serotonin stimulates protein phosphorylation in the cestode hymenolepis diminuta

Camp. Biochem. Physiol. Vol. lOOC, No. 3, pp. 483-489, 1991 Printed in Great Britain

0306-4492/91 $3.00 + 0.00

0 1991 Pergamon Press plc

SEROTONIN STIMULATES PROTEIN PHOSPHORYLATION IN THE CESTODE HYMENOLEPIS DIMINUTA

PAULA RIBEIRO* and RODNEY A. WEBB?

Department of Biology, York University, North York, Ontario, Canada M3J lP3 (Telephone: 416-736-2100, Ext. 33187)

(Received 14 December 1990)

Abstract-l. Serotonin stimulated the incorporation of a2P from [Y-‘~P] ATP into crude membrane preparations (P2) of Hymenoleph diminuta in a dose-dependent manner (Et& of -0.79 PM).

2. This response was seen with several serotonin agonists, and was inhibited by several serotonin antagonists, which were identical to the previously described activation and inhibition of serotonin- sensitive adenylate cyclase.

3. Cyclic AMP produced a dose-dependent stimulation of 32P incorporation into the P, fraction, with an ECU of -2.51 PM.

4. The targets for the serotonin stimulated incorporation of 32P were found to be in trypsin-labile proteins with M,‘s of 134,000, 110,000, 82,000, 80,000 and 31.000.

INTRODUCrION

Serotonin has been found to play an important role in the regulation of platyhelminth physiology. For example, application of serotonin to several platy- hehninths was found to stimulate motor activity and to promote carbohydrate metabolism (see Mansour, 1979, 1984; Mettrick and Cho, 1981; Rahman et al., 1982; Thompson and Mettrick, 1984). Furthermore, a high-a8inity transport mechanism for serotonin has been documented in the cestode Hymenolepis dzkninuta (Webb, 1985). Moreover, serotonin was shown by immunohistochemistry to be specifically localized in neurons in this animal, which not only innervated muscles, but also was present in varicosi- ties which terminated in the intercellular spaces (Webb and Mizukawa, 1985). Most recently we have found the quasi-physiological release of endogenous serotonin to be by a Ca*+-dependent process (Gor- don and Webb, 1989). Collectively, these findings implicate scrotonin as an important neuroactive agent in the helminths.

The mechanism of action of serotonin in the helminths is presently the subject of intense investi- gation (see Mansour et al., 1987; Webb, 1988, for reviews). We have recently shown four specific serotonin binding sites in a crude membrane fraction (P2) of H. diminuta, each site showing distinct affinities for the amine and pharmacological heterogeneity (Ribeiro and Webb, 1986, 1987). One such site has been correlated with the elevation of CAMP by serotonin, and has been tentatively characterized as an adenylate cyclase-linked serotonin receptor (Ribeiro and Webb, 1987). While the physiological role of this specific receptor is not yet known, it is tempting to speculate that such a site would be involved in the modulation of CAMP-sensitive pro-

*Present Address: NIH, Laboratory of Neurochemistry, Building 36, Room 3D-30, Bethesda, MD 20892. U.S.A.

tTo whom correspondence should he addressed.

tein kinase(s), thereby controlling the specific enzymatic phosphorylation of specific target proteins (see Nestler et al., 1984). Protein kinase(s), which are sensitive to serotonin and CAMP, and adenylate cyclase have been reported in several trematodes (see Mansour et al., 1987; Webb, 1988, for references). While the presence of protein kinase in H. diminuta has been suggested (Webb, 1988), demonstration of CAMP stimulated protein phosphorylation has not been shown in this animal, and the significance of protein phosphorylation as a mechanism of serotonin action remains unresolved.

The objectives of the present study were to establish a link between the characterized serotonin receptor-adenylate cyclase complex and the phosphorylation of specific proteins in the membranes of the cestode H. diminuta. The present findings demon- strate a role for serotonin in the regulation of phosphorylation of phosphoproteins in H. diminuta and clearly point to CAMP as the most probable second messenger involved in the regulation of phosphorylation of these specific phosphoproteins.

