Báo cáo khoa học: Acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity in cortical and medullary human renal tissue docx

Acetyl-CoA:1- O -alkyl- sn -glycero-3-phosphocholine acetyltransferase lyso-PAF AT activity in cortical and medullary human renal tissue Tzortzis N.. The latter proposal comes from our p

Acetyl-CoA:1- O -alkyl- sn -glycero-3-phosphocholine acetyltransferase (lyso-PAF AT) activity in cortical and medullary human

renal tissue

Tzortzis N Nomikos1, Christos Iatrou2and Constantine A Demopoulos1

1

National and Kapodistrian University of Athens, Faculty of Chemistry, Panepistimioupolis, and2Center for Nephrology

‘G Papadakis’, General Hospital of Nikea-Pireaus, Athens, Greece

Platelet-activating factor (PAF) is one of the most potent

inflammatory mediators It is biosynthesized by either the

de novobiosynthesis of glyceryl ether lipids or by remodeling

of membrane phospholipids PAF is synthesized and

catabo-lized by various renal cells and tissues and exerts a wide range

of biological activities on renal tissue suggesting a potential

role during renal injury The aim of this study was to identify

whether cortex and medulla of human kidney contain the

CoA:1-O-alkyl-sn-glycero-3-phosphocholine

acetyl-transferase (lyso-PAF AT) activity which catalyses the last

step of the remodeling biosynthetic route of PAF and is

activated in inflammatory conditions Cortex and medulla

were obtained from nephrectomized patients with adeno-carcinoma and the enzymatic activity was determined by a trichloroacetic acid precipitation method Lyso-PAF AT activity was detected in both cortex and medulla and dis-tributed among the membrane subcellular fractions No statistical differences between the specific activity of cortical and medullary lyso-PAF AT was found Both cortical and medullary microsomal lyso-PAF ATs share similar bio-chemical properties indicating common cellular sources Keywords: platelet-activating factor; biosynthesis; remode-ling; acetyltransferase; human kidney

Introduction

1-O-Alkyl-2-acetyl-sn-glycero-3-phosphocholine

(platelet-activating factor, PAF) [1] represents a class of highly active

lipid mediators It is produced and released by various cells

such as leukocytes, lymphocytes, endothelial cells, neurons,

myocytes, hepatic cells and it is known to elicit a variety of

biological responses participating in the pathogenesis of

many inflammatory and noninflammatory diseases [2]

PAF is produced by two distinct biosynthetic pathways

The first pathway, the de novo pathway, starts with the

acetylation of 1-O-alkyl-sn-glycero-3-phosphate (ALPA)

by the CoA:1-O-alkyl-sn-glycero-3-phosphate

acetyl-transferase (ALPA AT) (EC 2.3.1.105) followed by the

sequential action of a phosphohydrolase and a

dithiothre-itol-insensitive cholinephosphotransferase The second

pathway, the remodeling pathway, involves the hydrolysis

of preexisting plasma membrane phospholipids to 1-O-alkyl-sn-glycero-3-phosphocholine (lyso-PAF) which

is then acetylated by the acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine acetyltransferase (lyso-PAF AT; EC 2.3.1.67) leading to the formation of PAF The de novo pathway seems to be responsible for the constitutive production of PAF at basal levels in cells, while the remodeling one is thought to be responsible for the increased production of PAF by inflammatory cells upon stimulation The latter pathway is regulated mainly by the level and the degree of lyso-PAF AT and 85 kDa cPLA2 activation The latter enzyme hydrolyses plasma membrane phospholipids serving the substrates for lyso-PAF AT [3] Lyso-PAF AT is found in the microsomal fraction of cells and tissues It has a rather broad substrate specificity, concerning the alkyl/acyl group at the sn-1 position and the polar head group at the sn-3 position of the glycerol backbone, it is Ca2+-dependent and is activated by phosphorylation through the action of cAMP-dependent protein kinases, calcium-calmodulin dependent protein kinases, protein kinase C [4,5] and mitogen-activated protein kinases [6,7] According to our knowledge only partial purification of lyso-PAF AT from rat spleens, leading to a very low yield of pure enzyme, has been reported [8,9]

The possibility that the kidney could produce PAF, even under normal physiological conditions, has been proposed

in view of its activity in human urine [10,11] PAF production in the kidney is originated either by blood-born cells (neutrophils, platelets and macrophages) or by resident glomerular (messangial and endothelial) and medullary cells [12,13] Both biosynthetic pathways have been exhibited in renal cells of different animal species and human and several

Correspondence to C A Demopoulos, 39 Anafis Str., Athens,

GR-113 64, Greece.

Fax: + 32 10 7274265, Tel.: + 32 10 7274265,

E-mail: demopoulos@chem.uoa.gr

Abbreviations: ALPA, 1-O-alkyl-sn-glycero-3-phosphate; ALPA AT,

acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphate acetyltransferase;

ESI, electron spray ionization; [H 3 ]PAF, 1-O-hexadecyl-2-[H 3

]acetyl-sn-glycero-3-phosphocholine; PAF, platelet-activating factor;

lyso-PAF AT, acetyl-CoA:1-O-alkyl-sn-glycero-3-phosphocholine

acetyltransferase; PAF-AH, platelet-activating factor acetylhydrolase.

