Molecular and Biochemical Parasitology, 48 ( 1991 ) 151-162 (~) 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-6851/91/$03.50 A D O N I S 016668519100286N

MOLBIO 01584

151

Phospholipids and protein kinase C in acetylcholine-dependent signal transduction in A s c a r i s s u u m

Javier A r e v a l o and H o w a r d J. Saz

Department o f Biological Sciences. Universi O' o f Notre Dame, Notre Dame, IN. U.S.A.

(Received 7 November 1990; accepted 22 April 1991)

Quantitatively, the major phospholipid in the muscle of the nematode Ascaris suum was found to be phosphatidylcholine (lecithin). Stimulation of Ascaris muscle with acetylcholine or the agonists carbachol and levamisole increased the levels of phosphorylcholine, 1,2-diacylglycerides and phosphatidic acid. Increased levels of these compounds, together with the demonstration of phospholipase C activity, suggest that phospholipid hydrolysis may be associated with the ACh response of the muscle via second messenger pathways. In other tissues, diacylglycerides and phosphatidic acid have been reported to regulate protein kinase C activity. Protein kinase C activity also was demonstrated in the muscle of Ascaris. For optimal activity the kinase was dependent upon Ca 2 +, unsaturated 1,2-diacylglyceride and phospholipid. All of the data are in accord with the possible involvement of a second messenger system being operative in the ACh-stimulated contraction of Ascaris muscle.

Key words: Ascaris; Acetylcholine; Phospholipid; Protein kinase C; Second messenger; Signal transduction; Phosphatidylcho- line; Phospholipase

Introduction

Hydrolysis of phosphatidylinositol 4,5-bis- phosphate by phospholipase C gives rise to inositol 1,4,5-trisphosphate and 1,2-diacylgly- cerides, both of which serve as second messengers in mammalian tissues [1,2]; the former to mobilize Ca 2+, and the latter to activate protein kinase C. However, recently, the role of the phosphatidylinositols in the formation of the diacylglycerides has been questioned, since activation of phospholipase C results in the hydrolysis not only of the inositols, but also of phosphatidylcholine, which, quantitatively, results in the formation of considerably higher quantities of the

Correspondence address: Howard J. Saz, Department of Biological Sciences, University of Notre Dame, Notre Dame, 1N 46556, U.S.A.

Abbreviations: ACh, acetylcholine; GPC, glycerophosphoryl- choline; PMSF, phenylmethanesulfonylfluoride~ TLC, thin layer chromatography.

diacylglycerides. The compound glycerophosphorylcholine

(GPC) is present in many mammalian tissues [5-9]. It also has been reported to be present in Ascaris suum muscle in concentrations as high as 20 mM [10], and also in Fasciola hepatica [11]. GPC is presumed to be an intermediate in the metabolism of phosphatidylcholine (leci- thin; Fig. 1). It has been reported that either aerobiosis or the administration of the anthel- mintic closantel, produced a decrease in the level of GPC in both Fasciola and Ascaris prior to any change in ATP concentration [11]. In view of the elevated levels of GPC in Ascaris muscle and the possible importance of phos- phatidylcholine in signal transduction, analy- ses of the phospholipids, phospholipases and protein kinase activities in the muscle of this nematode were performed.

Pharmacologically, Ascaris muscle differs from mammalian muscle in that it will contract in response to acetylcholine (ACh), but exhibits no or little response to some other

152

PHOSPHATIOYLIINOSITOL • INOSITOL PHOSPHATE

Nc DIGLYCERIDE--,- (Activates PK C) k

PLC

PHOSPHATIDYLCHOUNE J ~ PHOSPHORYLCHOLINE

PL A2 PHOSPHATASE

LYSOPHOSPHATIDYLCHOLINE

+ FATTY ACID

CHOLINE + PHOSPHATE

GLYCEROPHOSPHORYLCHOLIN E

+ FATI'Y ACID

l GPC-DIESTERASE

GLYCEROL-3-P + CHOLINE

Fig. 1. Pathways of catabolism of phosphatidylcholine and phosphatidylinositol. PL, phospholipase; GPC, glycerophos-

phorylcholine; PK, protein kinase.

commonly employed ACh agonists and an- tagonists [12,13]. To examine the possible involvement of phospholipids in signal trans- duction in Ascaris body wall preparations, the effects of ACh, carbachol and levamisole on phosphatidylcholine and its metabolites were analyzed. Findings were related to possible signal transduction pathways. In addition, activities of the phospholipases which would be necessary for the conversion of lecithin to diacylglycerides, as well as protein kinase C activity, were assayed.

Materials and Methods

Materials. Phenylmethanesulfonylfluoride (PMSF), phorbol dibutyrate, phorbol myris- tate acetate, EGTA, EDTA, choline chloride, glycerophosphorylcholine, phosphatidic acid, lipid standards, 1,2-diolein, phosphatidylser- ine, and Triton X-100 were purchased from Sigma Chemical Co., St. Louis, MO. Glycerol dehydrogenase, phospholipase C, alkaline phosphatase, leupeptin, and trypsin inhibitor were obtained from Boehringer-Mannheim,

Indianapolis, IN. DEAE-cellulose (DE-52), phosphocellulose paper (P-81), LK5 and LK6 TLC plates (5 x 20 and 10 x 20 cm) were from Whatman, Clifton, NJ. [~,-32p]ATP was purchased from ICN Biomedicals, Costa Mesa, CA.

