Schistosoma mansoni: Possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae

EXPERIMENTAL

PARASITOLOGY

72, 311-320 (191)

Schistosoma mansoni: Linoleic Acid-Induced KENICHIRO Department

MATSUMURA, of Parasitology,

Possible Involvement of Protein Kinase C in Proteolytic Enzyme Release from Cercariae

YOSHINORI MITSUI, KATSUYUKI AND YOSHIKI AOKI Institute

of Tropical Nagasaki,

Medicine, Nagasaki 852 Japan

SATO, MAKOTO University,

SAKAMOTO,

Sakamotomachi,

K., MITSUI, Y., SATO, K., SAKAMOTO, M., AND AOKI, Y. 1991. SchistoPossible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae. Experimenta/ Parasitology, 72, 31 l-320. The possible involvement of protein kinase C and Ca*+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 mA4 calcium in the small glass petri dish and exposed to the stimuli(linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca” metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 uM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 pM of phorbol-12,13dibutyrate (PDBu), cercariae ceased the swimming movement, began arhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(B-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by I-(5isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 PM. H-7 alone, at a concentration of 100 p1%4,did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 u&Z) of the Ca2’ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 t&f) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 m&f EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-l-naphthalenesulfontide (W-7), a calmodulin antagonist, trifluoperazine (TFP), a better calmodulin antagonist on schistosome, or by verapamil, a Ca*’ channel blocker. Linoleic acid-induced release of enzyme was partially inhibited by 0.5 and 5 m&f of EGTA and by 1 to 100 t.o%fof H-7. While it was not inhibited by 1Y-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and N-(2guanidinoethyl)-Sisoquinolinesulfonamide (HA-1004), inhibitors of cyclic nucleotidedependent protein kinase which were used as negative controls of H-7, W-7, TFP, 8(N,N-diethylamino)octyl3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca2+ antagonist, and verapamil. These results strongly suggest the involvement of protein kinase C in the proteolytic enzyme release from cercariae which come into contact with skin lipid. The mechanisms by which phorbol esters and linoleic acid stimulate cercariae to release the proteolytic enzyme and the necessity of Ca*+ in release of proteolytic enzyme were discussed. Q 1991Academic Press,Inc. INDEX DESCRIPTORS AND ABBREVIATIONS: Schistosoma mansoni; Trematode; Cercaria; Secretion of proteolytic enzyme from cercariae; Azocoll assay; Calcium ion; Ethylene glycol-bis(g-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA); Verapamil; Linoleic acid; Calcium ionophore, A23187; Phorbol esters, 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol-12,13,dibutyrate (PDBu); Intracellular Ca’+ antagonist, 8(N,N-Diethylamino)octyl3,4,5-trimethoxybenzoate (TMB-8); Protein kinase C inhibitor, l(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); Cyclic nucleotide-dependent protein kinase inhibitors, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and NMATSUMURA, soma mansoni:

311 0014-4894191 $3.00 Copyright0

1!391byAcademicPress,Inc.

Au rights of reproduction

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312

MATSUMURA

ET AL.

(2-guanidinoethyl)-5isoquinolinesulfonamide (HA-1004); Calmodulin inhibitors, N(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W-7) and tritluoperazine (TFP); Cyclooxygenase inhibitor, esculetin; Eicosanoids; Dimethyl sulfoxide (DMSO).