Note: a web site is available at http://www.chem.uoa.gr/Personel/

Laboratories/FoodChem/CVS/demopoulos.htm

(Received 13 February 2003, revised 18 April 2003,

accepted 16 May 2003)

of their enzymes have been characterized in both cultures

and tissues [14–18] However, as in other cases, the

activation of lyso-PAF AT (to wit, of the remodeling

pathway) is mainly responsible for the rapid and increased

synthesis and release of PAF from the kidney, which

occurred after stimulation with inflammatory mediators

[19,20] Increased levels of PAF have been found in blood,

urine and kidney tissue of animals and humans with renal

inflammatory diseases, such as glomerulonephritis and

could be or due to the enhanced biosynthesis or decreased

degradation or to a combination of increased production

and decreased degradation of PAF in nephritic tissue

[21,22]

The latter proposal comes from our previous studies in

which we have demonstrated: (a) the existence of PAF

acetylhydrolase, PAF-AH, the degradative enzyme of PAF,

in human kidney tissue (cortex and medulla) [23]; (b) the

diminished activity of PAF-AH in renal tissue (mainly

cortex) received from patients with primary

glomerulo-nephritis compared to normal ones [24]; and (c) increased

levels of PAF in plasma and urine as well as increased

PAF-AH activity in serum in patients with primary

glomerulo-nephritis in comparison to normal volunteers [24]

As our previous works demonstrated the presence of the

degradative enzyme of PAF (PAF-AH) in human kidney

tissue, in this work an attempt is made to investigate the

presence of the biosynthetic enzyme of PAF, lyso-PAF AT,

in the same kind of tissue Additionally, we characterized

and compared the main biochemical properties of the two

enzymatic activities (cortical and medullary lyso-PAF AT),

utilizing a modified trichloroacteic acid precipitation

method for the lyso-PAF AT assay Our results

demon-strate the existence of lyso-PAF AT activity in both cortex

and medulla of human kidneys and show that cortical and

medullary lyso-PAF AT share similar biochemical

proper-ties indicating common cellular sources

Materials and methods

Materials and instrumentation

All solvents were of analytical grade and supplied by Merck

(Darmstadt, Germany) HPLC solvents were from

Rath-burn (WalkerRath-burn, Peebleshire, UK) The separation of the

lipid products of the assays was performed at room

temperature on a HP HPLC Series 1100 liquid

chromato-graphy model (Hewlett Packard, Waldbronn, German)

equipped with a 100-lL Rheodyne (7725 i.d.)

injector, a degasser G1322A, a quat gradient pump G1311A

and a HP UV spectrophotometer G1314A as a detection

system The spectrophotometer was connected to a

Hewlett-Packard (Hewlett-Hewlett-Packard, Waldbronn, German) model

HP-3395 integrator-plotter The separation of lipids was

performed on a Partisil 10 lm C18column (250· 4.6 mm

i.d.) from Analysentechnick (Wo¨ehlerstrasse, Mainz,

Germany) with an C18(20· 4.0 mm i.d.) precolumn

cart-ridge Chromatographic material used for TLC was silica

gel H-60 (Merck) The platelet aggregation was measured in

a Chrono-Log (Havertown, PA, USA) aggregometer

cou-pled to a Chrono-Log recorder (Chrono-Log) Electrospray

ionization (ESI) mass spectrometry experiments were

per-formed on a Q-Tof (Micromass UK Ltd, Manchester, UK)

orthogonal acceleration quadrupole time-of-flight mass spectrometer equipped with nano-electrospray ionization Radioactivity was measured in a 1209 RackBeta-Flexivial a-Counter (LKB-Pharmacia, Turku, Finland) A Virsonic

50 Ultrasonic Cell Disruptor was used for the homogeni-zation of our samples (Virtis Co Gardiner, USA) All centrifugations were performed in a Sorvall RC-5B refri-gerated Superspeed centrifuge (Sigma) apart from the centrifugation at 100 000 g, which was performed in a Heraeus Christ, Omega 70 000 ultracentrifuge (Hanau, Germany) For the precipitation of the BSA

201 M microcentrifuge was used (Sigma, St Louis, MO, USA)

1-O-Hexadecyl-2-[H3 ]acetyl-sn-glycero-3-phosphocho-line, [H3]PAF (specific activity 6 CiÆmmol)1) was purchased from DuPont NEN (Boston, MA, USA) [H3]acetyl-CoA (specific activity 200 mCiÆmmol)1) was obtained from ICN (Costa Mesa, CA, USA) Unlabeled PAF, lyso-PAF (1-O-hexadecyl-2-lyso-sn-glycero-3-phosphocholine) and acetyl-CoA were from Sigma Chemicals Co 2,5-Diphenyloxazole (PPO) and 1,4-bis(5-phenyl-2-oxazolyl)benzene (POPOP) were purchased from BDH Chemicals (Dorset, UK) 4-[2-Aminoethyl]benzenesulfonyl fluoride (pefabloc) was kindly offered by A Tselepis, University of Ioannina, Greece

3 All other reagents were from Sigma Chemicals

Human renal tissues Human renal tissues were obtained from 20 nephrectomized patients with adenocarcinoma Immediately after the neph-rectomy the kidneys were perfused with normal saline and

a speciment of the apparently normal parenchyma was separated under stereoscopic microscopy into cortex and medulla and placed immediately in cold saline Subse-quently, all homogenization and subcellular fractionation procedures were completed in less than 3 h

Homogenization of renal tissues and preparation

of subcellular fractions Homogenization of cortical and medullary samples and preparation of subcellular fraction is carried out by a modification of the method described by Lenihan and Lee [25] Briefly, cortical and medullary tissues were rinsed with ice-cold 0.25Msucrose, minced and homogenized with six strokes of a motor-driven Potter-Elvehjem homogenizer

in 0.25Msucrose, 10 mMEDTA, 5 mMmercaptoethanol,

50 mM NaF, 50 mM Tris/HCl (pH 7.4) (homogenization buffer) The final concentration of the tissue in the homogenization buffer was 10% w/v Further homogeni-zation of the tissue by sonication (4· 20 s with intervals of