Parasites. A. suum adult females were ob- tained from the abattoir, washed in a salt solution [14] and used immediately upon arrival at the laboratory. When crude mem- brane or cytosolic preparations were needed, the worms were dissected and the muscle homogenized in 0.25 M sucrose/20 mM Tris- HC1 buffer, pH 7.5/10 mM 2-mercaptoetha- nol/0.1 mM PMSF in a tissumizer (Tekmar, Cincinnati, OH) at medium speed for 1 min, then in a Teflon homogenizer with 10 strokes. Homogenates were centrifuged at 10000 x g for 20 min and the supernatant obtained was centrifuged at 100 000 x g for 1 h to separate the crude membrane (pellet) from the cytosolic (supernatant) fraction.

The effects of ACh, carbachol and levami- sole were examined employing body wall preparations (muscle plus cuticle) from which the intestinal and reproductive tissues had been removed. Such preparations were equilibrated in isotonic salt solution for 2 h and bubbled with N2 to maintain an anaerobic environment prior to the addition of ACh, carbachol or levamisole to a final concentration of 1 mM. The isotonic solution contained: 3 mM KC1/ 135 mM NaC1/6.4 mM MgC12/4 mM CAC12/3 mM glucose/5 mM Tris-HC1, pH = 7.1. After incubation, the preparations were extracted with chloroform/methanol (2:1). Phospholi- pids and diglycerides were determined in the organic phase, while choline, phosphorylcho- line and GPC were quantitated from the aqueous phase.

Chemical determinations. Choline was quan- titated enzymatically with choline oxidase by a modification of the procedure described by Gurantz et al. [15]. The choline assay con- tained in a final volume of 1 ml: 30 mM Tris- HCI buffer, pH 8.0/0.2 mg ml - l phenol; 0.12 mg m1-1 aminoantipyrine; 12.5 #g ml - ]

horseradish peroxidase and 0.5 units of choline oxidase. The vessels were incubated at 37°C for 30 min. The absorbance at 500 nm was proportional in the range of 5 200 nmol choline.

For the determination of phosphorylcho- line, an aliquot of 200 ktl of sample was preincubated with 3.8 units ml-~ of alkaline phosphatase at 37'~C for 30 min in a final volume of 350 ~1. At the termination of the incubation, the sample was made up to 800/~1 with the above-mentioned Tris-HCl assay buffer, and choline assay was performed. The concentration of phosphorylcholine was calcu- lated by subtracting the absorbance at 500 nm without the addition of alkaline phosphatase.

GPC determinations were performed in a similar manner on 200 ~1 after boiling in 1 N HC1 for 20 rain in a final volume of 400 ~tl. The mixture was neutralized, cooled, made up to 800 l~l with Tris-HC1 assay buffer and the choline oxia-%~ assay was carried out. GPC concentral,on~ were obtained after subtracting control choline and phosphorylcholine con- centrations.

1,2-Diacylglycerides were assayed with gly- cerol dehydrogenase according to Pandol and Schoeffield [16]. Phosphatidic acid was deter- mined by means of densitometry after TLC separation and staining with Coomassie blue [17,181.

Phosphatidylcholine was determined as cho- line following Folch lipid extraction [19] and digestion with phospholipase C and alkaline phosphatase incubated for 30 min at 37°C. Alternatively, phosphatidylcholine as well as other phospholipids were separated by TLC and the individual classes quantitated by the inorganic phosphate released after digestion in H2SO4/HNO3 (2:1) at 140°C for 3 h [20,21].

Phospholipase assays. Phospholipase C was determined using a phosphatase coupled assay [22] by measuring the release of inorganic phosphate. In a final volume of 40 pl the assay contained: 50 mM Tris-HC1, pH 7.5/6.3 mM CAC12/0.13 mg ml 1 BSA/2.5 mM phosphati- dylcholine (or other phospholipids)/70 mM ammonium sulfate/0.15 units of alkaline

153

phosphatase and the enzyme source. Incuba- tions were carried out for 15 min at 37°C. Reactions were terminated by the addition of 40 #1 of 20% SDS, followed by 200 /~1 of ascorbate molybdate reagent. After incubation for 1 h at 37°C, color was determined at 820 nm. One nmol of Pi released was equivalent to 0.045 net units of absorbance. To distinguish phospholipase C from phospholipase D activ- ity, the samples also were subjected to the determination of choline released in the presence and absence of alkaline phospha- tase, since phospholipase D hydrolyzes phos- phatidylcholine without the liberation of inorganic phosphate.

