A specific prostaglandin E2 receptor and its role in modulating salivary secretion in the female tick, Amblyomma americanum (L.)

Insect Biochem. Molec. Biol. Vol.27, No. 5, pp. 387-395, 1997

PII: S0965-1748(97)00010-6

Pergamon

© 1997ElsevierScienceLtd All rightsreserved.Printedin GreatBritain 0965-1748/97 $17.00+ 0.00

A Specific Prostaglandin E2 Receptor and its Role in Modulating Salivary Secretion in the Female Tick, Amblyomma americanum (L.) Y|NG QIAN,t RICHARD C. ESSENBERG,$ JACK W. DILLWlTH,t ALAN S. BOWMAN,t JOHN R. SAUER*t Received 24 September 1996; revised and accepted 15 January 1997

Prostag!andins of the 2-series (e.g. PGEz) are typically synthesized from arachidonic acid (AA) after AA is released from cellular phospholipids after activation of an intraceilular phospholipase A2 (PLA2). Treatment of isolated salivary glands with PLAz inhibitor oleyloxyethyl phosphorylcholine (OPC) or prostaglandin synthetase inhibitors reduced dopamine-induced fluid secretion and cyclic AMP (cAMP) levels in isolated salivary glands. PGE2 and its analog, 17-phenyi trinor PGE2, partly reversed the inhibition of secretion and cAMP level by OPC, suggesting that prostaglandins may have an autocrine effect in modulating tick salivary gland function. A specific PGE2 receptor was identified in the plasma membrane fraction of the salivary glands. The receptor exhibits a single, high affinity PGEz binding site with a KD~ 29 riM, is saturable, reversible, and specific for PGE2 and coupled to a cholera toxin-sensitive guanine nucleotide regulatory protein. Assay of adenylate cyclase activity in salivary gland membranes showed that PGE2 neither stimulated nor inhibited adenylate cyclase activity, indicating that the PGE2 effects on cAMP levels and possibly secretion are indirect, and that the PGE2 receptor stimulates an alternate "second messenger" pathway. © 1997 Elsevier Science Ltd Prostaglandin Receptor Phospholipase A2 Salivaryglands CyclicAMP

INTRODUCTION

tick salivary glands by activating a phospholipase A 2 Tick salivary glands serve as the major osmoregulatory (PLA2) (Bowman et al., 1995a). Free arachidonic acid organs during feeding. As feeding progresses, the rate can be converted into a series of bioactive compounds of salivary fluid secretion increases greatly, enabling the called eicosanoids via the cyclo-oxygenase, lipoxygenase ixodid tick to concentrate the bloodmeal by returning and cytochrome P-450 pathways. Prostanoids such as excess water and ions to the host. Salivary secretion is prostaglandin E2 (PGE2), PGF2,, PGD> PGI2 and TxA2 the route for transmission of most tick-borne pathogens are produced via the cyclo-oxygenase pathway. PGE2, to the host. Saliva from feeding ticks also contains many PGF2~, and PGD2 are in the saliva of A m b l y o m m a a m e r bioactive protein and lipid components (Sauer et al., i c a n u m (L.), with PGE2 at the highest concentration 1995). The salivary glands are controlled by nerves and (Ribeiro et al., 1992; Bowman et al., 1995b). The prostadopamine is the neurotransmitter at the neuroeffector glandins in tick saliva are believed to be important in junction. Dopamine stimulates fluid secretion at the neur- tick feeding (Bowman et al., 1996). However, they could oeffector junction by increasing intracellular levels of also have a local effect (autocrine or paracrine) on salicAMP (Schmidt et al., 1982). Dopamine also stimulates vary gland physiology. PGE2 has shown a wide spectrum of physiological and the release of arachidonic acid from phospholipids in the pharmacological actions in diverse tissues. The role of PGE2 in the regulation of fluid transport has been demon*Author for correspondence. Tel.: +1(405) 744-5435; Fax: +1(405) strated in invertebrates as well as vertebrates (Wollin et 744-6039; E-mail: [email protected] al., 1979; Soil, 1980; Bentley and McGahen, 1982; Mac+Department of Entomology,OklahomaState University,Stillwater, Donald, 1986; Bjerregaard and Nielsen, 1987; Smith, OK 74078-0464, U.S.A. ~:Departmentof Biochemistryand MolecularBiology,OklahomaState 1989; Brown et al., 1991; Petzel and Stanley-Samuelson, University, Stillwater,OK 74078-0464, U.S.A. 1992). In the mammalian kidney, PGE2 mediates water 387

388

YING QIAN el al.

and NaC1 reabsorption through cAMP metabolism (Smith, 1989). In insect Malpighian tubules (MTs), PGE2 regulates fluid secretory rates (Petzel and StanleySamuelson, 1992; Kerkhove et al., 1995). PGE~ inhibits 5-hydroxytryptamine-stimulated fluid secretion in Calliphora e~throcephala salivary glands via an inhibitory effect on adenylate cyclase (Dalton, 1977). In vertebrates, PGE2 exerts its effects by interacting with specific receptors on cell membranes (Coleman et al., 1990). PGE-specific binding sites are in the plasma membranes of various tissues including the corpora lutea, myometrium, kidney, intestinal epithelium, liver, heart, brain, circulating immune ceils and platelets (Coleman et al., 1990; Virgolini et al., 1992). Because PGE2 can induce diverse cellular responses through multiple signal transduction pathways, PGE2 receptors are classified into four subtypes in mammals, namely EP1, EP2, EP3, and EP4, on the basis of their responses to natural prostanoids and various synthetic PGE2 analogs (Coleman et al., 1990, 1994). Stimulation of EP1 receptors induces Ca2+ mobilization, EP2 and EP4 receptors increase intracellular cAMP, and EP3 receptors inhibit cAMP levels in cells (Negishi et al., 1993). Although there are many reports of the physiological effects of prostaglandins in invertebrates, there is little evidence for prostaglandin receptors. An exception is a receptor of a prostaglandin A in the gill tissue of a marine bivalve Modiolus demissus (Freas and Grollman, 1981). The present work was undertaken to examine if prostaglandin receptors exist in the tick salivary gland and, if so, do prostaglandins have an effect on fluid secretion by isolated salivary glands?