1 min) was followed The homogenates were centrifuged at

500 g for 10 min The pellets were discarded, a small portion of the supernatants was kept for protein and lyso-PAF AT determination and the rest of them were centrifuged at 20 000 g for 15 min The resulting pellets (mitochondria fraction) were suspended in 1 mL of 0.25M sucrose, 1 mM dithiothreitol, 50 mM Tris/HCl (pH 7.4) (suspension buffer) (5–10 mg of proteinÆmL)1) while the supernatants were centrifuged at 100 000 g for 1 h The

100 000 g pellet (microsomal fraction) was suspended in

1 mL of suspension buffer (1–5 mg of proteinÆmL)1) Total

membranes were obtained by centrifugation of the 500 g

supernatant at 100 000 g for 1 h All fractions were

aliquoted and stored at – 30C All homogenization and

fractionation procedures were carried out at 0C

Lyso-PAF AT activity assay

Unless stated otherwise, subcellular fractions of cortex and

medulla, containing 10–40 lg of total protein, were

incu-bated with 4 nmol of lyso-PAF and 40 nmol of [H3

]acetyl-CoA (100 BqÆnmol)1) for 30 min at 37C in a final volume

of 200 lL of 50 mMTris/HCl buffer (pH 7.4) containing

0.25 mg mL)1BSA The final volume of the suspension

buffer in all incubation mixtures was 50 lL By the end of

the incubation time, 2 lL of BSA 100 mgÆmL)1were added

and the reaction was stopped by addition of 64 lL of a 40%

cold trichloroacetic acid solution The reaction mixture was

kept in ice for 30 min and centrifuged at 10 000 g for 2 min

The supernatant was discarded and the pellet containing the

[H3]PAF bound to the denaturated BSA is dissolved in

the scintillation cocktail (dioxane-base) and the radioactivity

was determined by liquid scintillation counting Matching

controls were run in the absence of lyso-PAF in order to

subtract the radioactivity of the endogenously produced

[H3]PAF

Characterization of the lipid products of the lyso-PAF

AT assay

In order to identify the lipid products of the lyso-PAF AT

assay, the enzymatic reactions were terminated by lipid

extraction using the method of Bligh and Dyer [26],

modified by the addition of 1M HCl to the methanol

The lipid products were separated on TLC plates precoated

with Silica Gel H using a solvent system of chloroform–

methanol–ethanoic acid–water (100 : 57 : 16 : 8, by

vol-ume) The distribution of the label was determined by zonal

scraping and measuring the radioactivity by liquid

scintil-lation spectrometry Standards of [H3]PAF and [H3

]acetyl-CoA were ru n with the samples

Lipid products were further analyzed by HPLC on a

reversed-phase C18using an isocratic elution system

consis-ted of methanol 90% and water 10% (v/v) and a flow rate of

1 mLÆmin)1 Fractions of 0.5 mL were collected and their

radioactivity was measured by liquid scintillation counting

The retention time of authentic [H3]PAF and [H3]acetyl-CoA

was compared with the retention time of the radioactive

peaks obtained from the separation of assay products

The above procedures were also applied for the extraction

and chromatographic analysis of the radioactive products

bound to the BSA precipitates after the addition of

trichloroacetic acid to the reaction mixture

The radioactive product, co eluted with authentic PAF at

either TLC or HPLC separation, was further characterized

by bioassay using the washed rabbit platelet aggregation

method as described previously [1] We estimated the

biological activity of the product by measuring its

aggre-gatory activity towards washed rabbit platelets and

com-paring it with the aggregatory activity of known

concentrations of synthetic PAF The biological activity of

the hydrolyzed product, obtained by mild alkaline

hydro-lysis as well as the biological activity of the reacetylated

product obtained by acetylation of the hydrolyzed product was also estimated as described previously [27] Finally, the percentage inhibitory activity of 0.7 mMcreatine phosphate/ creatine phosphate kinase, 10 lM

4,5

BN 52021 towards the biologically active product was compared with their respective activity towards standard PAF of the same aggregatory activity with the product The biologically active lipid was also analyzed by ESI mass spectrometry Samples were dissolved in a small volume of HPLC grade methanol–water (70 : 30, v/v) 0.01M in ammonium acetate Electrospray samples are typically introduced into the mass analyzer at a rate of 4.0 lLÆmin)1 The positive and negative ions, generated by charged droplet evaporation, entered the analyzer through

an interface plate and a 100-mm orifice, while the declus-tering potential was maintained between 60 and 100 V to control the collisional energy of the ions entering the mass analyzer The emitter voltage was typically maintained at

4000 V

Analytical methods Protein was determined by the method of Lowry et al [28] Statistical analysis

Unless otherwise stated, data are expressed as mean values ± SD Differences between groups were assessed

by Mann–Whitney U-test A P-value <0.05 was considered

as significant

Results

Characterization of the trichloroacetic acid method for the determination of lyso-PAF AT activity Preliminary experiments were carried out in order to determine the best experimental conditions for the quanti-tative recovery of [3H]-PAF, the product of the lyso-PAF