MATERIALS AND METHODG

Materials Adenosine 5’ [$2P] triphosphate triethylammonium salt

(5209 Ci/mmol) was obtained from Amersham Corp. (Ontario, Canada). Methylsergide, ketanserin and phentolamine were gifts from Sandoz (Canada) Jannsen Pharma- ceutica (Canada) and Smith, Rline and French (Canada) respectively. Adenosine-5’-triphosphate (ATP), dibutyryl cyclic adenosine 5’ monophosuhate (CAMP). 3-isobutyl- llmethylxanthine (IBMX); ethylene glycol-dk (B-amino- ethyl ether) N,N,N’.N’-tetraacetic acid (EDTA). trvpta- mine hydrbchlbride,~ 5-hydroxytryptamine hydrochloride (serotonin), 5-methoxytryptamine hydrochloride, 5,6-dihydroxytryptamine hydrochloride, propanolol, yohimbine, imipramine, cyprohepatadine, trypsin, soybean trypsin inhibitor, ribonuclease (RNase) and deoxyribonuclease (DNase) were all purchased from Sigma Chemical Co. (St. Louis, U.S.A.). Gel electrophoresis reagents were obtained

483

484 P. RIBEIRO and R. A. WEBB

from Terochcm Laboratories Ltd., (Toronto, Canada). Pro- tein markers for electrophoretic estimations of relative molecular weights of H. diminuta phosphoproteins included amylase (200,000), hemocyanin (subunits 280,000; 21O,ooO, 140,ooO; 70,000), phosphorylase b (92,500), bovine albumin (66,000), egg albumin (45,000), pepsin (34,700) and trypsino- gen (24,000) were purchased from Sigma Chemical Co., (St. Louis, U.S.A.). The Bio-Rad Assay Kit was used for protein determinations. All other chemicals were of reagent grade.

Preparation of tissues and fractionation of homogenates

Twenty to 25 day-old H. diminuta were removed from the intestines of male Wistar rats and washed in saline containing antibiotic as described previously (Ribeiro and Webb, 1983). The teguments were removed according to the method of Rahman et al. (1981) and the wo&s were subsequently homogenized in ice-cold Tris-HCl (pH 7.4, 50mM) containing 1 mM EGTA. Fractionation of homogenates was achieved by centrifugation at 3000g for 10 min followed by a centrifugation at 30,OOOg for 20 min. The pellet of the latter (Pr) was washed through three cycles of resuspension in Tris-HCl (pH 7.4, 50 mM) and centrifu- aation at 30.000~ for 20min. Followine a final centrifugation the memb&e preparations (Pr fractions) were frozen at -8O”C, and showed no apparent loss of activity over 48 hr.

[32P]-phosphorylation of whole homogenates, supernatants and the P, and Pz fractions

Aliquots of whole homogenates, supernatants, and P, and P2 pellets rehomogenized in Tris-HCl buffer immedi- ately before use (0.05-0.07 mg protein) were incubated in 50~1 Tris-HCl (pH 7.4, 50mM) containing 60mM NaCl, 25mM KCl, 1OmM MgCI,, 5 x 10e6M [32P] ATP (1-5 x 106dpm/sample), and test drugs or buffer. Incu- bations were routinely conducted at room temperature for periods of 8min, unless otherwise indicated. Following incubation the reaction was stopped by the addition of 4 tni ice-cold 5% TCA and the precipitates rapidly separated from free [‘*P]ATP by filtration under vacuum through Whatman GF/F glass fiber tilters. The latter, which had been treated previously with 10 mM ATP to minimize filter binding artifact, were subsequently washed in 4 ml ice-cold 5% TCA, dried and radioassayed in ACS. Statistical analy- ses were performed using the unpaired t-test at P < 0.05.

SDS-PAGE and autoradiography

Aliquots of P, containing 0.5-0.7 mg protein/sample were phosphorylated as described above with 20-25pCi (5 x 10V6 M) [32P] ATP/sample. The reaction was terminated with 500 ~1 ice-cold 20% TCA, followed by centrifugation at 8000g for 8 min. The resulting pellets were washed twice with 10% TCA and once with Tris-HCI to remove TCA. Following a final centrifugation the pellets were resuspended in 250 ~1 boiling SDS-gel electrophoresis buffer (see Laemmli, 1970), and heated at 100°C in a sealed ampule for 5 min. Aliquots of the samples, containing 0.08 mg protein were subjected to SDS-PAGE on 1.5-mm thick gels. The gels consisted of 3-cm long stacking gels (4% acrylamide, 0.1% N,N’-methylene-bis-acrylamide lMBA1, 0.1% SDS, 0.08% N,N,N’,N’-tetramethyienediamine [TMED], 0.05% ammonium persulfate in 125 mM Tris-HCl, pH 6.8) and a IO-cm long resolving gel (10% acrylamide, 0.31% MBA, 1% SDS, 0.05% TMED, 0.03% ammonium persulfate in 375 mM Tris-HCI. nH 8.7). Electrouhoresis was carried out in 25 mM Tris, ‘192 mM glycine, 0.1% SDS buffer, pH 8.3, at a constant current of 20 mA. Following electrophoresis the gels were stained with 0.04% Coomassie Blue in 7.5% acetic acid for up to 24 hr and destained in acetic acid: methanol: water (2: 13 : 15 v/v). The aels were dried and subjected to autoradiography’ at - SO’C for 5-7 days using Curix RP Agfa-Gevaert 8lm with an intensifying screen.