MATERIALS AND METHODS

Materials

Reagents were obtained from the following sources: M-199 medium (Sigma catalogue no. M 0393), oleyloxyethyl phosphorylcholine, esculetin, clotrimazole, indomethacin, acetylsalicylic acid, diclofenac, PGE2, 17-phenyl trinor PGE2, GTP, GDP, ATP, App(NH)p, ouabain, creatine phosphate, creatine phosphokinase, dithiothreitol (DTT), cholera toxin, NAD, Dowex 50W-H + cation exchange resin (200-400 mesh, 8% cross-linkage), neutral alumina (type WN-3), GTPTS, pertussis toxin, cholera toxin, and other chemicals were acquired from Sigma Chemical Company, St Louis, MO, U.S.A. The cyclic AMP assay kit was obtained from Amersham International plc (Arlington Heights, IL). [5,6,8,11,12,14,153H(N)]-PGE2 (171 Ci/mmol), [oflZP]ATP (3000 Ci/mmol), and [2,8-3H]cAMP (30 Ci/mmol) were from Du Pont New England Nuclear (Boston, MA, U.S.A.); PGF2~, PGD2 and U-46619 were from Cayman Chemical Co. (Ann Arbor, MI, U.S.A.); GF/F microglass filters (25 mm) were from Fisher (St Louis, MO, U.S.A.).

Ticks A. americanum female ticks were reared on sheep according to the methods of Patrick and Hair (1975). Partially fed ticks weighing from 100 to 150 mg were removed from the host and were immediately used for all the assays. Assay o f salivar 3' secretion

Salivary glands were dissected and secretion was measured as described by Harris and Kaufman (1984) and McSwain et al. (1992). Briefly, the dorsal cuticle of a tick was carefully removed by making a circular lateral incision around the tick with a single-edge razor blade. M-199 buffer (pH 7.0) was then added to the tick haemocoel. The gut, reproductive, nervous and tracheal systems, along with any adhering tissues, were carefully removed to expose the salivary glands and ducts fully. The main duct was tied off with a silk thread (size 4-0) and then carefully cut in front of the knot. In each pair of isolated glands, one was used for the treatment and the other as the control. In assays for examining the effects of various eicosanoid inhibitors on secretion, the isolated salivary glands were weighed and then incubated with the various eicosanoid synthesis inhibitors (treatment gland) or a solvent carrier (control gland) for 15 min, followed by incubation for 5 min in 10~M dopamine dissolved in the previous solutions. After incubation, the glands were rinsed in buffer for 3 rain, weighed, and placed back into the incubation solutions for another 5 min. The process was repeated. After each designated time interval, the glands were reweighed. The weight changes before and after treatment represent the secretory abilities of the isolated salivary glands. In examining the ability of PGE2 to reverse the inhibition of secretion by inhibitors of PLA2 or cyclo-oxgenase, isolated salivary glands were pretreated with the inhibitor for 15 rain by the same procedure as in the previous assays, followed by incubation in the same buffer containing 10/xM dopamine, 100~M prostaglandin, and cAMP phosphodiesterase inhibitor, 10 mM theophylline or 1 mM 3-isobutyl-l-methyl-xanthine (IBMX). A control consisted of the same reaction mixture without prostaglandin. c A M P assay

Salivary glands were prepared as in the secretion assays. Glands were incubated in M-199 buffer, pH 7.5, containing 1 mM IBMX plus the desired effectors (treatment) or solvent carriers (control) for 32 min. The reaction was terminated by placing glands in 100/xl of 50raM Tris/4mM ethylenediamine tetraacetic acid (EDTA) buffer, pH 7.5, on ice. Levels of cAMP were measured with a cAMP kit by Amersham using the protein binding method according to the manufacture's instructions.

MODULATINGSALIVARYSECRETIONIN THE FEMALETICK Preparation of membranes Salivary gland membrane fractions were prepared according to Watanabe et al. (1986) and McSwain et al. (1987) with modifications. Briefly, salivary glands were dissected at 4°C in a solution of 50 mM Tris-HCl, 100/zM indomethacin, and 2 mM EDTA, pH 7.5. The excised glands were homogenized in an all-glass homogenizer. The crude homogenate was centrifuged at 325 g for 5 rain at 4°C. The supernatant was then centrifuged at 11 500g for 10 min at 4°C. The resulting pellet was resuspended in the dissection buffer and membranes collected by centrifugation at 11 500g for 10min twice more. The washed membrane pellet was resuspended in the dissection buffer for use in PGE2 binding assays. If membrane preparations were not used immediately, they were stored at -70°C. Protein was determined by the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin as a standard. Gradient purification of plasma membranes Gland membrane fractions were further purified by sucrose gradient centrifugation in experiments when the effects of guanine nucleotides and exotoxins on the PGE2-binding activity were investigated. Membranes prepared as above were suspended at a protein concentration of 2-5 mg/ml in 50 mM Tris-HCl, 2 mM EDTA, pH 7.5, containing 1.95 M sucrose. The suspension was overlayed with 0.25 M sucrose and the sample centrifuged at 100 000 g for 60 min at 4°C. Material at the interface of 0.25 M and 1.95 M sucrose was withdrawn with a syringe, diluted with approximately 5 volumes of 50 mM Tris-HC1, 2 mM EDTA, pH 7.5 and centrifuged at 100 000 g for 60 min at 4°C. The pellet was washed twice with 50 mM Tris-HC1, 2 mM EDTA, pH 7.5, and suspended in 50 mM Tris-HC1, 2 mM EDTA, pH 7.5. Purified membrane preparations were stored at -70°C until used. Binding assays Direct binding assays of PGE2 to the plasma membrane fraction were performed with radiolabelled PGE2 according to Williams and Sills (1990). The standard assay mixture contained 50mM Tris-HCl, 3.84mM MgC12, pH 7.5, 3 nM [3H]PGE2 and 50/xg membrane protein in a final volume of 200 ixl. Some variations are indicated in the legends. Incubations proceeded for 3 h at 30°C and were stopped by the addition of 5 ml of icecold binding assay buffer and the solution filtered rapidly through a Whatman GF/F microglass filter. Each filter was washed three times with 5 ml of ice cold buffer. In all experiments control assays were performed in parallel with 100 txM PGE2 included in the assay mixture to determine non-specific binding. PGE e metabolism The possibility that [3H]PGE2 might be degraded during the assay was examined by the incubation of radiolabelled PGE2 with the gland membrane in the same incu-