proteolytic enzyme from cercariae depends on protein kinase C activation and/or Ca2+ Penetration to host skin is the first step of mobilization. schistosome infection. Although the skin is an excellent mechanical and immunological MATERIALS AND METHODS barrier, cercariae have a unique capability CaCl,, EGTA, verapamil and linoleic to penetrate and transform, to finally be- acidChemicals. were purchased from Wako Pure Chemical Induscome schistosomules within a few hours tries, Ltd. (Osaka, Japan), Azocoll and A23187 from (Stirewalt 1974). Schistosoma cercariae Calbiochem Co. (La Jolla, CA), TPA, PDBu, TMB-8, penetrate the skin by using both mechanical TFP and esculetin from Sigma Chemical Co. (St. force and their proteolytic enzyme (Stire- Louis, MO), H-7, H-8, HA-1004, and W-7 from MedBiological Laboratories (Nagoya, Japan). wait 1966). It is known that essential fatty icalEGTA, TMB-8, H-7, H-8, HA-1004, Wacids evoke the penetration response which 7, and TFPverapamil, were dissolved in distilled water. A23187, consists of the cessation of swimming TPA, and PDBu were dissolved in DMSO. Esculetin movement, a rhythmic thrusting of the an- was dissolved in 99.5% ethanol. The concentrated terior tip of the parasites which is associ- stock solutions of the reagents were diluted by distilled immediately before the experiments and then ated with the release of proteolytic enzyme, water added to the cercarial suspension to finally give the and tail loss (Stirewalt and Hackey 1956; desired concentrations. The fmal concentrations of Stirewalt 1974). DMSO were 0.5% and less and that of ethanol was Recently it has been disclosed that the 0.1%. Parasites. The life cycle of Schistosoma mansoni enzyme released by cercariae is evolution(NIH-Sm-PR-1) been maintained in our laboratory ally related to eukaryotic serine proteases in Biomphalaria hasglabrata NIH Puerto which have broad substrate specificity Rican/Brazilian M-line) and (Newton’s GN hamsters. Cercariae (Mckerrow et al. 1989). However, the exact were shed in dechlorinated tap water by exposure to mechanism by which essential fatty acid light. They were then concentrated by filtration orstimulates cercariae to release proteolytic through a 8-pm millipore filter to yield 2w ganisms per milliliter of tap water. enzyme is unknown, although some related Proteolytic enzyme release. Cercariae (2w) studies have been done (Salafsky and were placed in dechlorinated tap water in the small Fusco 1987; Haas and Schmitt 1982a,b). glass petri dish (S = 5.7 cm*) and exposed to the stimRecently it has become evident that the ulus (phorbol esters, Ca2+ ionophore and linoleic acid) breakdown of inositol phospholipid occurs for a specific period of time at room temperature Calcium concentration was measured by a in many cell types in response to a wide (23-27°C). Polarized Zeeman Atomic Absorption Spectrophovariety of external signals. And the release tometer (Hitachi, Z-8000). Our dechlorinated tap wareaction, secretion, and exocytosis from a ter contained 0.37 + 0.06 m&f (mean f SD, n = 12) of wide variety of cell types depend on protein calcium. An aliquot of concentrated stock solution of kinase C activation and/or Ca” mobiliza- phorbol esters (TPA and PDBu) and Ca*’ ionophore 187) were added to the cercarial suspensions. The tion (Nishizuka 1984). Under appropriate (A23 effect of removal of Ca2’ from medium on phorbol conditions, protein kinase C activation and ester-induced and A23187-induced release was examCa2+ mobilization can be induced selec- ined. Linoleic acid was applied on the inside surface of tively and independently by the application the bottoms of the dishes at 10 ug/cm* by the method of permeable phorbol esters or a Ca2+ ion- of Stirewalt (1978). The final volume of incubation mewas 1 ml. ophore, respectively (Kikkawa and Nishi- dium To examine the effect of the inhibitors of Ca*’ mezuka 1986). By using this procedure and in- tabolism, protein kinase C and lipoxygenase on the hibitors of Ca2’ metabolism and protein ki- enzyme release, cercariae were incubated with linoleic nase C, we examined whether release of acid and one of the following reagents; EGTA (a Ca” INTRODUCTION