Characterization of the electrophoreticaliy separated ban& in the Pz fraction

Aliquots of phosphorylated P, preparations were subjected to lipid extraction, and trypsin, DNase and RNase digestion. Delipidation was carried out according to the procedure of Goy et al. (1984). Briefly, following the second wash in 10% TCA, the resulting pellets were rehomogenized in 500 ~1 ice-cold chloroform:methanol:water (5: IO:4 v/v), the organic phase was separated by centrifugation and removed. The aqueous phase was centrifuged at 10,OOOa for 20 min. Trypsin digestion was conducted prior to-and following phosnhorvlation with l’*Pl ATP. Between 0.5 and 0.7 mg oi’ &ypsfin was added to &sue samples containing an equivalent amount of protein and the samples were incubated at room temperature for 10min. The digestion was terminated by an equal amount of soybean trypsin inhibitor, and subsequently either r*P] ATP was added to proceed with phosphorylation or 20% TCA was introduced to terminate the reaction (see Shisheva an Imamura, 1986). Digestion of [32P] phosphorylated samples with DNase or RNase were performed following the technique of Penman (1966). The products of delipidation, and trypsin, DNase and RNase digestion, and control incubations were sub- iected to SDS-PAGE as described above.

For the quantitative determination of 32P incorporation, the electrophoretic bands were excised from dried gels, digested with protosol and radioassayed.

RESULTS

The effects of serotonin and CAMP on ‘*P incorporation from ATP into proteins were examined initially in whole worm homogenates and in homogenates that were fractionated by centrifugation. Aliquots of the unfractionated homogenates (H), of the pellet (P,) and supematant (S,) of the 3000g centrifugation, and of the pellet (P2) supematant (S,) of the 30,OOOg centrifugation, were assayed for 32P incorporation in the presence or absence of 10e4M serotonin or 10T4M CAMP. The data (see Fig. 1) revealed that both serotonin and CAMP significantly stimulated the incorporation of radiolabel from [“PI ATP into whole homogenates of H. diminuta. Following 3000 g centrifugation, the serotonin and CAMP-dependent phosphorylation were concentrated in the supematant (S,) fraction. The pellet (PI) did not show a significant increase in 32P incorporation in response to either serotonin or CAMP. Further fractionation of S, by centrifugation at 30,OOOg yielded a pellet (P2) in which both serotonin- and CAMP-stimulated phosphorylation were enriched, and a supematant (S,) wherein the phosphorylation was sensitive to CAMP but not to serotonin. Because the data demonstrated no significant serotonin-induced phosphorylation in the S2 fraction, only the P, fraction was subsequently investigated in this study.

32P labelling of aliquots of P, increased linearly with increasing amounts of protein (r = 0.99; data not shown). Furthermore ‘*P incorporation was sat- urable and reversible (see Fig. 2). Equilibrium was reached by approximately 7 min, while dephosphorylation, determined by the introduction of IOmM unlabeled ATP, proceeded at a similar rate as phosphorylation such that by 8 min after addition the level of phosphorylation was asymptotic. Thus the times to half-maximal phosphorylation and dephosphorylation were 4 min and 3 min respectively.

SHT-phosphorylation in H. diminuta 485

-+ -+ - 5-HT

Fig. 1. The effects of serotonin and CAMP on “P incorporation into whole homogenates and fractions of H. diminuta. The incorporation of “P was measured in homogenates (H), the supematant and pellet (P, and S, respectively) of the 3000g centrifugation, and the supematant and pellet (P2 and S, respectively) of the 30,000 g centrifugation following incubation in [y-32P] ATP in the presence ( + ) or absence ( - ) of 10m4 M serotonin or 10e4 M CAMP. Data shown are the mean k SE of 4-5 separate determinations.

*Significantly different from untreated ( - ) (P Q 0.05).