389

bation medium. After incubation, the suspension was acidified to pH 3-5 with 3% formic acid and extracted with 3 volumes of ethyl acetate. The extracted material was then analysed by thin layer chromatography (TLC) with radio scanning (Bowman et al., 1995b). Treatment of gland membranes with pertussis toxin or cholera toxin Purified membranes were treated with the exotoxins described by McKenzie (1992) and Watanabe et al. (1986). The membranes were incubated with 10 Ixg of activated pertussis toxin at 30°C for 40 min in 0.5 ml of a mixture containing 100 mM potassium phosphate, 5 mM MgCI2, pH 7.5, 10 mM NAD, 1 mM ATP, 1 mM EGTA, 10 mM thymidine, 2 mM DTT, 100/xM GTP, 3 mM potassium phosphoenolpyruvate and 10 mg/ml of pyruvate kinase. For cholera toxin treatments, membranes were incubated with 100 txg of activated cholera toxin in the same medium as was used for the pertussis toxin treatment. Controls were performed without treatment with toxins. Treated membranes were incubated with 10 mM N-ethylmaleimide at 0°C for 30 min. Finally, the treated membranes were washed twice with 50 mM Tris-HC1, pH 7.5, containing 2 mM EDTA and suspended in binding assay buffer. Binding of 3 nM [3H]PGE2 was then measured in the presence and absence of 1 mM GTP3,S. Assay of adenylate cyclase activity Adenylate cyclase activity was measured as described by Farndale et al. (1992) and Schmidt et al. (1982) with some modifications. The basic assay mixture contained 25 mM Tris-HCl, pH 7.5, 100 p,M[a32p]ATP (106 cpm), 5 mM MgC12, 0.77 mM IBMX, 0.1 mM EDTA, 500 mM cAMP, 20 mM creatine phosphate, 50 units/ml creatine kinase, and 1 mM DTT. Assays were initiated by the addition of PGE2 and gland membranes (40/xg protein). Reactions were allowed to proceed at 37°C in a water bath for 15 rain and were halted by adding stop solution containing 2% sodium dodecyl sulphate, 40 mM ATP and 1.4 mM cAMP. Tracer amounts of [3H]cAMP were added to monitor the recovery of biosynthetic [o~32p] cAMP that was isolated by sequential chromatography of the samples on Dowex 50W-X4 and alumina columns (Farndale et al., 1992). cAMP recoveries were 70-80% using this assay procedure. Statistical analysis The differences between the control and experimental treatment were tested for significance by paired Student's t-test. A P value of less than 0.05 was considered to be significant. RESULTS

The effects of eicosanoid synthesis inhibitors on dopamine-induced secretion by salivary glands in vitro Various inhibitors of eicosanoid synthesis, including the PLA2 inhibitor oleyloxyethyl phosphorylcholine

390

YING QIAN el a /

~- (A) OPC

(OPC), the lipoxygenase inhibitor esculetin (ESC) and the cytochrome P-450 inhibitor clotrimazole (CTM) as well as the cyclo-oxygenase inhibitors indomethacin, acetylsalicylic acid (ASA), and diclofenac, were tested to determine if they caused the inhibition of secretion induced by dopamine in isolated salivary glands. Dopamine is known to stimulate the release of arachidonic acid in isolated salivary glands (Bowman et al., 1995a), and arachidonic acid may be metabolized under these conditions to prostaglandins. The results showed that the treatment of isolated salivary glands with the PLA 2 inhibitor OPC or the cyclo-oxygenase inhibitor ASA at 10/xM inhibited dopamine-stimulated secretion (Fig. 1). An inhibitor of lipoxygenase, esculetin ESC, at 100/xM and an inhibitor of cytochrome P-450, CTM, at 10/xM had no significant effect on dopamine-stimulated secretion (Fig. 1). The inhibition of in vitro salivary secretion by OPC and ASA was dose dependent (Fig. 2). Other cyclo-oxygenase inhibitors, indomethacin and diclofenac, blocked dopamine-stimulated secretion almost completely at I mM, but not at low concentrations (data not shown)• The results suggest that cyclo-oxygenase products may be synthesized and may play an important role in enhancing dopamine-stimulated salivary secretion.

= ~o ~ ~ .~ ~

Reversal by PGEz of the inhibition of dopamine-stimulated salivao' secretion by inhibitors of PLA, and cyclo-o©'genase

~ oN -60 ~ ~ -80

Isolated salivary glands in the presence of 1 mM OPC treated with 100/xM PGE2 or its analog, 17-phenyl trinor PGE~, increased secretion above the level observed in glands in OPC alone (Fig. 3). However, neither 100/xM PGE2 nor its analog, 17-phenyl trinor PGE,, stimulated secretion above that noted when secretion was inhibited

-100

40

m

20

0.0

III

-20

gr, -40

-60 FIGURE I. Comparison of the effects of various eicosanoid synthesis inhibitors on secretion by isolated salivary glands after 32 rain. The control glands were treated with dopamine (10/xM) alone. The experimental glands were treated with dopamine (10/xM) plus the inhibitors as indicated. OPC, PLA2 inhibitor oleyloxyethyl phosphorylcholine; ASA, cyclo-oxygenase inhibitor acetylsalicylic acid; CTM, cytochrome P-450 inhibitor clotrimazole; ESC, lipoxygenase inhibitor esculetin. *Significantly different from zero (P<0.05).

