www.elsevier.com/locate/ibmb
Prostaglandin E
2-stimulated secretion of protein in the salivary
glands of the lone star tick via a phosphoinositide signaling
pathway
Jing Yuan
a, Alan S. Bowman
b, Majd Aljamali
c, Matthew R. Payne
a, James S. Tucker
a,
Jack W. Dillwith
a, Richard C. Essenberg
c, John R. Sauer
a,* aDepartment of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078-3033, USAbDepartment of Zoology, University of Aberdeen, Aberdeen AB24 3TZ, UK
cDepartment of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078-3033, USA
Received 24 May 1999; received in revised form 17 April 2000; accepted 19 April 2000
Abstract
Previous studies identified a prostaglandin E2 (PGE2) receptor in the salivary glands of partially fed female lone star ticks,
Amblyomma americanum (L.). In the present studies, protein secretion from dispersed salivary gland acini was shown to be specific
for PGE2, as compared with PGF2αor the thromboxane analog U-46619, in accordance with their respective binding affinities for
the PGE2 receptor. Furthermore, the selective PGE2 EP1 receptor agonist, 17-phenyl trinor PGE2, was as effective as PGE2in
stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 (1 to 100µM) stimulated secretion of anticoagulant protein in a dose-dependent manner but the voltage-gated Ca2+-channel blocker verapamil (1 to 1000µM) and the receptor-mediated
Ca2+-entry antagonist, SK&F 96365 (1 and 10µM), and 5 mM ethylene glycol bis(β-aminoethyl ether)-N,NN
9,N9-tetraacetic acid (EGTA) had no appreciable effect on inhibiting PGE2-stimulated secretion of anticoagulant protein. PGE2(0.1µM) and the
non-hydrolyzable analog of guanosine triphosphate (GTP), GTPγS (10µM), directly activated phospholipase C (PLC) in a membrane-enriched fraction of the salivary glands after PLC was first incubated with the PGE2 EP1 receptor antagonist AH-6809, which
presumably antagonized endogenous PGE2(0.3µM) in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of
intra-cellular inositol trisphosphate (IP3) receptors, inhibited PGE2-stimulated secretion. The results support the hypothesis that PGE2
stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca2+.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Tick salivary glands; Exocytosis; Calcium; PGE2receptor
1. Introduction
The prolonged attachment of ticks to host animals and the extensive fluid exchange that occurs during feeding contribute to the ixodid tick’s ability to serve as an important vector of medical and veterinary pathogens. The tick’s salivary glands are crucial in its ability to feed successfully (Sauer et al. 1995, 2000). As feeding pro-gresses, the rate of salivary fluid secretion increases gre-atly, enabling the tick to concentrate the bloodmeal by
* Corresponding author. Tel.:+1-405-744-5435; fax:+ 1-405-744-6039.
E-mail address: [email protected] (J.R. Sauer).
0965-1748/00/$ - see front matter2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 0 8 7 - 4
returning excess water and ions to the host. At the same time, numerous bioactive proteins (e.g., anticoagulants, anti-inflammatory and immunosuppressants) and prosta-glandins (PGs) are secreted in the saliva, modulating interactions of the host with the tick (Bowman et al., 1997).
Prostaglandins (PGs) are biologically active lipid mediators in both mammals (Smith, 1992) and insects (Stanley-Samuelson, 1994). In mammalian cells, PGs typically serve as local hormones acting on the same or nearby cells, whose effects are elicited by binding to specific transmembrane receptors coupled to signaling pathways that either mobilize Ca2+ or increase or
tick-derived PGs has focused on the function of PGs in facilitating tick feeding through interactions with host cells (Bowman et al., 1996), a G-protein-linked and PGE2-specific receptor has been identified in the salivary
glands of the female lone star tick (Amblyomma americanum), suggesting a functional role for PGE2 in
tick salivary gland physiology (Qian et al., 1997). The PGE2 receptor’s binding affinities for various
prostano-ids are PGE2.PGF2α.PGD2.U-46619 (thromboxane
A2 analog) (Qian et al., 1997). PGE2 stimulates release
of anticoagulant protein from dispersed salivary gland acini, suggesting a linkage between PGE2 stimulation
and exocytosis of bioactive proteins during tick feeding (Qian et al., 1998). In the present study we compared the effectiveness of various prostanoids in releasing anti-coagulant protein from dispersed salivary gland acini to determine if there is a correlation between prostanoid binding affinities to the PGE2receptor and the ability of
prostanoids to stimulate secretion of anticoagulant pro-tein. Since the selective PGE2 EP1 receptor antagonist
AH-6809 affects the stimulatory effect of PGE2on
anti-coagulant release (Qian et al., 1998), we tested the effect of 17-phenyl trinor PGE2(a selective EP1 agonist;
John-son et al., 1980) in stimulating secretion of anticoagulant protein to further determine if the exocytosis mechanism is via the EP1-like PGE2 receptor. We also determined
whether endogenous PGE2 was present in the dispersed
salivary gland preparation and tissue fraction because of the ability of low concentrations of AH-6809 to inhibit secretion of protein from isolated salivary glands in the absence of exogenous PGE2 (Qian et al., 1998). PGE2
does not affect adenylate cyclase activity in membrane preparations of tick salivary glands (Qian et al., 1997), but does increase IP3 in dispersed salivary gland acini
and the efflux of intracellular Ca2+in whole glands (Qian
et al., 1998). Qian et al. (1998) demonstrated that PGE2
mobilizes intracellular Ca2+. These results suggest the
existence of a signal transduction pathway involving activation of a plasma membrane-associated phospho-lipase C (PLC) and metabolism of phosphatidylinositol 4,5-bisphosphate to diacylglycerol and IP3 in response
to PGE2. We sought to determine if PGE2directly
acti-vates a salivary gland PLC and the relative importance of intracellular and extracellular sources of Ca2+in
reg-ulating secretion of anticoagulant protein.