The serotonin-stimulated increase in phosphorylation of the P, fraction was dose-dependent (Fig. 3). A marked increase in 32P incorporation was observed by 10mg M serotonin. The concentration required to elicit half maximal stimulation (EC&,) was found to be 0.79 f 0.27pM. Similarly an increase in CAMP stimulated ‘*P incorporation into the P, fraction was seen at lo-‘M CAMP (Fig. 4) but the EC& at -2.51 PM was considerably higher than that for serotonin.

Magnesium was found to potentiate both the serotonin- and CAMP-stimulated phosphorylation of the P, fraction in a dose-dependent manner (Fig. 5). Maximal stimulation of the serotonin- and CAMP-potentiated phosphorylation was achieved at 10 and 25 mM Mg2+ respectively. A smaller but

lb lb lo 15

TIME ( min 1

Fig. 2. The time course of 32P incorporation and dephosphorylation in the P, fraction. Incorporation of r2P into aliquots of the P, fraction was measured at various times over a 15-min period. Dephosphorylation was produced after a ‘I-min incubation in [Y-~*P] ATP by the addition of 10 mM unlabeled ATP and the levels of 32P associated with the TCA-precipitated proteins measured at different time inter- vals for up to 18 min. Data are the mean f SE of 4 indepen-

dent determinations.

significant increase in ‘*P incorporation was produced by Mg*+ in the absence of serotonin or CAMP.

Three agonists of serotonin, methoxytryptamine, tryptamine and dihydroxytryptamine, were able to stimulate 32P incorporation into the P, fraction in a dose-dependent manner (Fig. 3), but, with ECs values of 5.43 + 2.53 PM, 11.6 + 4.5 pM and 13.9 + 5.5 PM respectively, were less potent than serotonin or CAMP.

Three serotonin antagonists, methysergide, ketanserin and cyproheptadine significantly reduced the incorporation of 32P into the P, fraction by 26, 29 and 40% respectively (Fig. 6). Other agents including the serotonin transport inhibitor imipramine or the adrenergic blockers, phentolamine, propranolol and

0

A -1 x

z E” 2 8

_I f

w: EC50 0.79kO.27 pM

u: EC50 5.43_+2.53pM

I: EC60 11.60f4.50@ 5-HT

p: EC50 13.90%50,,,., MX T

D

O-v- -LOG (druO)

Fig. 3. Dose-dependent increases in the incorporation of 32P from [y-‘*PI ATP into the P2 fraction following treatment with serotonin (5-HT), methoxytryptamine (MX), tryptamine (T) and dihydroxytryptamine (D). Data are the mean f SE for 69 determinations. The EC, (concentration required to elicit half-maximal incorporation of ‘*P) is expressed as the mean + SE of three separate experiments.

486 P. WEIR0 and R. A. WEBB

Y EC60 2.51@

6-sl’ -LOG (CAMP)

Fig. 4. Dose-dependent increase in the incorporation of 32P from [Y-~~P] ATP into the P, fraction following treatment with CAMP. Data are the mean& SE of 4 independent determinations. EC% is the concentration of CAMP required

to elicit half-maximal 32P incorporation.

yohimbine failed to significantly influence the effect of serotonin on ‘*P incorporation.

Aliquots on the phosphorylated P, fractions were subjected to gel electrophoresis and autoradiography. A typical result is shown in Fig. 7. The molecular weight scale was generated by determining the relative positions of the marker proteins.

Autoradiographic analysis revealed five radio- labeled bands with relative molecular weights of 134,000, 110,000, 82,000, 80,000 and 31,000, which were consistently enhanced in 32P incorporation by 10m4M serotonin (Fig. 7). While some other bands displayed small apparent changes in radioactivity following treatment of the P, fraction with lo-‘M serotonin, these changes were not reproducible.

To determine that the serotonin-enhanced radio- labeled bands were proteins, the P, fraction were subjected to a variety of treatments (see Table 1). In control samples, approximately 10, 12, 7 and 16% of the total radioactivity recovered from gels was associated with the 134,000, 110,000, 31,000, and the combined 82,000 and 80,000 bands respectively. When the phosphorylated P, fraction was subjected

CAMP

6-HT

CT

6 lb0 MoCIZ. mM

Fig. 5. The effect of MgCl, on the incorporation of 32P from [Y-~~P] ATP into the P, fraction. Samples were assayed for 32P incorporation in various concentrations of MgCI, , in the presence or absence of 10e4 M serotonin or lo-’ M CAMP.