AT assay, to the BSA precipitate [3H]-PAF of known specific activity (20 000 c.p.m., 20 lM)

various concentrations of BSA, ranging from 0 to

8 mg mL)1, in 50 mM Tris/HCl buffer (pH 7.4) and the radioactivity of the pellet obtained by the addition of trichloroacetic acid was measured Practically, all [3H]-PAF

is obtained in the protein pellet at BSA concentrations of 0.5 mgÆmL)1and higher while only 0.6% of [3 H]-acetyl-CoA was coprecipitated with [3H]-PAF the rest of which remained in the supernatant This indicates an efficient separation of the substrate, [3H]-acetyl-CoA and the reaction product, [3H]-PAF Incubation of [3H]-PAF with different concentrations (0–0.5 mgÆmL)1) of various subcel-lular fractions of human kidney, such as the 100 000 g pellet, the 100 000 g supernatant, the 20 000 g pellet and the homogenates could not alter the recovery of [3H]-PAF in the BSA pellet indicating that apart from BSA no other endogenous protein could affect the precipitation of PAF after the addition of trichloroacetic acid None of the other constituents of the lyso-PAF AT assay, such as the type of buffer solution, the type and concentration of the enzymatic preparation, the temperature and the pH had any major effect on the recovery of the [3H]-PAF at the BSA pellet

Characterization of the assay products

The products of the lyso-PAF AT assay, with either cortex

or medulla microsomes, were extracted by the Bligh-Dyer

method and separated by TLC The main radioactive

product comigrated with authentic PAF at RF0.2 When

the lyso-PAF AT assay was carried out in the absence of

lyso-PAF, a minimal formation of [3H]-PAF was observed

suggesting that either endogenous lyso-PAF or lyso-PC may

be used as acetyl acceptors A small radioactive peak at RF

0.8 was also observed in the presence or absence of

lyso-PAF, which is probably due to by products of the reaction

between [3H]-acetyl-CoA and microsomal constituents The

same profile was obtained when the products of the assay

were analyzed by HPLC (Fig 1)

The distribution of the radioactivity between the BSA

pellet and the supernatant, after the addition of

trichloro-acetic acid to the incubation mixture, was also determined

Incubation of cortical or medullary microsomes with

[3H]-acetyl-CoA in the absence of lyso-PAF or incubation

of [3H]-acetyl-CoA with lyso-PAF in the absence of

micro-somes resulted in the precipitation of 0.8% of the

radio-activity to the BSA pellet All the precipitated radioradio-activity

was found in the water phase of the Bligh–Dyer extraction of

the pellet indicating that it was due to the small fraction of

[3H]-acetyl-CoA that was precipitated to the BSA pellet

under the assay conditions Incubation of microsomes with

both lyso-PAF and [3H]-acetyl-CoA resulted in a six- to

sevenfold increase of radioactivity in the pellet, most of which

was distributed to the organic phase of the Bligh–Dyer

extraction of the pellet and it comigrated with authentic PAF

after TLC analysis No [3H]-PAF was found in the

super-natant of the incubation mixture after the addition of

trichloroacetic acid indicating that all [3H]-PAF produced in

the lyso-PAF AT assay is precipitated in the BSA pellet

The lipid product, comigrated with PAF, and could

activate the aggregation of washed rabbit platelets The

biological activity of the product was proportional to its

radioactivity indicating that the radioactive product and the

biologically active compound are the same molecule Moreover, inhibitors of platelet aggregation such as BN-52021, indomethacin and the enzymatic system of creatinine phosphate and creatinine phosphate kinase exerted the same inhibitory effect on both standard PAF and the lipid product of similar aggregatory activity Mild alkaline hydrolysis of the product resulted to a complete loss

of its biological activity, which is re-obtained by reacetyla-tion of the hydrolyzed product

The positive ESI spectra of the assay product showed [M + Na]+at m/z 546 The ions at m/z 184 and 147 cor-responding to fragments of the phosphocholine moiety were also present (Fig 2) All the above findings show that the only product of the lyso-PAF AT assay is indeed C16-PAF

Effect of BSA on the microsomal lyso-PAF AT activity

of human cortex and medulla

As shown in Fig 3, a plateauof maximum activity, for both cortical and medullary lyso-PAF ATs, was observed for BSA concentrations ranging from 0 mgÆmL)1 to 0.5 mgÆmL)1 BSA concentrations above 0.5 mgÆmL)1have

a slight inhibitory effect on both acetyltransferases activities

We routinely used 0.25 mgÆmL)1BSA for the determina-tions of lyso-PAF AT activity However, this concentration

is insufficient for the quantitative precipitation of [3H]-PAF

so an excess of BSA (final concentration 2 mg mL)1) was added at the end of the incubation time in order to achieve the maximum recovery of PAF at the BSA precipitate

Effect of Tris concentration and pH on the microsomal lyso-PAF AT activity of human cortex and medulla

We studied the dependence of the enzyme activity with pH

at 20, 50, 100 mM

7 of Tris-buffered solution The pH–activity profile was bell-shaped for both enzymes and the optimum

pH was in the range of 7.4–7.7 Lyso-PAF AT activity was also dependent on Tris concentration showing maximum activity at 50 mM

8 of Tris Subsequent experiments were carried out with 50 mM

Kinetics of PAF formation The kinetics of PAF synthesis in relation to time and microsomal protein are shown in Fig 4 Linearity for up to

20 min of incubation and 0.2 mgÆmL)1 of cortex micro-somal protein and for u p to 20 min and 0.12 mgÆmL)1of medulla microsomal protein was observed Apart from the experiments concerning the influence of substrate concen-trations on lyso-PAF AT activity, we routinely used 0.1– 0.2 mgÆmL)1of microsomal protein for the lyso-PAF AT assays and incubated the reaction mixture for 30 min in order to achieve the maximum yield of the reaction product Dependence of lyso-PAF AT activity on divalent cations Cortical and medullary microsomes were incubated with various concentrations of exogenous added CaCl2 and MgCl2 in the presence or absence of EDTA Results are shown in Table 1 Low CaCl2and MgCl2concentrations,

u p to 10)5Mdid not have any significant effect on lyso-PAF

AT activity while higher concentration inhibited lyso-PAF

Fig 1 Separation of the lipid products produced by the incubation of

cortical microsomes (0.192 mg proteinÆmL)1) with [ H 3 ]acetyl-CoA in the

absence of lyso-PAF (m) or in the presence of lyso-PAF (e) The

retention time of authentic [H 3 ]PAF (d) and [H 3 ]lyso-PAF (s) was

compared with the retention time of the radioactive peaks obtained

from the separation of the assay products The extraction and

separ-ation of the products was carried out as described in materials and

methods.