0 ,-e ~ ~

T

-20

~ ~ .=. ~ "= .~ ~ ,~ ~ ~'~ ~ ~a g=

-40 10-SM -60

-80

-100

IO-3M i

0

60 ,-~

40

~

20

a~ "'

0

I

i

i

10

i

[

'

i

20 30 Time(min)

I

i

40

50

(B) ASA I i

"I'll

10-7M

-2o

~

.~,._ ~ , ~

-40

lO-SM IO'3M

0

10

20 30 Time(rain)

40

50

FIGURE 2. Inhibition of in vitro salivary secretion by different concentrations of (A) PLA_, inhibitor oleyloxyethyl phosphorylcholine (OPC) and (B) cyclo-oxygenase inhibitor acetylsalicylic acid (ASA) (n->3). The control glands were treated with dopamine (10/xM) alone. The experimental glands were treated with dopamine (10/xM) plus the inhibitors as indicated.

by the cyclo-oxygenase inhibitors indomethacin, ASA, and diclofenac. PGE2 was unable to stimulate secretion at low concentrations. PGF2~ (100/xM) was ineffective at stimulating secretion inhibited by OPC (data not shown). Arachidonic acid was unable to recover the secretion inhibited by 1 mM OPC (data not shown). In the absence of dopamine, PGE2 had no effect on salivary secretion. The effects of inhibitors of PLA 2 and cyclo-oxygenase, and PGE2 on intracellular cAMP accumulation Because dopamine stimulates salivary secretion through an increase in cAMP (Schmidt et al., 1982), the effect of PGE2 on the regulation of the fluid secretion stimulated by dopamine was examined by investigating the role of PGE2 in cAMP metabolism. The control glands were treated with a mixture of dopamine (10 p.M) plus 1BMX (1 mM). The experimental glands were treated with an inhibitor of PLA2 or cyclo-oxygenase

391

MODULATING SALIVARY SECRETION IN THE FEMALE TICK "3

601

=

50

~-

40

o

30

L

o

20

=

10

~

0

60

..=

so

.0 ~., ~'~

40

30 20 O

lo PGE 2

17-phenyl trinor PGE 2

FIGURE3. The stimulatory effects of PGE2 and 17-phenyl trinor PGE2 at 100/xM on secretion and intracellular cAMP formation in isolated salivary glands in the presence of 1 mM OPC. The control glands were treated with OPC (1 raM) plus dopamine (10/xM) in the presence of theophylline (10 raM) or IBMX (1 raM) for 16 rain. The experimental glands were treated with PGE2 (100/xM) or 17-phenyl trinor PGE2 (100/xM) in addition to the same components as the control glands for 16 min. PGE2 (100/xM) stimulated secretion approximately 23% above the control level in the presence of theophylline (P<0.05); 17-phenyl trinor PGE2 (100/xM) increased secretion approximately 40% above the control level in the presence of IBMX (P<0.05). PGE2 and 17-phenyl trinor PGE2 increased cAMP accumulation approximately 20% and 40% above the control in the presence of 1 mM IBMX, respectively (P<0.05).

plus the same components as in the control glands. The PLA2 inhibitor OPC inhibited dopamine-stimulated c A M P accumulation approximately 25% at 1 mM. In addition, the cyclo-oxygenase inhibitor indomethacin inhibited c A M P accumulation approximately 27% at 1 raM. Verapamil, which inhibits dopamine-stimulated secretion (Needham and Sauer, 1979) and also blocks arachidonic acid release ( B o w m a n et al., 1995a), also reduced the c A M P accumulation approximately 65% at 1 m M (data not shown). In the presence of OPC, PGE2 and its analog, 17-phenyl trinor PGE2, stimulated c A M P accumulation approximately 20% and 40% above the OPC-inhibited control, respectively (Fig. 3). However, neither PGE2 nor its analog, 17-phenyl trinor PGE2, stimulated c A M P accumulation above that inhibited by the cyclo-oxygenase inhibitor indomethacin or veraparail. PGF2~, did not stimulate an accumulation of c A M P above that observed in the presence o f 1 m M O P C (data not shown). Again, PGE2 on its own had no effect on c A M P accumulation.

Evidence of a specific PGEe receptor in tick salivary gland plasma membranes To see whether the effects of PGE2 on secretion and level of c A M P are physiological, we sought evidence of a receptor in the salivary glands through the use of binding assays with labelled PGE2. T L C analysis indicated no detectable metabolism of [3H]PGE2 during the binding assays (data not shown). Comparison of specific binding of PGE2 to different fractions of the salivary glands, i.e. 11 5 0 0 g pellet, 1 0 0 0 0 0 g supernatant and pellet, found that specific binding o f PGE2 to the 11 500 g fraction was at least three times that in other

0

11soog Pellet

t oo, ooog l oo, ooog Pellet Supernatant

FIGURE 4. Comparison of specific [3H]PGE2 binding to different fractions of tick salivary gland homogenates. 13H]PGE2binding assays were performed at 24°C for 3 h using 2.75 nM [3H]PGE2 and 128/xg protein (n>4). Asterisks indicate the values significantly different from zero (P<0.05). tissue fractions (Fig. 4). This fraction is known to contain the highest percentage of plasma membranes after tissue fractionation (McSwain et al., 1987). Electron micrographs of this fraction also showed rich contents of membrane (data not shown). The 11 500 g membrane fraction was therefore used in most of the subsequent binding assays. Specific binding of [3H]PGE2 to the 11 500 g membrane fraction was directly related to the membrane protein amount up to 2 0 0 / x g (data not shown). The specific binding of [3H]PGE2 to salivary gland membranes was also related to increasing concentrations of [3H]PGE2 (Fig. 5). The optimum pH for the [3H]PGE2 binding was 8.5. In addition, the specific PGE2 binding activity was enhanced twice by 300 mM MgC12 compared to the binding activity in the presence of 3.84 m M MgC12 in the assay buffer. The specificity of [3H]PGE2 binding was investigated by adding increasing concentrations of various unlabelled prostaglandins (PGE2, PGF2~, PGD2 and TxA2) to the 140 120

,oo ~

~

80

~--- ~

60

~. ~

40

~

20

C~

0

10

I ' 20

'

; ' 30

' ' : ' 40

' ' I 50

13I-IIPGE2(nM)

FIGURE 5. Specific binding of [3H]PGEzto tick gland membranes as a function of concentrations of [3H]PGE2.The [3HIPGE2 binding assay was performed in duplicate for 3 h at 24°C using 140 txg membrane protein with the indicated concentrations of [3H]PGE2.The curve was fitted using KaleidaGraph.