Phospholipase A activity was determined by measuring the release of fatty acids to the incubation media. Aliquots were taken from the membrane and cytosolic preparations and incubated in the presence of the substrate dipalmitoyl phosphatidylcholine, prepared in absolute ethanol, to a final volume of 1 ml. The reaction vessel contained 50 mM Tris- HCI, pH 7.3/6.3 mM CAC12/0.13 mg ml - l BSA/2.5 mM phosphatidylcholine/100 /tg of protein. The assay was incubated at 37°C for 15 to 30 min. Reactions were terminated by the addition of 50 /A of 1 N HC1 followed by extraction 3 times with 3 volumes of ether. The pooled extracts were evaporated, resuspended in 50 /~1 of chloroform: methanol ( l : l ) and aliquots were subjected to TLC on LK6 plates developed with heptane/diethyl ether/acetic acid (60:40:2). Plates were stained with Coo- massie blue [17] and fatty acid released determined by densitometry. Alternatively, fatty acids were determined by a colorimetric method using diethyldithiocarbamate and the production of the copper salt of the fatty acid, a complex that absorbs at 440 nm [23].

Protein was determined by the Bradford procedure with bovine serum albumin (BSA) as standard [24].

Protein kinase C determination. Protein ki- nase C was obtained from Ascaris muscle homogenates after DEAE-cellulose chromato- graphy according to the procedure of Kikkawa et al. [25,26]. The muscle was homogenized in

154

20 mM Tris-HC1 buffer, pH 7.5/0.25 M sucrose/10 mM EGTA/2 mM EDTA/0.1 mM PMSF. The homogenate was centrifuged at 3 000 × g for 10 min to remove cellular debris. The resulting supernatant was centrifuged at 100000 x g and the new supernatant was designated as the crude cytosolic extract. The crude membrane extract was obtained from the pellet by a 1-h extraction with the same buffer, but containing 0.4% Triton X-100. After recentrifugation at 100000 x g, the extracts were applied to DEAE-cellulose columns and the fractions which possessed activity were pooled prior to assay.

The protein kinase C assay mixture con- tained, in a final volume of 60/A: 20 mM Tris- HC1 buffer, pH 7.5/10 mM MgCI2/5 mM NaF/ 0.1 mg ml "histone H1 and 50 #M [732p]ATP (specific activity 15 x 10 4 dpm nmo1-1) plus 0.5 mM EGTA or a mixture of 0.5 mM CaC12/ 60 #g m l - 1 phosphatidyl serine and 3 #g m l - 1 diolein. Lipids were added either as mixed micelles in 1% Triton X-100 [27] or as sonicated vesicles in 20 mM Tris-HC1, pH 7.5. Vessels were incubated at 30°C for 10 min. Reactions were stopped by spotting 50 #1 of the reaction mixture on P-81 phosphocellulose filter paper squares (2 x 2 cm), which were then washed 4 times in 75 mM H3PO4 [28]. Filters were dried and radioactivity was determined with a Packard Tricarb liquid scintillation spectrometer.

Western blots. Samples from Ascaris muscle membrane and cytosolic extracts obtained from the homogenates, as well as the corre- sponding peaks from DE-52, were subjected to PAGE and then blotted onto nitrocellulose paper with the Bio-Blot system (Bio-Rad, Richmond, CA). A rat brain cytosolic extract was included routinely as a positive control for the antibody. The blot was treated with protein kinase C antibody (Oncogene Sciences, Man- hasset, NY) and visualized with the alkaline phosphatase linked secondary antibody. To indicate the specificity of the reaction and recognition of the homologue proteins only, the following 3 preparations of the nitrocellu- lose blot were run: (1) with the primary

antibody against the kinase C and the secondary antibody; (2) with normal rabbit serum and the secondary antibody; (3) with the secondary antibody alone. The primary anti- body was used at 10 /~g ml ~ and the secondary antibody at 0.2/~g m l -

Results

Ascaris muscle phospholipids. The relative concentrations of the phospholipid compo- nents of the adult parasite's muscle were determined (Table I). In agreement with previous reports [29], phosphatidylcholine plus phosphatidylethanolamine constituted approximately 85% of the phospholipids in the parasite's muscle. The phospholipid frac- tion represented approximately 50% of the total lipids, which in turn, represented approxi- mately 1% of the total wet weight of the organism's muscle.

Of particular interest, the inositol phospho- lipids are present and account for approxi- mately 3% of the total phospholipid fraction. This value is within the concentration levels found in other organisms where the inositol phosphates participate in second messenger associated responses. Phosphatidyl serine also was detected, which could be significant, since it may serve as an activator of protein kinase C [3].

TABLE 1

Major phospholipids in Ascaris muscle

Components Inorganic Phospholipids phosphate (% of total) recovered (/~g)

Phosphatidylcholine 33.2 45 (lecithin)

Phosphatidylethanolamine 29.6 40 Phosphatidylinositol 2.1 3 Phosphatidylglycerol 4.3 6 Phosphatidylserine 2.2 3 Others ~ 2.2 3

Lipids were separated on TLC plates by developing with chloroform/methanol/ammonium hydroxide (65:25:4). aOthers represent unidentified phospholipids such as sphingomyelin, phosphatidic acid, and lysophospholipids.