AT activity in a dose-dependent manner Mg2+was a more

potent inhibitor of both cortical and medullary lyso-PAF

AT than Ca2+ Both divalent cations showed an increased

inhibitory activity against medulla lyso-PAF AT The

chelating agent, EDTA (1 mM) inhibited the cortex and

medulla acetyltransferase activity by 80 and 90%,

respect-ively, in the absence of divalent cations Addition of 10)3M

CaCl2totally reversed the inhibition caused by EDTA This

reversion was more significant for the cortical lyso-PAF AT

activity than the medullary one

Influence of various compounds on lyso-PAF AT activity

The influence of various compounds on the activity of both

cortical and medullary lyso-PAF AT activity was tested

Fig 2 Positive ion electrospray mass spectrum

of the lyso-PAF AT assay product The isola-tion of the assay product and the electrospray analysis was performed as described in Mate-rials and methods.

Fig 3 Effect of BSA concentration on microsomal lyso-PAF AT.

Microsomal fractions of human cortex (0.14 mgÆmL)1) (s) or hu man

medulla (0.12 mgÆmL)1) (d) were incubated with various

concentra-tions of BSA and the lyso-PAF AT activity was determined as described

in the materials and methods section Results are expressed in

per-centage related to control (incubation in the absence of BSA) and are the

averages of two experiments with different enzyme preparations.

Fig 4 Time course of [H3]PAF formation as a function of cortical and medullary microsomal protein concentration (A) Kinetics of [H3]PAF formation using 0.04 (d), 0.1 (s) and 0.2 (h) mg proteinÆmL)1of human cortex microsomes (B) Kinetics of [H 3 ]PAF formation using 0.02 (d), 0.06 (s) and 0.12 (h) mg proteinÆmL)1of human medulla microsomes Lyso-PAF AT activity was determined as described

in Materials and methods Results are representative of three experiments.

(Table 2) Neither NaF, a phosphatase inhibitor, nor

phenylmethylsulfonyl fluoride, a serine esterase inhibitor,

had any significant effect on both enzymatic activities The

same results were obtained after the incubation of cortical

and medullary microsomes with the reducing agents

dithiothreitol and mercaptoethanol On the other hand,

lyso-PAF AT activity was completely abolished by the

incubation of microsomes with

5,5¢-dithio-bis(2-nitroben-zoic acid) (DTNB), a potent inhibitor of -SH enzymes

Pefabloc, a very potent, irreversible inhibitor of the PAF

degradative enzyme, PAF-AH, had no significant effect on

lyso-PAF AT activities, indicating that the possible presence

of PAF-AH in the lyso-PAF AT assay does not influence

the product formation

Substrates of lyso-PAF AT

When the activity of PAF ATs was determined at

lyso-PAF concentrations ranging from 2 to 100 l at a fixed

concentration of acetyl-CoA (200 lM) both cortical and medullary lyso-PAF AT exhibited classical Michaelis– Menten kinetics up to 60 lM Higher concentrations of lyso-PAF resulted in a drop of the activity possibly due to the detergent effects of the substrate against the enzyme Simple saturation kinetics were also observed up to 400 lMof acetyl-CoA when the activity of the lyso-PAF ATs was determined

at acetyl-CoA concentrations ranging from 25 to 800 lMat a fixed concentration of lyso-PAF (20 lM) When the concen-tration of acetyl-CoA exceeded 400 lMa reduction of the lyso-PAF AT activity was also observed (Fig 5) The kinetic parameters derived from these experiments are summarized

in Table 3 In all experiments, the KM,appand Vmax,appvalues for medullary lyso-PAF AT were higher than the respective values of cortical lyso-PAF AT However, a statistical analysis for the comparison of the values could not be conducted because of the small number of samples Subsequently, the specificity of the microsomal enzymes for ester/ether substrates was investigated Microsomes of

Table 1 Effect of divalent cations and EDTA on cortical and medullary lyso-PAF AT activity Lyso-PAF AT was assayed in the presence of varying concentration of CaCl 2 , MgCl 2 and EDTA as described in Materials and methods Results are expressed in percent related to non added control (100%) are the averages of two determinations in different enzyme preparations.

a

nd, no lyso-PAF AT activity was detected.