2. Materials and methods
2.1. Chemicals
Chemicals were obtained from the following sources. Medium-199 (M-0393), lyophilized sheep plasma, acti-vated partial thromboplastin time (APTT) reagent, cal-cium ionophore A-23187, verapamil, phosphatidylinosi-tol (PI), deoxycholate (DOC), Triton X-100,
3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester (TMB-8), GTPγS and PGE2antiserum were from Sigma
Chemical Company (St. Louis, MO, USA). 6-Isopro-poxy-9-xanthone-2-carboxylic acid (AH-6809) and 1-[β -[3-(4-methoxyphenyl)propoxy-4-methoxyphenethyl]-1H-imidazole HCl] (SK&F 96365) were from Biomol (Plymouth Meeting, PA, USA); PGE2, PGF2α, U-46619
and 17-phenyl trinor PGE2were from Cayman Chemical
(Ann Arbor, MI, USA).l-α-[myo-2-3
H(N)]-Phosphatid-ylinositol (11 Ci/mmol) and [5,6,8,11,12,14,15-3
H(N)]-prostaglandin E2 (200 Ci/mmol) were from NEN Life
Science Products (Boston, MA, USA). ScintiSafe Econo 2 scintillation cocktail was from Fisher Scientific (St. Louis, MO, USA). Ethereal diazomethane, methoxy-amine and pyridine were from Aldrich (Milwaukee, WI, USA) and N,O-bis[trimethylsilyl]trifluoroacetamide (BSTFA) was from Pierce (Rockford, IL, USA).
2.2. Tick rearing
Amblyomma americanum (L.) ticks were reared at Oklahoma State University’s Central Tick Rearing Facility, according to the methods of Patrick and Hair (1975). Immature ticks were fed on rabbits and adult ticks on sheep. All unfed ticks were maintained at 27– 28°C and 90% relative humidity under 14 h light/10 h dark photoperiod before infestation of the hosts. Partially fed female A. americanum ticks were used within 4 h of being removed from the host.
2.3. Salivary gland preparation and dispersion
The technique for preparing dispersed salivary gland acini was as described previously (Qian et al., 1998). Briefly, tick salivary glands were dissected out in Medium-199 containing 20 mM morpholinopropanesul-fonic acid (MOPS) buffer (pH 7.0), then one of two intact glands from each tick was put in the control group, while the other was placed in the experimental group. Four glands per group were used in the study. Salivary glands were gently teased apart with fine-tipped forceps. The dimensions of the dispersed tissue were smaller than 0.5 mm2. Dispersion was confirmed by viewing under a
microscope. The viability of the dispersed gland acini and cells assessed by trypan-blue exclusion was esti-mated to be about 60%. Dispersed tissue was transferred to a microcentrifuge tube, and rinsed five times with the same buffer prior to treatment.
2.4. Treatment of salivary glands
5 min at room temperature except as stated otherwise. Dispersed tissue was gently agitated twice during each incubation. After incubation, the tissue was centrifuged for 1 min at 1000g, and two 20µl aliquots of the super-natant (containing salivary secretion products) were removed and tested for anticoagulant content via the sheep plasma coagulation assay. The coagulation assay was performed as described by Zhu et al. (1997) with some modifications. Activated partial thromboplastin time (APTT, intrinsic pathway assay) was used to exam-ine anticoagulant activities (Evatt et al., 1992). Sheep plasma (50µl) was incubated with 20µl samples in a 96-well plate at room temperature for 30 min. The coagulation assay was initiated by addition of 100µl APTT reagent:20 mM CaCl2 (1:1, v/v) and the
absorbance monitored at 405 nm. Time to initiation of coagulation (Vmax), determined by SOFT Max PRO
software (version 1.1) using a Thermo Max plate reader (Molecular Devices, Sunnyvale, CA, USA), was taken as the clotting time. The change in anticoagulant activity was expressed as the percentage of the clotting time dif-ference over the control.