TABLE II

Concentrations of choline compounds in Ascar i s s u u m muscle and perienteric fluid

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Compound Muscle Perienteric fluid (/tmol (g wet weight)-i)a (#mol (mg protein) i),

Phosphatidylcholine 1.39 (0.50) 0.04 (0.002) GPC 5.76 (1.27) 0.11 (0.010) Phosphorylcholine 3.44 (0.82) 0.06 (0.010) Choline 0.39 (0.17) 0.03 (0.005)

'tEach figure represents an average of 5 separate determinations. The figures in parentheses indicate the standard deviation.

TABLE Ill

Phospholipase activities in Ascar i s membrane and cytosol fractions

Enzyme source Fatty acid content of Phospholipase C activity Phospholipase A activity ~ phosphatidylcholine substrate (nmol min 1 mg 1)

Membrane 1,2-dipalmitoyI 0.00 0.95 l-oleoyl-2-stearoyl 6.67 l-stearoyl-2-oleoyl 7.82

Cytosol 1,2-dipalmitoyl 1.31 O. 14 I-oleoyl-2-stearoyl 11.70 1 -stearoyl-2-oleoyl 12.01

~gg fatty acid (f.a.) released min ~ mg protein 1.

Analysis of choline esters. Ascaris muscle and perienteric fluid were analyzed for choline and various choline esters (Table II). In corlfirma- tion of previous findings, glycerophosphoryl- choline was present at high levels in the muscle. The perienteric fluid also contained this and other phospholipids. Of particular interest, phosphorylcholine was present in high con- centrations in the muscle, while free choline was detected at approximately one tenth the level of phosphorylcholine. Phosphatidylcho- line constituted one of the major phospholipid components of the parasite muscle.

These findings suggest the presence of phospholipases and possibly GPC diesterase which presumably would be required for the formation of some of these metabolites from lecithin. Also, the presence of choline kinase is suggested by the high ratio of phosphorylcho- line over choline. Generally, phosphorylcho- line accumulates in cells rather than free choline [30], since phosphorylcholine would then be more readily available for phospholi- pid synthesis.

Phospholipase activities. Phospholipases A

and C were found to be present in both cytosol and crude membrane fractions from Ascaris muscle (Table Ill). The enzyme showed a decided preference for the presence of an unsaturated fatty acid in the substrate. When the saturated dipalmitoyl ester was employed as substrate, phospholipase C activity was not detected in the membrane fraction and was only of h~w activity in the cytosol fraction. Howeve' , when the oleoyl ester was employed as substrate, the activities were greatly stimu- lated in both fractions. No significant differ- ence in the phospholipase C activity was found when the unsaturated fatty acid of the phospholipid substrate, was in the 1 or 2 positions of the glycerol moiety (Table Ill).

It is of interest that the phospholipase C activity was considerably higher on phosphati- dylcholine than it was on the corresponding inositol or ethanolamine esters when employing the cytosol fraction (Table IV). However, this relationship did not hold with the membrane fraction. In neither case was there demonstra- ble activity with phosphatidylserine. Specific phospholipase A1 o r A2 activities could not be distinguished on the basis of the assay used. It

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TABLE IV

Relative activities of cytosolic and membrane phospholipase C activities from A. s u u m

Enzyme source Substrate ~b Activity (nmol Pi min ~ mg i)

Cytosol Phosphatidylcholine 2.40 Phosphatidylethanolamine 1.50 Phosphatidylinositol 1.30 Phosphatidylserine 0.00

Membrane Phosphatidylcholine 1.11 Phosphatidylethanolamine 1.26 Phosphatidylinositol 1.26 Phosphatidylserine 0.00

Phospholipids were added as sonicated micelles in the assay buffer. Activity was followed by the release of inorganic phosphate in the presence of alkaline phosphatase. Incubations were carried out for 15 min at 3T'C. b All substrates consist of crude preparations of the isolated species that contain various chain lengths and double bonds (Sigma Chemical Co,, St. Louis). Qualitatively, similar results were obtained when using the specific phospholipids (See Table ll~).

only accounts for total phospholipase A activity, including both A~ and Az.

To determine the individual phospholipases A1 and A2, the membrane fraction was incubated with l-palmitoyl, 2-oleoyl phospha- tidylcholine as above. After incubation, the fatty acids were extracted with ether and dried. The dry acids were converted to the corre- sponding methyl esters with boron trifluoride in methanol and subjected to GLC. Two peaks were obtained which corresponded to the methyl esters of known palmitate and oleo- ate, respectively. These findings indicate the presence of both phospholipases AI and A2. Employing the methodology described under Materials and Methods, phospholipase D was not detected in any of the fractions obtained from the parasite tissue preparations.

Ejfects of acetylcholine, carbachol and levami- sole. The pharmacology of Ascaris' muscle is clearly different from that of its mammalian counterpart. In an attempt to determine the effect of acetylcholine and its agonists on the activation of the phospholipases, Ascaris body wall preparations were incubated with acet- ylcholine, carbachol or levamisole for the indicated periods of time and the levels of phosphorylcholine, 1,2-diglycerides, phospha- tidic acid, choline, GPC, lecithin, 1,3-diglycer- ides and triglycerides were determined.