392

YING

QIAN

assay mixture. [3H]PGE2 was displaced in the rank order of PGE2>PGF2,>PGD2>U-46619 (non-hydrolysable TxA2 analog) (Fig. 6). The binding characteristic of [3H]PGE2 to the gland membranes was analysed by a Scatchard plot using the LIGAND program (Munson and Rodbard, 1980). The best fit of the data indicated a single site model with a KD~29 nM and B ......-~1.2 pmol/mg protein. The time course of specific binding of [~H]PGE2 to membranes was studied at various temperatures (Fig. 7(A)). The rate of specific binding of [3H]PGE2 to membranes increased with increasing temperature and reached equilibrium after 4 h at 37°C and 6 h at 30°C. Subsequent displacement experiments were carried out at 37°C. Unlabelled PGE2 (100 o,M) was added after [3H]PGE2 had incubated with membranes in assay tubes for 4 h. Bound [3H]PGE2 was rapidly dissociated from the membrane fraction during the first 30 rain and then the rate of dissociation slowed (Fig. 7(B)). It is unclear why the bound labelled PGE2 was only partly displaced from membranes.

~ 100 --.. o

tl

9o -

•r,

"~

~ \ \ T~,,,.

• °~0

eo

~'"-.

so 4o

_

\ \

PGE2

3o

31.6nM

2o

PGF2~,.,

u~e~ I

J

. "'-

I

*

I

'~.. "|

8500nM -9

~ '\

"'.

\

k k • ~

0 0

.. \

\

PGD 2

D. u~

"•..

3~6nM PGE2~k x 2820nM

PGF2~

30°C

1oo.

et ~

10-

¢,,,I

e~

1-

C~

.~

illlllllllll,llll,lll,lllll,l,,,ll

320

100 200 300 400 500 600 700 Time (rain)

'

(B)

300

.~ ~ ~.

280 260

.~ ~ t= $ o

220 200 180

0 2'0 41) 6'0 S0 100120140 Time(min)

FIGURE 7. (A) Time coursesof specificbinding of [ 3 H ] P G E 2 to gland membranes. Binding assays were performed in triplicate at the indicated temperatures for the indicated times using 3 nM [3H]PGE2and 50/xg of membrane protein (the assays used pH 8.5 and 300 mM MgC12). (B) Time courses of displacement of specific [3HIPGE2 binding to gland membranes. The binding assays were performedin triplicate at 37°C using 3 nM [3H]PGE2and 50/xg of membrane protein. Values represent the mean of triplicae determinations +_SE.

*

I

slightly reduced [-~H]PGE~ binding activity. Moreover, l mM GTPyS reduced specific [3H]PGE2 binding to membranes in a PGE2 dose-dependent manner (Fig. 8(B)). Results indicate that [3H]PGE2 binding to membranes is sensitive to GTP. To confirm if the PGE2 binding activity is associated with a guanine nucleotide regulatory protein, we made another observation. Pretreatment of gland membranes with cholera toxin and NAD prevented GTPTS from inhibiting PGE2 binding activity (Table 1). A similar treatment of membranes with pertussis toxin and NAD had no effect on the ability of GTPTS to inhibit PGE2 binding activity. These results strongly suggest that the PGE2 receptor in tick salivary glands is linked to a G~-like rather than a G~-like GTPbinding protein.

"~ U-46619 PGD~\\

== 70 - - ~ ' " . . . ,

_

~L

•

xk"'" ~x~.:,

8o

1,000-

._=

Evidence that the PGE2 receptor is associated with a guanine nucleotide regulatory protein A series of purine nucleotides was chosen to detect their effects on [3H]PGE2 binding activity. Data showed that both GTP and its non-hydrolysable analog GTPTS significantly reduced specific [3H]PGE2 binding to gland membranes (Fig. 8(A)). In constrast, ATP and GDP had no significant effects on the binding activity• ATPvS

eta/.

*

I

-8 -7 -6 Prostanoid (log M)

*

I

-5

FIGURE 6. Inhibition of specific 13H]PGE2binding to gland membranes by various prostaglandins. The binding assays were performed in triplicate using 3 nM [3H]PGE2and 50 p,g of membraneprotein for 3 h at 24°C (the assays used pH 8.5 and 300 mM MgCI2) in the presence of the indicated concentrations of prostaglandins. Displacement curves were fitted using SigmaPlot (Jandel), and the ICso values were determined as the concentrationof prostaglandins necessary to cause 50% inhibition of binding of 3 nM [3H]PGE2to gland membranes.

Adenylate cyclase activity Because PGE2 and its analog, 17-phenyl trinor PGE2, were shown to affect intracellular cAMP formation and the PGE2 receptor is linked to a G~-like protein, we determined whether PGE2 had an effect on adenylate cyclase activity in the tick salivary glands. Prostaglandin E2 receptors in mammals linked to G~ GTP-binding proteins (EP2 and EP4 receptors) stimulate adenylate cyclase activity. Even though PGE2 affects cAMP con-

393

MODULATING SALIVARY SECRETION IN THE FEMALE TICK

1207

1oo

(A)

TABLE 2. Comparison of the effects of PGE2 and dopamine on adenylate cyclase in salivary gland membranes Treatment

6o-

PGE2

4o_

Dopamine

..