PROTEIN

KINASE

C AND PROTEOLYTIC

chelator), verapamil (a Ca” channel blocker), W-7 and TFP (a calmodulin antagonist), TMB-8 (an intracellular Ca*+ antagonist), H-7 (a protein kinase C inhibitor), H-8 and HA-1004 (cyclic nucleotidedependent protein kinase inhibitor) which were generally used as negative controls of H-7, esculetin (lipoxygenase inhibitor). The effect of H-7 on phorbol ester-induced enzyme release and the effect of verapamil, W-7, and TFP on A23187-induced enzyme release were also examined. The penetration response of cercariae thus treated was observed under dissecting microscope. Measurement of proteolytic activity. Upon termination of the incubation period, cercarial suspensions from the test dishes were poured into a test tube, and cercariae were removed by centrifugation (SOOg for 5 min at 4°C). The supematant was examined for proteolytic enzyme activity by the azocoll assay described by Campbell et al. (1976) with a slight moditication. Briefly, the assay mixture contained 3 mg of azocoll, 0.8 ml of 0.05 M glycine-NaOH buffer, pH 8.5, 0.7 ml of supematant, and 0.1 ml of supplementary fluid by which the final concentration of each of the chemicals used in an experiment was made identical. The test tubes were incubated on a shaker at 35°C for 36 hr and then transferred to a water bath (65°C) for 15 min to inactivate the enzyme. Nonreacted azocoll was centrifuged out (800g for 5 min) and the absorbance of the supematant was read at 520 nm. Two types of negative controls were always run in parallel. One contained similar numbers of cercariae but lacked enzyme release stimuli. The other contamed the enzyme release stimuli in water in which cercariae had emerged from snails, but lacked cercariae. Statistical analysis. Statistical comparison between groups was performed by the Duncan’s multiple range test. The sensitivity of different cercarial population to penetration stimuli varies from day to day and furthermore depends on the age of the parasites (Haas and Schmitt 1982a). Therefore, only fresh cercariae (3 hr after shedding) were used in our study. Only values which were obtained simultaneously from the cercarial population are quantitatively comparable. RESULTS

Results Effect of phorbol esters on proteolytic enzyme releasefrom cercariue. Phorbol esters are intercalated into the membrane, directly activate protein kinase C without interaction with cell surface receptors, and produce various biological and biochemical changes in a wide variety of biological systems (Castagna et al. 1982; Kikkawa and

ENZYME

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313

Nishizuka 1986). The cercariae were exposed to phorbol ester in the presence or absence of Ca2’. One hour or more after exposure to a high concentration of TPA in the dechlorinated tap water containing 0.37 mM calcium, some cercariae ceased swimming, dropped to the bottom of the dish, and showed a rhythmic thrusting of the anterior tip of the parasites. Two hours later, all cercariae showed exploratory movement which is similar to the penetration reaction induced by linoleic acid, but they did not shed their tail. The tap water in which cercariae were incubated with TPA for 2 hr showed significantly high proteolytic enzyme activity (Fig. la). TPA-stimulated release of enzyme was dose dependent over the concentration range of 0.02-2 PM. When cercariae were stimulated by PDBu, similar cercarial penetration responses, which were accompanied by enzyme release, were observed, although a low concentration of PDBu (0.02 @4) did not elicit the enzyme release (Fig. lb). In the tap water containing 5 mM EGTA, cercariae showed penetration response after exposure to a high concentration (2 pM) of TPA and PDBu for 2 hr but the incubation medium of cercariae did not show proteolytic enzyme activity (data not shown). Effect of calcium ionophore on proteolytic enzyme release from cercuriue. Since the increase in cytosolic Ca2+ is essential for the release reactions in variety cell types, Ca2 + ionophore was used, at concentrations of 0.1-l ~44, to stimulate the release reactions (Kikkawa and Nishizuka 1986). When cercariae were exposed to Ca2+ ionophore (A23187) at 0.5 and 1 pM in the presence of 0.37 mM calcium, cercariae did not change the swimming movement nor release the enzyme. When cercariae were exposed to high concentrations (10 and 100 p.44) of Ca2’ ionophore (A23 187) in the tap water containing 0.37 mM calcium, they ceased swimming and showed strong muscle contraction all over the body. One hour later they became completely nonmo-

314

MATSUMURA Incubation condition of cercariae

Enzyme (Azocoll

activity assay;

of incubation absorbance

0.2

0 Fil. Cer Cer

ET AL.

at

medium 520 M)

0.4

0.6

a . .