Table 2 Effect of various chemicals on cortical and medullary lyso-PAF AT activity Lyso-PAF AT was assayed in the presence of varying concentration of chemicals as described in Materials and methods Results expressed in percent related to non-added control (100%) are the average

of two determinations in different enzyme preparations.

a

nd, not detected.bND, not determined.

either cortex or medulla were incubated with [H3

]-acetyl-CoA and lyso-PAF or the ester analog

lyso-phosphatidyl-choline (lyso-PC) and the kinetics of the product formation

was determined The velocities of both acetyltransferases

were twice as high in the presence of lyso-PAF (10–20 lM)

as in that of their ester analogs In fact, no radioactive

product was recovered in the BSA precipitate when

microsomes were incubated with lyso-phosphatidylcholine

concentrations exceeding 20 lM In order to rule out the

possibility of the degradation of lyso-phosphatidylcholine

by non specific PLA1, the experiment was repeated in the

presence of phenylmethylsulfonyl fluoride (1 mM and

5 mM), a well-known PLA1 inhibitor No increase of the radioactivity recovered in the BSA pellet was observed suggesting a low specificity of acetyltransferases for ester analogs

The ability of lyso-PAF ATs to acetylate ALPA, which is

su bstrate for the acetyltransferase of the de novo biosyn-thetic route, was also tested Cortex or medulla microsomes were incubated with either lyso-PAF (20 lM), ALPA (20 lM) or with a mixture of lyso-PAF (20 lM) and ALPA (20 lM) and the products of the reactions were extracted by

a modified Bligh–Dyer extraction and separated by TLC The radioactivity of the areas with the same Rfas PAF, AAPA and alkylacetylglycerol, was determined The results showed that PAF was the only product of the incubation of the microsomes with lyso-PAF No lipidic products were found when ALPA served as the substrate in the lyso-PAF ATs assay Moreover, addition of ALPA to the lyso-PAF ATs assay had no significant effect on the production of PAF indicating that ALPA cannot serve as substrate for the lyso-PAF AT of cortex or medulla

Subcellular localization of lyso-PAF ATs activity The specific activity of lyso-PAF AT was determined in all subcellular fraction obtained by the subcellular fraction-ation of the tissues Microsomes exhibited the higher lyso-PAF AT activity The total activity of the enzyme in all subcellular fractions was also determined and the subcellu-lar distribution of the activity was calculated considering the total activity of the 500 g supernatant as 100% (Table 4) The acetyltransferase activity was distributed between the membrane fractions of the 20 000 g and the 100 000 g pellets (microsomes) The recoveries of lyso-PAF ATs activity for cortex and medulla were 27.8 ± 8.5% (n¼ 5) and 28.4 ± 8.1% (n¼ 5), respectively No statistical difference between the cortical and medullary lyso-PAF

AT specific activities of the same fractions was observed

Discussion

This study demonstrates the existence of an acetyltrans-ferase activity, capable of transferring the acetyl moiety of acetyl-CoA to lyso-PAF, in both cortical and medullary human renal tissue As far as we know this is the first study concerning with the biochemical characterization of lyso-PAF AT in human renal tissues The determination of the acetyltransferase activity was carried out by a modified trichloroacetic acid precipitation method described previ-ously [29] Assessment of the method indicates that it can be used for the routine determination of lyso-PAF AT activity

Fig 5 Influence of substrate concentration on microsomal lyso-PAF

AT activity of human cortex and medulla (A) Activity of cortical (s)

and medullary (d) lyso-PAF AT as a function of lyso-PAF at fixed

concentrations (200 l M ) of acetyl-CoA (B) Activity of cortical (s) and

medullary (d) lyso-PAF AT as a function of acetyl-CoA at fixed

concentrations (20 l M ) of lyso-PAF Lyso-PAF AT activity was

determined as described in Materials and methods Results are

rep-resentative of four experiments.

Table 3 Kinetic parameters of cortical and medullary microsomal lyso-PAF AT Kinetic data were obtained from experiments shown in Fig 3 Results are the averages of two determinations or the mean ± SD of four determinations in different enzyme preparations.

Enzyme preparation

Substrates

K M,app (l M ) V max (nmolÆmin)1Æmg)1) K M,app (l M ) V max (nmolÆmin)1Æmg)1) Cortex, microsomes 15.6 ± 2.6 (n ¼ 4) 2.1 ± 0.5 (n ¼ 4) 90.4 ± 14.7 (n ¼ 4) 1.49 ± 0.25 (n ¼ 4) Medulla, microsomes 24.9 (n ¼ 2) 3.72 (n ¼ 2) 100.6 (n ¼ 2) 2.92 (n ¼ 2)

as [H3]PAF, the only radioactive product of the

acetyl-transferase reaction, is quantitatively precipitated to the

BSA pellet, readily separating from [H3]acetyl-CoA, which

remains in the supernatant This method is faster and more

convenient than the TLC methods routinely used for the

determination of lyso-PAF AT activity [30]

Both cortical and medullary lyso-PAF AT activities share

similar biochemical characteristics indicating that they are

originated from common cellular sources As previous

studies have shown that mesangial and endothelial cells

possess the higher acetyltransferase activities in rat and

human renal tissue [13,15], we can hypothesize that the

lyso-PAF AT activity found, in this study, in homogenates

of renal cortex and medulla is mainly derived by this kind

of cells

The lyso-PAF AT activity is associated with the

mem-branous fractions of the renal cells No lyso-PAF AT

activity could be detected in the cytoplasmic fraction The

higher specific activity of lyso-PAF AT is found in the

microsomes Microsomal lyso-PAF AT showed an

opti-mum pH in the range of 7.4–7.7, similar to that found for

the acetyltransferases of other tissues [5] Lyso-PAF AT is

also sensitive to the concentration of Tris in the buffer

solution showing a maximum activity at 50 mM This

indicates that it is important to determine the concentration

of Tris solution in order to achieve the best experimental

conditions for the determination of acetyltransferase

BSA concentrations up to 0.5 mgÆmL)1had no

signifi-cant effect on lyso-PAF AT activity while higher

concen-trations inhibited lyso-PAF activity dose-dependently The

above results are in conflict with previous studies in human

polymorphonuclear neutrophils showing an activation of

microsomal lyso-PAF AT at BSA concentrations of

0.5 mgÆmL)1and higher The activating effect of BSA was

attributed to its ability to bind PAF and free the enzyme

from the product of the reaction, which has inhibitory

effects on lyso-PAF AT The same researchers had also

shown that microsomal fraction is much more effective than

BSA in binding PAF or lyso-PAF [31] In our assays, it

seems that the microsomal proteins can bind PAF

effect-ively preventing it from inhibiting lyso-PAF AT activity and

the addition of BSA up to 0.5 mgÆmL)1has no effects on the

lyso-PAF AT activity However, higher BSA concentrations

could prevent the enzyme to act on the substrate and an

inhibitory action is observed

Addition of divalent cations in the incubation mixture resulted in a dose-dependent inhibition of lyso-PAF AT A complete loss of lyso-PAF AT activity was also observed in the presence of chelating agents which was totally reversed