200" I

~

150-]

•~

6 PGE 2 (Log M)

FIGURE 8. (A) Effects of purine nucleotides on specific binding of [3H]PGE2 to gland membranes. Purified membranes were prepared and binding assays were performed in triplicate using 3 nM [3H]PGE2and 50 txg of membrane protein in the presence or absence of 1 mM or 5 mM (#) various purine nucleotides at 30°C for 4 h (the assays used pH 8.5 and 300 mM MgCI2). Values represent the mean of triplicate determinations _+SE. *Significantly different from control (no purine nucleotide) (P<0.05). (B) Inhibition of specific [3H]PGE2binding to gland membranes by 1 mM GTP3,S. Membrane preparations and binding assays were performed as in (A). Values represent the mean of triplicate determinations _+SE. TABLE 1. Effects of cholera toxin and pertussis toxin treatments on the inhibition by GTP3~S of PGE2 binding to tick gland membrane preparations Treatment

Control Pertussis toxin Cholera toxin

-9 -8 -7 -6 -5 -4.3 -5

97.6 97.6 98.6 98.3 98.6 94.6 325"

DISCUSSION

50.4 /

o/

% of basal activity

The data are means from three determinations. *Indicates significantly different from basal level of adenylate cyclase activity (P<0.05). Basal level of adenylate cyclase activity was 197 pmol/mg/min for measuring the effect of PGE2 and 112 pmol/mg/min for measuring the effect of dopamine.

,oo.I •: "

Concentration (log M)

Specific PGE2 binding (fmol/mg protein) -GTP3,S

+GTP3,S

70_+15 82_+14 5 I+13

39_+8* 38_+7* 50_+20

Salivary gland membranes were prepared and subjected to the indicated treatments and carried out in triplicate for [3H]PGE2 binding assay in the presence or absence of 1 mM GTP~/S. This experiment was performed twice with similar results. *Values were significantly different from values in the absence of GTP'yS (P<0.05) centrations in whole salivary glands, various concentrations of PGE2 (10 -9 M - 1 0 -5 M) had no effect on plasma membrane adenylate cyclase activity (Table 2). High adenylate cyclase activity was observed with dopamine, thus demonstrating that the assay system was valid.

Our secretion assays show consistent results with the effects o f the P L A 2 inhibitor OPC, and PGE2 and its analog 17-phenyl trinor PGE2 on salivary secretion. OPC can inhibit dopamine-induced secretion, and PGE2 or 17phenyl trinor PGE2 is capable of partly reversing the secretion inhibited by OPC. Furthermore, the increase of c A M P accumulation by PGE2 and its analog, 17-phenyl trinor PGE2, is about the same as the increase of salivary secretion (Fig. 3), which suggests a role for PGE2 in regulating secretion by isolated salivary glands. However, the results have certain ambiguities. Although a variety o f cyclo-oxygenase inhibitors inhibit dopamine-stimulated secretion and at least one (indomethacin) inhibits dopamine-stimulated accumulation of cAMP, their effects cannot be reversed by PGE:. W h y this is so for inhibitors of cyclo-oxygenase but not for an inhibitor o f P L A 2 is unclear. In addition, neither PGE2 nor its analog, 17-phenyl trinor PGE2, stimulates salivary secretion and intracellular c A M P formation on its own. Overall, the action of PGE 2 in salivary gland physiology seems to be related to events after salivary glands are first stimulated by dopamine and the magnitude of effects is modest. We performed [3H]PGE2 binding assays to determine if a specific PGE2 receptor exists in tick salivary glands. Our results show that there is a single, high affinity PGE2 binding site (KD~29 riM) in tick salivary gland plasma membranes. PGE2 binding is saturable, reversible, and specific for PGE2. The observation that PGE2 binding to gland membranes is reduced by GTP and its analog ( G T P y S ) indicates that the P G E : receptor is functionally associated with a guanine nucleotide regulatory protein. Further evidence shows that it is a cholera toxin-sensitive G protein because pretreatment of membranes with cholera toxin plus N A D eliminated GTP3,S inhibition of PGE2 binding activity but pertussis toxin did not. To our knowledge, this is the first evidence of a PGE2 receptor coupled to a cholera toxin-sensitive G protein existing in an invertebrate. Although PGE 2 has been shown to stimulate intracellular c A M P formation at a high concentration in isolated salivary glands, PGE2 did not stimulate nor