+ 0.1%

DMSO

Cer .

+ 0.02

@! TPA/O.l%

DHSO

Cer .

+

pl4 TPA/O.l%

DUSO

Cer. Fil.

+ +

2 @I TPA/O.l% 2 w TPA/O.l%

DUSO DUSO

0.2

Pil. Cer . Cer. + 0.1%

B

b DUSO

0

0.1

0.3

0.5

0.7

FIG. 1. Effect of phorbol esters on proteolytic enzyme release from Schisrosoma mansoni (NIHSm-PR-1) cercariae. Cercariae were incubated with TPA or PDBu in dechlorinated tap water containing 0.37 m&f calcium for 2 hr, and then centrifuged. The supematant was examined for proteolytic activity by azocoll assay (see text for details). A population of cercariae used for TPA experiment (a) was different from that of PDBu experiment (b). Values represent the means f SE for four experiments. FL, filtrate of dechlorinated tap water in which cercariae were shed from snails; Cer., cercarial suspension; TPA, 12-O-tetradecanoylphorbol-13-acetate; PDBu, phorbol-12,13-dibutyrate; DMSO, dimetbyl sulfoxide. *Columns with different letters in each figure have significantly different mean (P < 0.05; Duncan’s multiple range test).

tile and shed the tails. The medium in which cercariae were incubated with A23187 for 1 hr showed high enzymic activity (Fig. 2). The lowering the concentration of Ca*+ in the incubation medium of cercariae did not affect the effect of high concentration of A23187 on enzyme release of cercariae. The high proteolytic enzyme activity was observed in the incubation medium of cercariae which contained 0, 0.5, and 5 mM EGTA (Fig. 2b). Ability of various calcium and protein kinase modulators to inhibit linoleic acidinducedproteolytic enzyme release. The effects of H-7, H-8, HA-1004, EGTA, W-7, TFP, TMB-8, verapamil, and esculetin on

the release of enzyme induced by linoleic acid were studied. The cercariae were incubated in dechlorinated tap water containing 0.37 mM calcium with both linoleic acid and inhibitors for 1 hr. The ability of inhibitors to directly affect the swimming movement of cercariae were also observed. Linoleic acid-induced release of enzyme was partially inhibited by H-7 (Fig. 3a), although, the differences in cercarial behavior were not obvious when compared with those induced by linoleic acid alone. At a concentration of 100 fl, H-7 alone did not affect the cercarial swimming. H-8 (l-100 $l4) and HA-1004 (l-100 CLM), which were used as negative controls of H-7, did not

PROTEIN

KINASE C AND PROTEOLYTIC

Incubation condition of cercariae

Fil. Cer Cer

. .

Cer.

DIG0

+ 0.5%

DUSO

+ 0.5

Cer . Cer. Cer

+ 0.5%

*

Pil.

Enzyme fAzocol1

ENZYME

activity assay;

315

RELEASE

of incubation absorbance

at

medium 520 nm)

a

pH A23187/0.5%

DMSO

+ 1 FM A23187/0.5%

DI4SO

+ 10

JIM A2318710.5%

DBlSO

+ 100 + 100

pn A23187/0.5% PM A23187/0.5%

DIG0 DUSO

6 A*

JIG

b Fil. + 0.5% Cer . Cer . + 0.5%

DUSO DUSO

Cer .

+ 10

pi

A23187/0.5%

DMSO

Cer .

+ 10

Cer .