by the addition of CaCl2, thus a direct inhibitory effect of EDTA on the enzyme should be ruled out It seems that the endogenous microsomal stores of Ca2+are adequate for the proper function of the enzyme, which is inhibited by the chelation of the endogenous Ca2+by EDTA The same mode of action has been observed for the lyso-PAF AT from rat spleen [9] and mouse macrophages [32]

The substrate specificity of lyso-PAF determines the composition and the biological activity of the molecular mixture of PAF analogs that are produced under inflam-matory conditions Lyso-PAF AT specifically acts on ether analogs of PAF while its activity on ester analogs (acyl-PAF) is diminished by almost 70% These results are in agreement with previous studies showing that the major PAF species synthesized by rat glomerular mesangial cells is the ether analog of PAF [33] The inability of lyso-PAF AT

to act on ALPA indicates that the lyso-PAF AT activity is distinct from the acetylating activity of the de novo biosynthetic route

In conclusion, an acetylating activity, capable of trans-ferring an acetyl group from acetyl-CoA to lyso-PAF, was demonstrated in cortical and medullary human renal tissues for the first time The biochemical properties of both cortical and medullary acetylating activities are similar with the biochemical properties of lyso-PAF ATs characterized in other tissues or cells The existence of a lyso-PAF AT activity in human renal tissues indicates that human kidneys are able to produce PAF through the remodeling pathway However, the relative contribution of this pathway to the increased synthesis of PAF under pathological conditions needs further elucidation

Acknowledgements

This work was supported by grants from the General Secreteriat for Research and Technology, Ministry of Development.

References

1 Demopoulos, C.A & Pinckard, R.N & Hanahan, D.J (1979) Platelet-activating factor: evidence for

1-O-alkyl-2-acetyl-sn-Table 4 Subcellular distribution and specific activities of lyso-PAF AT in human cortex and medulla Lyso-PAF AT activity was determined as described in materials and methods Results of subcellular distribution are expressed as percent related to the total activity of lyso-PAF AT in the

500 g supernatant (100%) Results are the mean ± SD of 4–14 determinations in different enzyme preparations.

Fraction

Specific activity (nmolÆmin)1Æmg)1)

Distribution of total activity (%)

Specific activity (nmolÆmin)1Æmg)1)

Distribution of total activity (%)

500 g supernatant 0.37 ± 0.20 (n ¼ 6) 100 0.43 ± 0.31 (n ¼ 7) 100

20 000 g pellet 0.54 ± 0.33 (n ¼ 5) 15.6 ± 4.5 (n ¼ 5) 0.77 ± 0.42 (n ¼ 9) 18.3 ± 5.1 (n ¼ 5)

100 000 g pellet 1.14 ± 0.53 (n ¼ 14) 12.1 ± 3.5 (n ¼ 5) 1.45 ± 0.67 (n ¼ 13) 10.6 ± 3.8 (n ¼ 5) Total membrane fraction 0.80 ± 0.39 (n ¼ 4) ND 1.24 ± 0.28 (n ¼ 4) ND

a nd, not detected b ND, not determined.

glyceryl-3-phosphorylcholine as the active component (a new class

of lipid chemical mediators) J Biol Chem 254, 9355–9358.

2 Prescott, S.M., Zimmerman, G.A., Stafforini, D.M & McIntyre,

T.M (2000) Platelet-activating factor and related lipid mediators.

Annu Rev Biochem 69, 419–445.

3 Snyder, F (1995) Platelet-activating factor and its analogs,

metabolic pathways related intracellular processes Biochim

Bio-phys Acta 1254, 231–249.

4 Lee, T.C., Vallari, D.S & Snyder, F (1992)

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase Methods Enzymol.

209, 396–401.

5 Snyder, F (1995) Platelet-activating factor, the biosynthetic and

catabolic enzymes Biochem J 305, 689–705.

6 Nixon, A.B., O’Flaherty, J.T., Salyer, J.K & Wykle, R.L (1999)

Acetyl-CoA: 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine

acet-yltransferase is directly activated by p38 kinase J Biol Chem 274,

5469–5473.

7 Baker, P.R., Owen, J.S., Nixon, A.B., Thomas, L.N., Wooten, R.,

Daniel, L.W., O’Flaherty, J.T & Wykle, R.L (2002) Regulation

of platelet-activating factor synthesis in human neutrophils by

MAP kinases Biochim Biophys Acta 1592, 175–184.

8 Gomez-Cambronero, J., Velasco, S., Sanchez-Crespo, M.,

Vivanco, F & Mato, J.M (1986) Partial purification and

char-acterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:

acetyl-CoA acetyltransferase from rat spleen Biochem J 237,

439–445.

9 Seyama, K & Ishibashi, T (1987) Biochemical characterization

of CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine

acetyl-transferase in rat spleen microsomes Lipids 22, 185–189.

10 Billah, M.M & Johnston, J.M (1983) Identification of

phos-pholipid platelet-activating factor

(1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in human amniotic fluid and urine Biochem.