394

YING QIAN e/ aL

inhibit adenylate cyclase activity, indicating that the PGE2 effects on salivary gland cAMP levels are indirect. In functional assays, we found that 17-phenyl trinor PGE2 (EPI subtype agonist in mammalian tissues) has the same function as PGE2 in salivary secretion and cAMP formation. We suspect that the PGE~ receptor may be linked to Ca 2+ mobilization. The function of PGs in the regulation of fluid transport has been studied mostly in vertebrates. In the mammalian kidney, there is now overwhelming evidence that the PGs have important regulatory effects on the transport of water and many electrolytes across epithelia (Smith, 1989; Frazier and Yorio, 1992). Low concentrations of PGE~ (-<10 ~ M) are found to inhibit arginine vasopressin (AVP)-induced water and sodium reabsorption by interfering with AVP-induced cyclic AMP synthesis on the collecting tubule and thick ascending limb. However, higher concentrations of PGE2 (->10 7 M) cause stimulation of water reabsorption by activating adenylate cyclase in both the collecting tubule and thick limb cells. Morever, AVP-induced NaC1 reabsorption in the perfused mouse thick limb is attenuated by PGE2 (Smith, 1989). The opposite actions of PGE, are considered to result from the existence of both stimulatory and inhibitory PGE2 receptor subtypes in the kidney (Watanabe et al., 1986). It has been demonstrated that the inhibitory receptor exhibits a high affinity for PGE2 and sulprostone, and is coupled to a pertussis toxin-sensitive guanine nucleotide regulatory protein (Gi) (Watanabe et al., 1986; Sonnenburg et al., 1990). There is indirect evidence that suggests the existence of a lower affinity, pertussis toxin-insensitive stimulatory PGE2 receptor in the kidney (Sonnenburg et al., 1990). Recently, Sugimoto et al. (1994) demonstrated that a high level of EP1 receptor mRNA was expressed in the collecting ducts of the kidney where PGE2 attenuated the vasopressin-induced osmotic water permeability through Ca 2+ mobilization. The EP1 receptor is thus thought to be an important modulator of renal function through opening the Ca 2+ channel in plasma membranes. Compared to other animal taxa, very little is known about the physiological role of prostaglandins in invertebrates. PGs regulate sodium transport in the freshwater bivalve Ligumia subrostrata, probably through intracellular cAMP because PGE2 inhibition could be reversed by the addition of dibutyryl cAMP (Saintsing and Dietz, 1983). Dalton (1977) showed that PGE~ inhibited 5-hydroxytryptamine- and theophylline-stimulated cAMP production in the salivary glands of Calliphora erythrocephala by the inhibition of adenylate cyclase. Prostaglandins were suggested to be involved in the regulation of basal fluid secretion in Malpighian tubules in the yellow fever mosquito, because inhibition of PLA2 and cyclo-oxygenase substantially reduced basal fluid secretion rates (Petzel and Stanley-Samuelson, 1992). Kerkhove et al. (1995) also reported that inhibition of eicosanoid biosynthesis or prostaglandin biosynthesis in in vitro preparations of Malpighian

tubules from adult ants, Formica polyctena, strongly reduced basal fluid secretion and suggested that prostaglandins are involved in regulating fluid secretion rates in ant Malpighian tubules. A model for the action of PGE2 in modulating tick salivary secretion based on previous knowledge and present observations can be advanced (Fig. 9). Dopamine stimulates salivary secretion by binding to a Dj receptor, causing the activation of adenylate cyclase and the formation of cAMP (Schmidt et al., 1982). Secretion is dependent on an increase in salivary gland cAMP. Secretion also requires an influx of Ca 2+ because the deletion of calcium or the addition of the calcium antagonist verapamil to the bathing medium inhibits dopamine-stimulated fluid secretion by isolated salivary glands (Needham and Sauer, 1979). PLA2 is a Ca2+-requiring enzyme, and the influx of Ca -~+ stimulated by dopamine causes the release of arachidonic acid from salivary gland phospholipids, presumably by activating an intracellular PLA2 (Bowman et al., 1995a). The free arachidonic acid is believed to be converted into prostaglandins such as PGE2 by the cyclo-oxygenase pathway. Present results suggest that PGE2 interacts with a specific G proteinlinked receptor which stimulates an unknown effector system, possibly one that further mobilizes intracellular Ca 2+. The effect of PGE2 indirectly stimulates an increase in intracellular cAMP formation and salivary secretion in the presence of dopamine. Future work will focus on identifying the effector system of the signal transduction pathways and its subsequent role in controlling salivary gland function.

PG.E2~ .. DA Ca2÷ / ,, ] (~~ Haem~ym p h s ~ I}| I

@

\.9

9/

cAMP \ pk

"/

.a,* ,"

Pro~ 1 Protein-P

Lumenalside

T

•

/ AA t

PGE2

Secretion

FIGURE 9. Model for the action of PGE2 in modulating tick salivary secretion. Abbreviationsinclude: DA, doparnine;D l, dopamine receptor subtype; G, G-protein; AC, adenylate cyclase; PK, protein kinase; Protein-P, phosphorylated protein; R, prostaglandin Ee receptor; E, unknown effector system coupled to the prostaglandin E2 receptor: PLA> phospholipase A2; AA, arachidonic acid; PGE2, prostaglandin E2. Dashed line and "?" indicate uncertainty.

MODULATING SALIVARY SECRETION IN THE FEMALE TICK

REFERENCES Bentley P. J. and McGahen M. C. (1982) A pharmacological analysis of chloride transport across the amphibian comea. J. Physiok 325, 481-492. Bjerregaard H. F. and Nielsen R. (1987) Prostaglandin E~-stimulated glandular ion and water secretion in isolated frog skin (Rena esculenta). J. Membr. Biol. 97, 9-19. Bowman A. S., Dillwith J. W., Madden R. D. and Sauer J. R. (1995a) Regulation of free arachidonic acid levels in isolated salivary glands from the lone star tick: a role for dopamine. Arch. Insect Biochem. Physiol. 29, 309-327. Bowman A. S., Sauer J. R., Zhu K. C. and Dillwith J. W. (1995b) Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum. Insect Biochem. Molec. Biol. 25, 735-741. Bowman A. S, Dillwith J. W. and Sauer J. R. (1996) Tick salivary prostaglandins: presence, origin and significance. Parasitol. Today 12, 388-395. Brown J. A., Gray C. J., Hattersley G. and Robinson J. (1991) Prostaglandins in the kidney, urinary bladder and gills of the rainbow trout and European eel adapted to fresh water and seawater. Gen. Comp. Endocr. 84, 328-335. Coleman R. A., Kennedy 1., Humphrey P. P. A., Bunce K. and Lumley P. (1990) In Comprehensive Medicinal Chemistry. Vol. 3. Membranes and Receptors (edited by Emmett J. C.), pp. 643-714. Pergamon Press, Oxford. Coleman R. A., Grix S. P., Head S. A., Louttit J. B., Mallett A. and Sheldrick R. L. G. (1994) A novel inhibitory prostanoid receptor in piglet saphenous vein. Pros~glandins 47, 151-168. Dalton T. (1977) The effect of prostaglandin E~ on cyclic AMP production in the salivary glands of Calliphora erythrocephala. Experientia 15, 1329-1330. Farndale R. W., Allan L. M. and Martin B. R. (1992) Adenylate cyclase and cAMP. In Signal Transduetion (edited by Milligan G.), pp. 89-98. Oxford University Press, New York. Frazier L. W. and Yorio T. (1992) Eicosanoids: their function in renal epithelia ion transport. Proc. Soc. Exp. Biol. Med. 201, 229-243. Freas W. and Grollman S. (1981) Uptake and binding of prostaglandins in a marine bivalve, Modiolus demissus. J. Exp. Zool. 216, 225233. Harris R. A. and Kaufman W. R. (1984) Neural involvement in the control of salivary gland degeneration in the ixodid tick, Amblyomma hebraeum. J. Exp. Biol. 1119, 281-290. Kerkhove E. V., Pirotte P., Petzel D. H. and Stanley-Samuelson D. W. (1995) Eicosanoid biosynthesis inhibitors modulate basal fluid secretion rates in the Malpighian tubules of the ant, Formica polyctena. J. Insect Physiol. 41, 435-441. MacDonald B. R. (1986) Parathyroid hormone, prostaglandins and bone resorption. Worm Rev. Nutr. Diet. 47, 163-201. McKenzie F. R. (1992) Basic techniques to study G-protein function. In Signal Transduction (edited by Milligan G.), pp. 35-39. Oxford University Press, New York. McSwain J. L., Masaracchia R. A., Essenberg R. C., Tucker J. S. and Sauer J. R. (1992) Amblyomma americanum (L.): protein kinase C-independent fluid secretion by isolated salivary glands. Exp. Parasitol. 74, 324-331. McSwain J. L., Schmidt S. P., Claypool D. M., Esssenberg R. C. and Sauer J. R. (1987) Subcellular location of phosphoproteins in salivary glands of the lone star tick, Amblyomma americanum (L.). Arch. Insect Biochem. Physiol. 5, 29-43. Munson P. J. and Rodbard D. (1980) LIGAND: a versatile computerized approach for characterization of ligand-binding systems. Analyt. Biochem. 1117, 220-239. Needham G. R. and Sauer J. R. (1979) Involvement of calcium and