+ 10

pn A23187/0.5% + 0.5 pM A23187/0.5%

DUSO mU EGTA DUSO

l

5

ml4

A

EGTA

I 0

0.2

0.4

I 0.6 mansoni

FIG. 2. Effect of a calcium ionophore A23187 on proteolytic enzyme release from S. (NIH-Sm-PR-1) cercariae in the different concentrations of calcium. Cercariae were stimulated by A23187 for 1 hr in dechlorinated tap water containing 0.37 n~%4calcium (a) or in tap water to which 0.5 or 5 nu%4EGTA were added (b). DMSO was used as solvent for A23187. Cercariae were centrifuged and the supematant was examined for proteolytic activity by azocoll assay (see text for details). A population of cercariae used for (a) was different from that for (b). Values represent the means + SE for four experiments. Fil., fdtrate of dechlorinated tap water in which cercariae were shed from snails; Cer., cercarial suspension; A23187, a calcium ionophore; DMSO, dimethyl s&oxide; EGTA, ethylene glycol-bis(R-aminoethyl ether) N,N,N’,N’-tetraacetic acid. *Columns with different letters in each figure have significantly different mean (P < 0.05; Duncan’s multiple range test).

alter the ability of linoleic acid to induce the enzyme release (data not shown). These results suggest that protein kinase C is involved in the enzyme release induced by linoleic acid. The lowering of the Ca*’ concentration in the medium by EGTA reduced the release of enzyme (Fig. 3b). EGTA partially inhibited the cercarial tail loss induced by linoleic acid, although the exploratory behavior of cercariae during the incubation period was not different from that of control group. These results suggest the involvement of Ca*’ in linoleic acid-induced release of enzyme. However, linoleic acid-

induced release of enzyme was unaffected by W-7 (I-100 t&I), TFP (0.5-50 p&f), a better calmodulin antagonist on schistosome than W-7, verapamil (l-100 r-LM) or TMB-8 (0.2-20 ~m which perturbs Ca2+ metabolism, although the latter three chemicals, TFP, verapamil, and TMB-8, strongly inhibited cercarial swimming (data not shown). Linoleic acid-induced release of enzyme was not influenced by inhibition of lipoxygenase by 0. I-10 p&I esculetin (data not shown). Ability of various calcium and protein kinase modulators to inhibit phorbol esterinduced and A23187-induced proteolytic

316

MATSUMURA Incubation condition of cercariae

Enzyme (Azocoll

ET AL.

activity assay;

of incubation absorbance

at

medium 520 nm)

. Fil. Cer.

+ LA

+ LA

Cer.

+ LA

Cer.

l

Cer.

l

Fil. Cer. Cer. Cer Cer.

IA

LA

a

C

A+ + 1 pl( H-7 + 10 pn E-7 + 100

p?l H-7

b

+ LA

+ LA .

+ LA + 0.5 + LA

ml4 EGTA

+ 5 WI EGTA

0

0.2

0.4

3. Effect of H-7 (a) and EGTA (b) on release of cercarial proteolytic enzyme induced by linoleic acid. Cercariae were incubated with both linoleic acid and inhibitor in dechlorinated tap water containing 0.37 rn%f calcium for 1 hr and then centrifuged. The supematant was examined for proteolytic activity by azocoll assay (see text for details). A population of cercariae used for H-7 experiment was different from that of the EGTA experiment. Values represent the means 2 SE for four experiments. Fil., filtrate of dechlorinated tap water in which cercariae were shed from snails; Cer., cercarial suspension; LA, 10 &cm* linoleic acid as enzyme release stimulant; H-7, protein kinase C inhibitor; EGTA, ethylene glycol-bis(+uninoethyl ether) N,N,N’,N’-tetraacetic acid. *Columns with different letters in each figure have significantly different mean (P < 0.05; Duncan’s multiple range test). FIG.

enzyme release. Phorbol ester-induced enzyme release was reduced by high concentrations of H-7 (100 $t4 for TPA; 10 and 100 p,M for PDBu) (Fig. 4). The higher concentration of H-7 partially reduced phorbol ester-induced cercarial exploratory movements. The inhibitors or blockers of calcium metabolism, W-7, TFP and verapamil did not inhibit the A23187-induced enzyme release (data not shown). DISCUSSION

The signal-dependent breakdown of inosit01 phospholipids is directly linked to activation of protein kinase C (via elevated diacylglycerol level) and mobilization of calcium (via elevated levels of inositol triphosphate) and evokes subsequent cellular response such as release reactions