Phosphorylcholine, the 1,2-diacylglycerides

and phosphatidic acid were the only com- pounds of those analyzed which showed a significant increase upon treatment with ACh, carbachol or levamisole (Table V). Values were compared to controls (by paired Student's t- test) and considered significant if P<0.05. Increased levels of these compounds were seen consistently after 5 min incubation with either drug. The levels observed were comparable for each treatment, which would be in agreement with earlier evidence that levamisole acts at the cholinergic site [12,13]. The increased amounts of these compounds, suggest that stimulation at the cholinergic site, induces activation of phospholipase C which, in turn, hydrolyzes lecithin to give rise to the 1,2-diacylglycerides and phosphorylcholine. It appears unlikely that the increase in phosphorylcholine was due to a choline kinase activity, since the levels of free choline do not vary.

The increase in phosphatidic acid (Table V), is thought to be associated with tissues that respond through second messenger systems, since it is assumed to arise from diacylglycer- ides which are subsequently phosphorylated by diacylglycerol kinase [31,32]. This pathway serves to remove the second messengers and return them to their basal levels. After 10 min, the concentrations of the 1,2-diglycerides and phosphatidic acid remain relatively constant in the presence of the drug. However, phosphor- ylcholine continues to increase.

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TABLE V

Effects of acetylcholine and agonists on phosphorylcholine, diacylglyceride and phosphatidic acid levels in Ascari:s" muscle- body wall preparations

Expt. Treatment Phosphorylcholine 1,2-Diacylglyceride Phosphatidic acid

1 Control 243.5 (21.2) 13.1 (1.1) 6.5 (0.6)

ACh 1 min 221.3 (43.3) 16.1 (2.8) 6.9 (1.0) 5 min 338.6 (27.6)* 20.3 (1.3)* 10.7 (0.7)*

10 rain 384.5 (63.4)* 24.9 (0.6)* 12.1 (0.4)* 30 min 553.2 (96.3)* 25.7 (3.7)* 11.3 (1.0)*

Control 165.8 (28.4) 14.4 (1.3) 6.3 (0.7)

Carbachol 1 min 112.5 (69.8) 15.9 (2.1) 7.0 (1.3) 5 min 204.5 (32.6)* 19.5 (1.2)* 10.0 (0.1)*

10 min 273.5 (90.3)* 25.6 (0.3)* 11.8 (0.4)* 30 min 636.4 (98.7)* 26.5 (6.5)* 10.2 (1.0)*

Levamisole 1 min 136.0 (58.1) 13.6 (2.0) 8.0 (0.9)

10 min 369.4 (116.7)* 27.0 (1.1)* 12.0 (0.5)* 30 min 400.0 (108.8)* 26.9 (3.1)* 10.6 (0.1)*

Figures represent nmol of compound /tmol ~ of phospholipid phosphate and are average values of a typical experiment. Figures in parentheses represent the standard deviation where N = 3. This experiment was repeated at least 10 times with qualitatively similar results. However, values varied considerably with each batch of ascarids obtained from the abattoir. Tissues were pre-equilibrated as described under Materials and Methods. Controls were incubated for the same length of time as the longest time point and processed last, in other experiments it was processed first, this did not appear to change the behavior of the experimental groups. *P = < 0.05.

Protein kinase C activity. Cytosolic and membrane extracts of Ascaris muscle were obtained and applied to DEAE-cellulose columns as described under Methods. Protein kinase C activity was eluted with a linear gradient of NaC1. Both preparations yielded protein kinase C peaks that eluted at 100 mM NaC1. The membrane activity represented the major portion of the total kinase C activity in the tissue (Table VI) [28]. Binding calcium with EGTA, markedly prevented activation of the protein kinase which, in addition to Ca 2÷, required phospholipid and diglyceride for optimal activity. Neither phospholipid nor diglyceride alone had any effect on activa- tion. Both the dibutyrate and myristate-acetate phorbol esters substituted for diglyceride in producing activation of the protein kinase.

2+ Ca added an excess of 0.5 mM did not produce additional histone phosphorylation.

Protein kinase C Western blot. To further confirm the presence of protein kinase C in Ascaris muscle, the cytosol and membrane fractions were subjected to PAGE followed by electroblotting onto nitrocellulose for immu- nological detection with a specific antibody directed against the C4 conserved region of the catalytic site of human protein kinase C (Oncogene Sciences, Manhasset, NY). The antibody was polyclonal and recognized types ~,/~ and 7 of protein kinase C.

As a control, a sample of rat brain extract containing protein kinase C activity was isolated and subjected to electroblotting by the same procedures. The section of the blot where only the combination of anti-protein kinase C antibody and the chromogenic secondary antibody (Fig. 2) were included revealed the recognition of positive bands, which should correspond to the putative kinase from Ascaris muscle. As expected, 70- and 50-

158

TABLE VI

Protein kinase C activity in DE-52 fractions from membrane and cytosol o f Ascaris muscle

Fract ion Addi t ions Total 32p incorporated (pmol)

E G T A 0.5 mM Ca 2+ 0.5 mM

Cytosol

Membrane

None 12.57 Diolein 11.31 Phosphat idylser ine 9.64 Phosphatidylserine + diolein 10.14 Phosphatidylserine + phorboldibutyra te 12.20 Phosphatidylserine + phorbolmyris ta teaceta te 12.81

" ~ + . . . .