395

cyclic AMP in controlling ixodid tick salivary fluid secretion. J. Parasitol. 65, 531-542. Negishi M., Sugimoto Y. and Ichikawa A. (1993) Prostanoid receptors and their biological actions. Progr. Lipid Res. 32, 417-434. Patrick C. D. and Hair J. A. (1975) Laboratory rearing procedures and equipment for multihost ticks (Acarina: Ixodidae). J. Med. Entotool. 12, 389-390. Petzel D. H. and Stanley-Samuelson D, W. (1992) Inhibition of eicosanoid biosynthesis modulates basal fluid secretion in the Malpighian tubules of the yellow fever mosquito (Aedes aegypti). J. Insect Physiol. 38, 1-8. Ribeiro J. M. C., Evans P. M., McSwain J. L. and Sauer J. R. (1992) Amblyomma americanum: characterization of prostaglandin E2 and F2,~ by RP-HPLC bioassay and gas chromatography-mass spectrometry. Exp. Parasitol. 74, 112-116. Saintsing D. G. and Dietz T. H. (1983) Modification of sodium transport in freshwater mussels by prostaglandins, cyclic AMP and 5hydroxytryptamine: effect of inhibitors of prostaglandin synthesis. Comp. Biochem. Physiol. 76C, 285-290. Sauer J. R., McSwain J. L., Bowman A. S. and Essenberg R. C. (1995) Tick salivary gland physiology. Annu. Rev. Entomol. 40, 245-267. Schmidt S. P., Essenberg R. C. and Sauer J. R. (1982) Dopamine sensitive adenylate cyclase in the salivary glands of the lone star tick. Comp. Biochem. Physiol. 72, 9-14. Smith W. L. (1989) The eicosanoids and their biochemical mechanisms of action. Biochem. J. 259, 315-324. Sonnenburg W. K., Zhu J. and Smith W. L. (1990) A prostaglandin E2 receptor coupled to a pertussis toxin-sensitive guanine regulatory protein in rabbit cortical collecting tubule cells. J. Biol. Chem. 265, 8479-8483, Soil A. H. (1980) Specific inhibition by prostaglandins E2 and I2 of histamine-stimulated [~4C] aminopyrine accumulation and cyclic adenosine monophosphate generation by isolated canine parietal cells. J. Clin. Invest. 65, 1222-1229. Sugimoto Y., Namba T,, Shigemoto R., Negishi M,, Ichikawa A. and Narumiya S. (1994) Distinct cellular localization of mRNAs for three subtypes of prostaglandin E2 receptor in kidney. Am. J. Physiol. 266, F823-828. Virgolini I., Li S., Sillaber C., Majdic O., Sinzinger H., Lechner K., Bettelheim P. and Valent P. (1992) Characterization of prostaglandin (PG)-binding sites expressed on human basophils. J. Biol. Chem. 267, 12700-12708. Watanabe T., Umegaki K. and Smith W. L. (1986) Association of a solubilized prostaglandin E~ receptor from renal medulla with a pertussis toxin-reactive guanine nucleotide regulatory protein. J. Biol. Chem. 261, 13430-13439. Williams M. and Sills M. A. (1990) Quantitative analysis of ligandreceptor interactions. In Comprehensive Medicinal Chemistry. Vol. 3. Membranes and Receptors (edited by Emmett J. C.), pp. 58-80. Pergamon Press, Oxford. Wollin A., Soil A. H. and Samloff I. M. (1979) Actions of histamine, secretin, and PGEz on cyclic-AMP production by isolated canine fundic mucosal cells. Am. J. Physiol. 237, E437-E443.

Acknowledgements--The authors express their gratitude to D. Forest for help with the tick dissections, and their appreciation to Dr K. M. Kocan for the electron micrograph of tick gland membranes and Mr J. S. Tucker and Ms R. D. Madden for technical help. We thank Drs Robert Barker and Melanie Palmer for critically reviewing this manuscript, which was approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported by NIH grants AI-31460 and AI-26158.