(Nishizuka 1986). A similar pathway linking protein kinase C, protein phosphorilation, and calcium mobilization may exist in the proteolytic enzyme release reaction of cercariae which is initiated by contact with skin surface lipids. Physiological and biochemical evidence for the presence of protein kinase C in schistosome has been demonstrated. When S. mansoni adult worms were exposed to phorbol esters, they became unpaired and were contracted and inactive (Blair et al. 1988). Schistosome protein kinase C was phospholipid dependent, but not Ca2’ dependent. The present study demonstrated that the penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When ex-

ENZYME

PROTEINKINASECANDPROTEOLYTIC Enzyme (Azocoll

Incubation condition of cercariae

l-l + 0.1% + 0.1% TPA/O.l%

activity assay;

of incubation absorbance

+ 2 p

Cer.

+ 2 pI4 TPA/O.l%

DUSO

Cer .

+ 2 JIM TPA/O.l%

DllSO

Cer .

+ 2 pt4 TPA/O.l%

DIG0

at

medium 520 nm)

0.2

0.4 ,

a

Dl4SO DUSO DUSO

Fil. Cer . Cer .

317

RELEASE

l

+ 1 pl

H-7

10 pu

H-7

+ 100

A

PM H-7 I

Fil. Cer. Cer .

+ 0.1% + 0.1% + 2 pl4 PDBu/O.l%

DllSO DllSO DMSO

Cer .

+ 2 pl4 PDBu/O.l%

DHSO

Cer .

+ 2 J.IM PDBu/O.l%

DMSO

Cer.

+ 2 JIM PDBu/D.l%

DMSO

b

A

+ 1 @l H-7 + 10 )xM H-7 + 100

)IM B-7

0

0.2

0.4

FIG. 4. Effect of H-7 on release of cercarial proteolytic enzyme induced by phorbol esters. Cercariae were incubated with either TPA (a) or PDBu (b) and H-7 in dechlorinated tap water containing 0.37 mIt4 calcium for 2 hr. DMSO was used as solvent for phorbol esters. Cercariae were centrifuged, and the supematant was examined for proteolytic activity by azocoll assay (see text for details). Values represent the means 2 SE for four experiments. Fil., filtrate of dechlorinated tap water in which cercariae were shed from snails; Cer., cercarial suspension; TPA, 12-O-tetradecanoylphorbol-13-acetate; PDBu, phorbol-12,13-dibutyrate; DMSO, dimethyl sulfoxide; H-7, protein kinase C inhibitor. *Columns with different letters in each figure have significantly different mean (P < 0.05; Duncan’s multiple range test).

posed to phorbol esters, cercariae ceased swimming, began exploratory behavior, and released proteolytic enzyme. Treatment with phorbol ester rarely caused tail loss and cercariae required a longer time to respond. Phorbol esters are intercalated with the membrane and directly activate protein kinase C (Nishizuka 1986). Therefore, the change of cercarial movement and the release of enzyme from cercariae is probably closely related to the activation of protein kinase C. Phorbol ester-induced release of enzyme was almost completely inhibited by 100 t~A4 H-7, an inhibitor of protein kinase C. The linoleic acid-induced enzyme release was also inhibited by H-7, although the inhibition was not complete. These results further support the idea that

activation of protein kinase C is essential to induce the release of proteolytic enzyme. It is well known that cercarial body contractions do squeeze out small amounts of secretion from the acetabular glands (Stirewalt 1973; Samuelson et al. 1984). The muscle cells, nerve cells, and parenchymal cells closely invest the pre- and postacetabular glands which secrete proteolytic enzyme and mucus substance, respectively (Dorsey and Stirewalt 1971). Therefore, the closely associated muscle and nerve cells were considered to provide the mechanism for cercarial regulation of secretory process. Protein kinase C and Ca2’ play a leading part in the muscle contraction. The results of the present experiments, therefore, support the possible involvement of muscle

