarbon-associated gas exchange in gastric aspiration.Cfit Care Med 22:1445-1452, 1994 18. SchertelER, Pratt JW, Schaefer SL, et al: Effects of acid aspiration-induced lung injury on left ventricular function. Surgery 119:81-88, 1996