Ca ~ + phosphat ldyl serme + dloleln 13.30

None 12.31 Diolein 9.56 Phosphat idylser ine 12.06 Phosphatidylserine + diolein 11.21 Phosphatidylserine + phorboldibutyra te 12.00 Phosphatidylserine + phorbolmyris ta teaceta te 12.06 Ca 2+ + phosphat idylser ine + diolein 10.57

8.12 10.91 16.77 23.45 ~ 23.88 25.63 26.00

9.60 9.67

18.00 30.41 ~ 25.38 27.11 25.86

Incubat ions were carried out for 10 min, then aliquots were spot ted onto P-81 filter squares, washed in 75 m M H3PO 4 and counted for radioactivity. The pmol of phospha te incorporated were calculated from the total counts per vessel using the specific activity of the ATP added. ~The specific activity of these fractions in pmol o f 32p incorporated min i (mg protein) t was 0.26 for the cytosol and 1.76 for the membrane .

kDa bands were observed from the rat brain which corresponded to the native enzyme and the catalytic subunit, respectively. The cataly- tic subunit is produced by the action of calpain [25,26]. The Western blot of the Ascaris DE-52 fractions (Fig. 2) showed a double band of

A

approximately 60 kDa. These findings, in conjunction with the previous activity determi- nations constitute strong evidence in support of a protein kinase C activity in the muscle of the parasite.

B

9 2 . 5 - 6 4 -

4 5 -

2 9 -

1 2 3 4 1 2 3 4 Fig. 2. Western blot of Ascaris protein kinase C fraction (A) and rat brain extract (B), 10 ,ug of Ascaris protein kinase C fraction were run on an 8% PAGE, then blotted and probed with the antibodies. 5 #g of rat brain extract protein was run on a 12.5% PAGE, then blotted and probed with the antibodies. (1) Molecular weight markers. (2) Anti-protein kinase C antibody + anti-rabbit IgG alkaline phosphatase. (3) Normal rabbit sera + anti-rabbit IgG alkaline phosphatase. (4) Anti-rabbit lgG alkaline phosphatase.

Discussion

The enzymes necessary for the breakdown of membrane phosphatidylcholine to form cho- line and glycerophosphorylcholine appear to be present in Ascaris membrane and cytosol fractions. In accord with these findings, the expected major products of lecithin catabolism have been demonstrated in Ascaris muscle. These are 1,2-diacylglycerides, phosphorylcho- line, choline, glycerophosphorylcholine and phosphatidic acid. Phospholipase C and phospholipase A activities have been demon- strated in Ascaris muscle which is in accord with the products of lecithin metabolism found. Phospholipase activities are of impor- tance in other tissues, since they are associated with responses to hormones through G protein-activated receptors. They also appear to act in response to injury of muscle tissue [3,9].

Most interesting was the finding that incubation of the muscle with acetylcholine or its agonists, carbachol or levamisole, resulted in an elevation of both the 1,2- diacylglycerides and phosphorylcholine. These would be the products expected from phos- pholipase C activity on lecithin. It has been demonstrated in other tissues that 1,2-diacyl- glycerides serve as second messengers for the activation of protein kinase C, which also was found to be present in Ascaris muscle and which also required the diglyceride for optimal activity. Although the 1,2-diglycerides may arise also from the hydrolysis of phosphatidyl- inositol, the very low concentrations of the inositol derivatives in Ascaris could not account for the diglycerides which accumu- late. The considerably higher levels of phos- phorylcholine found in response to stimulation would indicate the breakdown of phosphati- dylcholine and the accumulation of consider- ably higher concentrations of the 1,2- diacylglycerides than could be accounted for on the basis of phosphatidylinositol hydrolysis. Recently, it has been suggested that the phospholipase C activity resulting from recep- tor activation would hydrolyze both phospha- tidylinositol and phosphatidylcholine, but the

159

latter would give rise to considerably higher concentrations of the 1,2-diacylglycerides [3,4]. This, in addition to the finding that the cytosol component of Ascaris phospholipase C ap- pears to have a preference for the choline ester is in agreement with this hypothesis.

Acetylcholine, carbachol and levamisole all produced a contraction of the parasite muscle and presumably all would occupy the acet- ylcholine receptor site. In addition, incubation of Ascaris muscle preparations with each of these compounds resulted in increased levels of phosphorylcholine, diacylglycerolide and phosphatidic acid. The first two presumably could result from the activation of phospholi- pase C and the following hydrolysis of lecithin. The possibility exists that phosphorylcholine may be arising from other sources such as the hydrolysis of glycerophosphorylcholine or the phosphorylation of choline. However, there are several findings which favor the hydrolysis of lecithin as the precursor of phosphorylcho- line. One is the failure to detect the presence of phospholipase D activity that would result in an increase of phosphatidic acid and free choline. Choline, in turn, could then be phosphorylated by choline kinase. During the experiment, however, the levels of free choline and GPC did not vary significantly. In the cases of GPC and lecithin, the pool sizes are large such that small differences may not be detected. Phosphatidic acid in most second messenger systems has two possible origins. The first is through the activation of phospho- lipase D, the second is through the densensi- tization pathway of protein kinase C that includes the conversion of the 1,2-diglyceride to phosphatidic acid through the action of diglyceride kinase. Attempts to demonstrate these activities in Ascaris were negative. However, most of the phospholipase D activities from other animal tissues, are difficult to demonstrate in crude preparations since they appear to be tightly bound to the membrane and most of the detergents used for solubilization are known to inhibit the activity of the enzyme [33]. Diglyceride kinase, on the other hand, is generally present in low concentrations in most animal tissues, but