318

MATSUMURA

contraction in release of proteolytic enzyme from cercariae. In addition to the present experiments, we previously reported that praziquantel stimulated S. mansoni cercariae to release the proteolytic enzyme and the praziquantel-induced release of enzyme was partially inhibited by EGTA (Matsumura et al. 1990). Praziquantel is also a potent drug which causes the sudden and violent contraction of muscle of schistosome in the presence of Ca*‘. A rise in the cytoplasmic Ca*+ content initiates the variety of cell function including release reaction (Douglas 1968). It is inconclusive as to whether calcium is important in the release of the proteolytic enzyme from cercariae. The results of some of our experiments suggested that the release of enzyme from cercariae is not dependent on the calcium metabolism. Although elevated Ca*+ concentration in cell cytoplasm can easily be induced by a low concentration of Ca*+ ionophore, A23 187, at 1 p.M, failed to stimulate the cercariae to release the enzyme, while the high concentrations of A23 187 (10 and 100 p&f) induced the release of enzyme. However, this may be due to the fact that A23 187 activates protein kinase C at concentrations above 1 fl (Kajikawa er al. 1983). W-7, TFP, verapamil, TMB-8 did not inhibit linoleic acid-induced release of the enzyme. On the other hand, some experiments suggested that Ca*+ plays an important role in release of proteolytic enzyme from cercariae. In the absence or presence of very low concentration of calcium, neither phorbol esters nor linoleic acid stimulated the cercariae to release the proteolytic enzyme. Very recently, Fusco et al. reported that if Ca*’ influx does not occur, proteolytic enzyme will not be released from schistosome cercariae, calcium uptake alone does not cause the release of enzyme though (personal communication). A high concentration of H-7 (100 l.~J4) almost completely inhibited the phorbol ester-induced release of enzyme, but only

ET AL.

partially inhibited the linoleic acid-induced release. The difference strongly suggests that the‘linoleic acid-induced release of enzyme is regulated by protein kinase C and some other biochemical or physiological factors such as Ca*’ metabolism. Fusco et al. observed the ability of linoleic acid to stimulate calcium uptake in cercariae (personal communication). We used relatively high concentrations of inhibitors in the present study. And most of the compounds used are not absolutely specific. Therefore the results of our experiments with inhibitors should be carefully considered. H-7, a potent inhibitor of protein kinase C, at 100 fl would also inhibits myosin light chain kinase (Hidaka et al. 1984). Therefore it is possible that the inhibition of myosin light chain kinase by H-7 leads to the reduction of magnitude of muscle contraction, and finally causes the reduction of linoleic acid-induced enzyme release. W-7, a potent calmodulin antagonist, at 100 PM, would also have a significant effect on protein kinase C (Tanaka et al. 1982). Our study showed that W-7 did not have any effect on the swimming movement of cercariae nor inhibit linoleic acidinduced release of enzyme. Schistosome calmodulin and protein kinase C may be less sensitive to W-7. In fact Thompson ef al. (1986) reported that TFP is a better calmodulin inhibitor on schistosome than W-7. In our study, TFP affected the swimming movement of cercariae; however, it did not inhibit linoleic acid-induced release of enzyme. Salafsky et al. (1984a,b) and Fusco et al. (1986) reported that successful cercarial penetration and transformation are dependent on both skin essential fatty acid levels and resulting cercarial eicosanoids production. And then they hypothesized that leukotriene B4 may be involved in proteolytic enzyme release reaction by virtue of its action as a Ca*’ secretagogue. Esculetin, a specific lipoxygenase inhibitor, inhibited eicosanoids production at lo-’ M and inter-