Data are the mean *SE of 4 separate experiments.

-- -R--c-xc-l-TFrTT CT 5-HT 5-HT

Fig. 6. The effect of various pharmacological agents on the serotonin stimulated 32P incorporation into the P2 fraction. Aliquots were assayed for 32P incorporation in the absence of drugs (CT), in the presence of 10m4 M serotonin (S-HT), and the presence of 10e4M serotonin and ketanserin (K), cyproheptadine (C), methylsergide (MG), imipramine (I), phentolamine (PH), propanolol (P) or yohimbine (Y). All agents were at 10e4 M. Data are the mean f SE of 4 separate determinations. Significant reductions in 32P incorporation (P d 0.05) are expressed as % inhibition of the stimulated

32P incorporation.

to DNase and RNase digestion, followed by lipid extraction, there was no significant reduction in the radiolabel associated with the bands. On the other hand, treatment of the P, fraction with trypsin prior to, or following, phosphorylation resulted in a very pronounced decrease in the radioactivity associated with the bands of interest, substantiating the proteinaceous nature of the bands.

DISCUSSION

This study has demonstrated serotonin- and CAMP-stimulated incorporation of 32P from [y-‘*PI ATP into whole homogenates, tissue fractions and specific proteins of H. diminutu. Using the filtration technique for the separation of 32P labelled tissue products from free (Y-~*P] ATP and 32P, most of the serotonin-stimulated labelling was detected in the post-30,OOOg crude membrane pellet (P2). This suggests that some of the sites of the serotonin-stimulated phosphorylation were membrane-associated. On the other hand, TCA precipitable 32P labelled proteins in the supernatant fractions were not significantly affected by serotonin, suggesting that there was no serotonin-stimulated phosphorylation of soluble sites. However, there may have been TCA soluble targets that were able to respond to serotonin but were not detected by the analytical technique utilized.

Some aspects of the incorporation of 32P into the P, fraction were comparable to other phosphorylation systems. For example, labelling of the tissue by [32P] ATP, and its reversal by unlabeled ATP oc- curred rapidly and at approximately the same initial rate. This is consistent with the enzymatic phosphorylation/dephosphorylation of specific components in the P2 fraction. Similarly the effect of Mgr+ on this system in elevating the incorporation of 32P in a dose-dependent manner correlated with the Mg*+-dependency of several kinases and phosphatases (Miyamoto et al., 1969; Kuo and Greengard, 1969; Cohen, 1982; Ingebritsen and Cohen, 1983).

SHT-phosphorylation in H. diminuta

MW. KDa

487

KDa

(-_) SHT Fig. 7. Electrophoretic separation of P2 proteins on a 10% SDS-PAGE gel, followed by staining (A) and autoradioaranhv (B) of 10v4 M serotonin stimulated (S-HT) and non-stimulated ( - ) 3ZP incornoration. The mole&l& weight scale was determined by comparison cf the relative mobilitids of proteins of known molecular weight (see Material and Methods). The arrows mark the positions of phosphoprotein bands

(with relative molecular weight noted) which were consistently enhanced by serotonin.

Serotonin stimulated the incorporation of 32P into the Pr fraction with a threshold of less than lO-9 M. The EC% characterizing this effect, 0.79 PM, was within the range of response to serotonin seen in both vertebrates and invertebrates, including H. diminutn (see Vandermaelen, 1985; Brown and Nestler, 1985; Walker, 1986; Ribeiro and Webb, 1987). Further- more, the action of serotonin on the labelling of the P, fraction was mimicked by several agonists of serotonin and was inhibited by antagonists. Other pharmacological agents, however, were without effect. The elevation of “P incorporation into the P, fraction was thus a specific response to serotonin, and thus likely to be of significance to the physiology of the cestode.

Table 1. Relative levels of [‘2P] labelling in electrophoretically separated P, proteins, following various treatments

% of total radioactivity recovered (A’ + SD)* in proteins

Treatments 134 kDa 110 kDa SO-82 kDa 31 kDa

Control 10.0?2.0 12.0?1.0 16.0+1.0 7.0+1.0 DNase/RNaset Chloroform: 10.0+2.0 11.0+1.0 17.0+6.0 7.Okl.O methanol Trypsin I$ 1.7 * 0.3 1.3 f 0.3 2.8 k 1.5 1.7 f 0.4 Trypsin II 1.6 f 0.1 1.4 + 0.4 2.6 k 0.1 1.7 * 0.1

*Percent of total radioactivity recovered from the gel. Data are shown as the means and standard deviations of three determinations.

t[‘2P]-labelled samples were subjected to RNase/DNase digestion followed by delipidation with chloroform:methanol. as indicated in the text.

fTrypsin was added prior to phosphorylation (I), and after phosphorylation (II).