160

extraction procedures may be difficult [31]. Of particular interest, it was reported recently that phosphatidic acid may serve to regulate Ca 2 + channels in other tissues [4,32]. If this were the case in Ascaris, the inositol phosphate may serve a minor or a triggering role which would be maintained via the phosphatidylcholine system.

Adult Ascaris muscle differs both pharma- cologically and anatomically from that of the higher animals. The nematode muscle is organized in a fashion similar to a mixture of smooth and skeletal muscle, but physiologi- cally it responds more like smooth muscle and is dependent on external Ca 2+ concentrations and flux for activity [34]. This is in agreement with the findings of a calcium dependent protein kinase C in the parasite muscle. Conflicting evidence still exists concerning the mechanism of smooth muscle contraction and the cellular events which lead to the sustained phase of contraction [35]. Although participa- tion of inositol trisphosphate, diacylglycerol, phosphatidic acid and protein kinase C have been suggested [32,35,36], the underlying mechanism remains elusive and the evidence is not definitive.

From these studies, it is still not possible to define precisely the second messenger respon- sible for the events following stimulation of muscle contraction. However, the turnover of lecithin appears to be increased under these conditions with the concomitant generation of 1,2-diglycerides, phosphorylcholine and phos- phatidic acid. The Ascaris protein kinase C may be similar to the protein found in Caenorhabditis elegans, coded by the tpa-1 gene [37]. The C. elegans enzyme is reported to have 526 amino acid sequence which would correspond to a peptide of 50 60 kDa. The enzymes necessary for the generation of the diglycerides and other metabolites of lecithin have been shown to exist in the muscle of Ascaris. This suggests that protein kinase C could be activated as a consequence of the increased levels of diglycerides, which would subsequently lead to protein phosphorylation and generation of a cellular response. The results are of particular interest, since they

establish the possibility of important second messengers being associated with the acetyl- choline response in the muscle of Ascaris suum.

Acknowledgements

Supported in part by research grant AI- 09483 from the National Institutes of Allergy and Infectious Diseases U.S. Public Health Service.

The authors thank Wilson Foods Corpora- tion, Logansport, IN for their cooperation in obtaining A. suum.

References

I Berridge, M.J. (1987) lnositol triphosphate and diacyl- glycerol: two interacting second messengers. Annu. Rev. Biochem. 56, 159 193.

2 Abdel-Latif, A.A. (1987) Calcium-mobilizing receptors, polyphosphoinositides and the generation of second messengers. Pharmacol. Rev. 38, 227 272.

3 Besterman, J.M., Duronio, V. and Cuatrecasas, P. (1986) Rapid formation of diacylglycerol from phos- phatidyl choline: A pathway for generation of a second messenger. Proc. Natl. Acad. Sci. USA 83, 6785 6789.

4 Extort, J.H. (1990) Hormonal regulation of phosphati- dylcholine breakdown. In: The Biology and Medicine of Signal Transduction (Nishizuka, Y., ed.), pp. 152 157. Raven Press, New York.

5 Cohen, S. (1983) Simultaneous I~C and 31p NMR studies of perfused rat liver. J. Biol. Chem. 258, 14294 14308.

6 Bagnasco, S., Baldsan, R., Fales, H.M., Yang, Y.M. and Burg, M. (1986) Predominant osmotically active organic solutes in rat and rabbit renal medulas. J. Biol. Chem. 261, 5872 5877.

7 Butt, C.T., Glonek, T. and Barany, M. (1976) Phosphorus 3~ Nuclear Magnetic Resonance detection of unexpected phosphodiesters in muscle. Biochemistry 15, 485~4853.

8 lnfante, J.P. (1985) Defective synthesis of GPC in murine muscular dystrophy; the primary molecular lesion'? FEBS Lett. 185, 205 210.

9 Billadelo, J.J., Gard, J.K., Ackerman, J.J.H. and Gross, R.W. (1985) Determination of intact tissue glyceropho- sphoryl choline levels by quantitative 31p NMR spectroscopy and correlation with spectrophotometric quantification. Anal. Biochem. 144, 269 274.

l0 Kubistova, J. (1970) Occurrence of high amounts of glyceryl phosphoryl choline in Ascaris lumbricoides. FEBS Lett. 9, 351 352.11.