PROTEIN

KINASEC

AND PROTEOLYTIC

fered with the ability of cercariae to respond to linolate (Salafsky and Fusco 1985). So, we examined whether esculetin at 10e5 M blocked the linoleic acid-induced release of enzyme. However, esculetin failed to block the enzyme release from cercariae. The discrepancy may suggest that the complex and diverse routes of signal transduction are involved in penetration response of cercariae which are the organisms of various cell types. There are several reported types of protein kinase C in the tissue of vertebrates (Nishizuka 1988). Among them, rat hypothalamic type I protein kinase C can be activated by unsaturated fatty acids in the absence of Ca*+ (Murakami and Routtenberg 1985; Naor ef al. 1988). Naor et al. (1988) described the differences in ability of fatty acids to activate type I protein kinase C; arachidonic and linoleic acid were active but nonfatty acids were less active (Table I). Interestingly enough, Haas and Schimitt (1982a) and Salafsky et al. (1984a) reported that arachidonic and linoleic acids were potent stimuli for penetration response of cercariae, whereas nonessential fatty acids were less potent (Table I). Since a protein kinase C-like enzyme of schistosome was TABLE1 Activation of Cercarial Penetration Response and Ca*+-Independent Hypothalamic Type I PKC by Fatty Acids Fatty acid None Arachidonic acid Linoleic acid Oleic acid Palmitic acid Stearic acid Elaidic acid

Penetration response* ++++ +++ + -

PKC activity** (pmol/min) 3.4 16.3 13.5 7.9 4.7 4.2 3.7

(100) (78) (35) (10) (6) (3)

Note. Modified from *Haas and Schmitt (1982a) and **Naor et al. (1988). The numbers in parentheses represent percentages of PKC activity obtained with arachidonic acid as 100.

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319

reported to be not dependent on Ca*+ (Blair et al. 1988), cercarial protein kinase C may be type I-like protein kinase. Essential fatty acids are permeant for cell membrane. Therefore it may be attractive to hypothesize that essential fatty acids are intercalated with cercarial membrane and directly activate cercarial protein kinase C; that is, essential fatty acids play the role of a second messenger. ACKNOWLEDGMENT We thank Dr. Noda (Kagoshima University, Japan) for providing M-line B.glabratu infected with S.mnnsoni (NIH-Sm-PR-1). We express special appreciation to Professor Buz Salafsky (University of Illinois) for his helpful criticism and suggestions in the preparation of the manuscript. This work was supported in part by Grant-in-Aid (02670170) for Scientific Research from Ministry of Education, Science, and Culture of Japan. REFERENCES BLAIR, K. L., BENNET, J. L., AND PAX, R. A. 1988. Schistosoma mansoni: Evidence for protein kinase C-like modulation of muscle activity. Experimental Parasitology 64, 243-252. CAMPBELL, D. L., FRAPPAOLO, P. J. F., STIREWALT, M. A., AND DRESDEN, M. H. 1976. Schistosoma mansoni: Partial characterization of enzyme(s) secreted from the preacetabular glands of cercariae. Experimental Parasitology 40, 3340. CASTAGNA, M., TAKAI, Y., KAIBUCHI, K., SANO, K., KIKKAWA, U., AND NISHIZUKA, Y. 1982. Direct activation of calcium-activated, phospholipiddependent protein kinase by tumor-promoting phorbol esters. The Journal of Biological Chemistry 257, 7847-785 1. DORSEY, C. H., AND STIREWALT, M. A. 1971. Schistosoma mansoni: Fine structure of cercarial acetabular glands. Experimental Parasitology 30, 199-214. DOUGLAS, W. W. 1968. Stimulus-secretion coupling: The concept and clues from chromaflin and other cells. British Journal of Pharmacology 34, 451-474. Fusco, A. C., SALAFSKY, B., AND DELBROOK, K. 1986. Schistosoma mansoni: Production of cercarial eicosanoids as correlates of penetration and transformation. The Journal of Parasitology 72,397-404. HAAS, W., AND SCHMITT, R. 1982a. Characterization of chemical stimuli for the penetration of Schistosoma mansoni cercariae. I. Effective substances, host specificity. Zeitschrif fir Parasitenkunde 66, 293-307. HAAS, W., AND SCHMITT, R. 1982b. Characterization of chemical stimuli for the penetration of Schisto-

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