Using electrophoretic and autoradiographic tech- niques, the targets of the serotonin-stimulated phosphorylation were identified as five bands of relative molecular weights of 134,000, 110,000, 82,000, 80,000 and 31,000. However, additional targets may exist in the P, fraction but because they were very labile, or because of their occurrence in very small quantities, they were not detected. Thus, while changes in labelling intensity appeared to be limited to the bands described above, the possible existence of additional sites of serotonin-stimulated phosphorylation cannot be ruled out.

The proteinaceous nature of the phosphorylated bands was substantiated in the present study. The bands were trypsin labile, as shown by the decline in the relative incorporation of 32P following exposure to the proteolytic enzyme prior to, or after, phosphorylation. Furthermore the bands were unaffected by DNase or RNase digestion or by delipidation. While the specific sites of phosphorylation in these proteins are likely to be’ at serine, threonine and/or tyrosine residues (see, for example, Cahill and Perl- man, 1987; Cohen, 1982; Yeaman et al., 1977) the target amino acids in the phosphoproteins of H. diminuta have not been elucidated.

Cyclic AMP acted as a stimulator of protein phosphorylation in the whole homogenate, P, and P, fractions, but, unlike serotonin, was also able to stimulate 32P incorporation into the final supernatant (S,). Furthermore, CAMP stimulated 32P incorporation into the P2 fraction at concentrations as low as 10m9 M. While the ECso for 32P incorporation (2.51 PM) was much higher than that for serotonin

488 P. RIBEIRO and R. A. WEBB

(0.79 PM), the latter was virtually identical to the ECsO

described previously in the P, fraction for the activation of adenylate cyclase by serotonin (0.76pM) (Ribeiro and Webb, 1987). Furthermore, the order of drug potency for the stimulation of “P incorporation by serotonin agonists, and its reversal by serotonin antagonists, was identical to that reported for the modulation of adenylate cyclase in H. diminuta (Ribeiro and Webb, 1987). Collectively these finding strongly support the concept that the serotonin- stimulated incorporation of 32P into the specific proteins of the P, fraction is mediated by the previously described adenylate cyclase-linked serotonin receptor. Furthermore, we have some preliminary evidence which shows the serotonin-stimulated 32P incorporation into several protein bands is reduced by an inhibitor of CAMP-dependent protein kinase (data not shown). Cyclic AMP protein kinases have been described in several trematode platylhelminths (see Mansour et al., 1987; Webb, 1988, for review). The enzyme from Fasciola hepatica has recently been purified and partially characterized (Mansour et al., 1987), and displays some characteristics different from the enzyme derived from mammals. Thus, CAMP-dependent protein kinases may be common components in the mechanism of transmembrane signaling in the parasitic helminths and may represent sites suitable for the development of specific anthelminthics.

Although cGMP has been linked to neurotransmitter action in the phosphorylation of proteins in a variety of organisms (see Greengard, 1976), and guanylate cyclase has been reported in some platyhelminths (Higashi et al., 1973) the activity of the latter is low in platyhelminths suggesting that this mechanism is not important in these organisms. Furthermore, there is no evidence for serotonin- stimulated phosphorylation being mediated by cGMP in these or any other organism (Goy et al., 1984; Vandermaelen, 1985; Walker, 1986; Brown and Nestler, 1985). On the other hand, pathways involv- ing Ca’+-dependent kinases are plausible targets for serotonin regulation of metabolic activities. While the direct modulation of such enzymes by serotonin has not been demonstrated, such activity could be at- tained indirectly through the activation of adenylate kinase (see Cohen, 1982). In this respect it is note- worthy that a protein with the characteristics of Ca*+/calmodulin has been reported in H. diminuta (White et al., 1984). However, it has not been estab- lished if this protein activates protein kinase or if it is regulated by serotonin.

In summary, the present study provides evidence for serotonin-stimulated phosphorylation of specific proteins of H. diminuta, and supports the concept that serotonin exerts its regulatory role through CAMP via the previously described serotonin- adenylate cyclase-linked receptor.

Acknowledgements-This work was supported by grant #A6508 from the Natural Science and Engineering Research Council of Canada to R. A. Webb.

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