Biophys Res Commun 113, 51–58.

11 Sanchez-Crespo, M., Inarrea, P., Alvarez, V., Alonso, F., Egido, J.

& Hernando, L (1983) Presence in normal human urine of a

hypotensive and platelet-activating phospholipid Am J Physiol.

244, F706–F711.

12 Schlondorff, D., Goldwasser, P., Neuwirth, R., Satriano, J.A &

Clay, K.L (1986) Production of platelet-activating factor in

glo-meruli and cultured glomerular mesangial cells Am J Physiol.

250, F1123–F1127.

13 Kester, M., Nowinski, R.J., Holthofer, H., Marsden, P.A &

Dunn M.J (1994) Characterization of platelet-activating factor

synthesis in glomerular endothelial cell lines Kidney Int 46,

1404–1412.

14 Neuwirth, R., Ardaillou, N & Schlondorff, D (1989) Extra and

intracellular metabolism of platelet-activating factor by cultured

mesangial cells Am J Physiol 256, F735–F741.

15 Pirotzky, E., Ninio, E., Bidau lt, J., Pfister, A & Benveniste, J.,

1984) Biosynthesis of platelet-activating factor VI Precursor of

platelet-activating factor and acetyltransferase activity in isolated

rat kidney cells Lab Invest 51, 567–572.

16 Zanglis, A & Lianos, E.A (1987) Synthesis of alkyl-ether

gly-cerophospholipids in rat glomerular mesangial cells, evidence for

alkyldihydroxyacetone phosphate synthase activity Biochem.

Biophys Res Commun 144, 666–673.

17 Woodard, D.S., Lee, T.C & Snyder, F (1987) The final step

in the de novo biosynthesis of platelet-activating factor.

Properties of a unique CDP-choline, 1-alkyl-2-acetyl-sn-glycerol

choline-phosphotransferase in microsomes from the renal inner medulla of rats J Biol Chem 262, 2520–2527.

18 Karasawa, K., Qiu, X.Y & Lee, T.C (1999) Purification and characterization from rat kidney membranes of a novel platelet-activating factor (PAF)-dependent transacetylase that catalyzes the hydrolysis of PAF, formation of PAF analogs, and C-2-cer-amide J Biol Chem 274, 8655–8661.

19 Lianos, E.A & Zanglis, A (1992) Effects of complement activa-tion on platelet-activating factor and eicosanoid synthesis in rat mesangial cells J Lab Clin Med 120, 459–464.

20 Biancone, L., Tetta, C., Turello, E., Montrucchio, G., Iorio, E.L., Servillo, L., Balestrieri, C & Camussi, G (1992) Platelet-activat-ing factor biosynthesis by cultured mesangial cells is modulated by proteinase inhibitors J Am Soc Nephrol 2, 1251–1261.

21 Camussi, G., Salvidio, G & Tetta, C (1989) Platelet-activating factor in renal diseases Am J Nephrol 9 (Suppl 1), 23–26.

22 Lopez-Novoa, J.M (1999) Potential role of platelet activating factor in acute renal failure Kidney Int 55, 1672–1682.

23 Antonopoulou, S., Demopoulos, C.A., Iatrou, C., Moustakas, G.

& Zirogiannis, P (1994) Platelet-activating factor acetylhydrolase (PAF-AH) in human kidney Int J Biochem 26, 1157–1162.

24 Iatrou, C., Moustakas, G., Antonopoulou, S., Demopoulos, C.A.

& Ziroyiannis, P (1996) PAF levels and PAF-acetylhydrolase activities in patients with primary glomerulonephritis Nephron 72, 611–616.

25 Lenihan, D.G & Lee, T.C (1984) Regulation of platelet activating factor synthesis, modulation of 1-alkyl-2-lyso-sn-glycero-3-phos-phocholine, acetyl-CoA acetyltransferase by phosphorylation and dephosphorylation in rat spleen microsomes Biochem Biophys Res Commun 120, 834–839.

26 Bligh, E.G & Dyer, W.J (1959) A rapid method of total lipid extraction and purification Can J Biochem Physiol 37, 911–917.

27 Karantonis, H.C., Antonopoulou, S & Demopoulos, C.A (2002) Antithrombotic lipid minor constituents from vegetable oils: comparison between olive oils and others J Agric Food Chem.

50, 1150–1160.

28 Lowry, O.H., Rosebrough, N.J., Farr, A.L & Randall, R.J (1951) Protein measurement with the folin phenol reagent J Biol Chem 193, 265–275.

29 Yamazaki, R., Sugatani, J., Fujii, I., Kuroyanagi, M., Umehara, K., Ueno, A., Suzuki, Y & Miwa, M (1994) Development of a novel method for determination of acetyl-CoA: 1-alkyl-2n-gly-cero-3-phosphocholine acetyltransferase activity and its applica-tion to screening for acetyltransferase inhibitors Biochem Pharmacol 47, 995–1006.

30 Lee, T.C., Vallari, D.S & Snyder, F (1992) 1-Alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase Methods Enzymol.

209, 396–401.

31 Ninio, E., Joly, F & Bessou, G (1988) Biosynthesis of PAF-acether XI Regulation of acetyltransferase by enzyme-substrate imbalance Biochim Biophys Acta 963, 288–294.

32 Ninio, E., Mencia-Huerta, J.M., Heymans, F & Benveniste, J (1982) Biosynthesis of platelet-activating factor I Evidence for

an acetyltransferase activity in murine macrophages Biochim Biophys Acta 710, 23–31.

33 Lianos, E.A & Zanglis, A (1987) Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells J Biol Chem 262, 8990–8993.