11 Rohrer, S.P., Saz, H.J. and Nowak, T. (1986) 3JP-NMR

studies of the metabolism of the parasitic helminths Ascaris lumbricoides and Fasciola hepatica. Arch. Biochem. Biophys. 248, 200 209.

12 Harrow, I.D. and Gration, K.A.F. (1986) Mode of action of the anthelmintics morantel, pyrantel and levamisolc on muscle cell membrane of the nematode Ascaris suum. Pestic. Sci. 16, 662 672.

13 Martin, R.J. (1982) Electrophysiological effects of piperazine and diethylcarbamazine on Ascaris suum somatic muscle. Br. J. Pharmacol. 77, 255 265.

14 Saz, H.J. and Lescure, O.L. (1969) The functions of phosphoenol pyruvate carboxykinase and malic enzyme in the anaerobic formation of succinate by Ascaris lumhricoides. Comp. Biochem. Physiol. 30, 49 60.

15 Gurantz, D., Laker, M.F. and Hofmann, H.F. (1981) Enzymatic measurement of choline-containing phos- pholipids in bile. J. Lipid Res. 22, 373 376.

16 Pandol, S.J. and Schoeffield, M.S. (1986) 1,2-diacylgly- cerol, protein kinase C, and pancreatic enzyme secre- tion. J. Biol. Chem. 261, 4438~4444.

17 Aqwu, D.E., McPhail, L.C., Wykle, R.L. and McCall, C.E. (1989) Mass determination of receptor-mediated accumulation of phosphatidate and diglycerides in human neutrophils measured by coomassie blue stain- ing and densitometry. Biochem. Biophys. Res. Com- mun. 159, 79 86.

18 Bockino, S.B., Blackmore, P.F., Wilson, P.B. and Exton, J.H. (1987) Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism. J. Biol. Chem. 262, 15309 15315.

19 Folch, J., Lees, M. and Sloane-Stanley, G.H. 0957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497 509.

20 Snyder, F. (1973) Thin-layer chromatographic behavior of glycerolipid analogs containing ether, ester, hydroxyl and ketone groupings. J. Chromatogr. 82, 7 14.

21 Bartlett, G. (1959) Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466 468.

22 Krug, E.D. and Kent, C. (1981) Phospholipase C Assays. Methods Enzymol. 72, 746 750.

23 Higgins, J.A. (1987) In: Biological Membranes. A Practical Approach (Findlay, J.B.C. and Evans, W.H., eds), pp. 103 137. IRL Press, Oxford.

24 Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein, utilizing the principle of protein-dye binding. Anal.

161

Biochem. 72, 248 254. 25 Kikkawa, U., Minokuchi, R., Tokai, Y and Nishizuka,

Y. (1983) Calcium-activated, phospholipid-dependent protein kinase (protein kinase C from rat brain). Methods Enzymol. 99, 288 298.

26 Yasuda, I., Kishimoto, A., Tanaka, S., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) A synthetic peptide substrate for selective assay of protein kinase C. Biochem. Biophys. Res. Commun. 166, 1220 1227.

27 Hannun, Y., Loomis, C.R. and Bell, R.M. (1985) Activation of protein kinase C by Triton X-100 mixed micelles containing diacylglycerol and phosphatidyl serine. J. Biol. Chem, 260, 10039 10043.

28 Gomez, M.L., Erijman, L., Arauzo, S., Torres, H.N. and Tellez-lnon, M.T. (1989) Protein kinase C in Trypanosoma cruzi epimastigote forms: partial purifica- tion and characterization. Mol. Biochem. Parasitol. 36, 101 108.

29 Subrahmanyam, D. and Venkatesan, (1968) On the phospholipids of Ascaris lumbricoides. Comp. Biochem Physiol. 25, 733 737.

30 Vance, D.E. and Vance, J.F. (1985) Biochemistry of lipids and membranes. Benjamin Cumings, California.

31 Kanoh, H., Yamada, K. and Sakane, F. (1990) Diacylglycerol kinase: a key modulator of signal transduction. Trends Biochem. Sci. 15, 47 50.

32 Ohanian, J., Ollerenshaw, J., Collins, P. and Heagerty, A. (1989) Agonist induced production of 1,2-diacylgly- cerol and phosphatidic acid in intact resistance arteries. J. Biol. Chem. 265, 8921 8928.

33 Taki, T. and Kanfer, J.N. (1981) Phospholipasc D of rat brain. Methods Enzymol. 71, 746 750.

34 Bird, A.F. ( 1971 ) The Structure of Nematodes. pp. 102 129. Academic Press, New York.

35 Rasmussen, H., Takuwa, Y. and Park, S. (1987) Protein kinase C in the regulation of smooth muscle contrac- tion. FASEBJ . 1., 177 185.

36 Cabot, M.C., Welsh, C.J., Cao, H. and Chabbott, H. (1988) The phosphatidylcholine pathway of diacylgly- cerol formation stimulated by phorboldiesters occurs via phospholipase D activation. FEBS Lett. 1, 153 157.

37Tabuse, Y., Nishiwaki, K. and Miwa, J. (1989) Mutations in a protein kinase C homologue confer phorbol ester resistance on Caenorhabditis eh, gans. Science 243, 1713 1716.