2.5. Tissue extraction and radioimmunoassay (RIA) of PGE2
Prostaglandins were extracted from samples according to the method of Powell (1982) using a 500 mg Sep-Pak C18 cartridge (Waters, Milford, MA, USA). A quantity (0.1 ml) of sample or standard concentrations of PGE2
in 0.01 M sodium-phosphate-buffered saline containing 0.1% bovine serum albumin and 0.1% sodium azide (RIA buffer) were incubated on ice with PGE2antiserum
for 30 min. A quantity (0.1 ml) of buffer containing 5 pg of 3H-PGE
2 (5500–6000 disintegration per minute
(dpm)) was added, and the mixture was incubated on ice for 90 min. Cold dextran (0.1%) coated and activated (1.0%, 100–400 mesh) charcoal suspension (0.2 ml) was added and incubated in ice water for a further 10 min. The tubes were centrifuged at 2000g at 4°C for 15 min, and the radioactivity of 800µl of supernatant measured by liquid scintillation counting. The sensitivity of the assay was 31 pg and unknown samples were diluted with RIA buffer to be in the working range of the standard curve (31–1000 pg).
2.6. Gas chromatography/mass spectrometry
Extracted saliva and salivary gland samples were pre-pared for gas chromatography/mass spectrometry (GC/MS) by derivatization using methods established by Barrow and Taylor (1989) and Ngan and Toofan (1991). The prostaglandins in the samples were sequentially derivatized using ethereal diazomethane, 2% methoxya-mine–HCl in pyridine and BSTFA. Derivatized samples were reconstituted in 20µl of 99% dodecane prior to
splitless injection. Analysis was performed with a Hew-lett-Packard GC model 6890 interfaced to an HP5973 Mass Selective Detector. Separation was performed on a 30 m×0.25 mm internal diameter, 0.25µm film thick-ness, HP-5MS capillary column with oven parameters set at 200°C for 5 min, ramping to 290°C at 10°C/min and holding for 8 min. Analysis utilized both total ion scans and selective ion monitoring for the identification and quantification of PGE2 using HP Chem Station
software and comparison with the same parameters for authentic PGE2standard. Varying quantities of authentic
PGE2 were derivatized and used to construct linear
regression analysis for the natural samples. The R2were
typically between 0.94 and 0.99. Authentic PGE2 was
subjected to the same extraction procedures and resulted in a 98% recovery. Natural samples were also divided and spiked with known amounts of authentic PGE2 to
check procedural efficiency. Separate derivatization and analysis of authentic PGE2were used as a quality control
check of the linear regression accuracy.
2.7. PLC assay
Phospholipase C (PLC, EC 3.1.4.10) activity was measured as described by Perrella et al. (1991) and Rad-allah et al. (1995) with some modifications. Substrates consisted of a mixture of PI (200µM) and [3H]-PI
(1µCi) in 100 mM N-[2-hydroxyethyl]piperazine-N9 -[2-ethanesulfonic acid] (HEPES) buffer containing 2 mg/ml DOC, sonicated three times for 1 min each with 30 s intervals between each sonication period (Fisher Sonic Dismembrator, model 300, 20 kHz, 35 W). The final concentrations in 100µl reaction mixture were: 50 mM HEPES buffer (pH 7.0), 100 mM KCl, 2 mM CaCl2,
1 mg/ml DOC, 100µM PI and 10,000 dpm of [3H]-PI.
tempera-ture, a 200µl aliquot of the upper methanolic phase con-taining inositol phosphates was counted for radioactivity by a Beckman LS 6000SC counter using 10 ml ScintiSafeEcono 2 scintillation cocktail. PLC activity was expressed as nmol of inositol phosphate released/min/mg protein.
2.8. Protein assay
Protein concentration was determined by the method of Bradford (1976) with Bio-Rad protein assay dye using bovine serum albumin as the protein standard.
2.9. Statistical analysis
The results are expressed as mean±standard error of the mean (SEM). The number of replicates is indicated in the figure legends. The differences of means between the control and experimental treatment were tested for significance by Student’s t-test. A P value of,0.05 was considered significant.
3. Results
3.1. Effects of different prostanoids on releasing anticoagulant protein
Incubation of dispersed salivary glands with 1 nM to 1µM PGE2 significantly stimulated secretion of
antico-agulant protein. The selective PGE2EP1 receptor agonist
17-phenyl trinor PGE2 also significantly (P,0.05)
stimulated anticoagulant release and was as potent as PGE2 at low concentrations (10 to 100 nM) (Fig. 1),
strongly suggesting that the exocytosis in tick salivary glands is via an EP1-like PGE2 receptor. PGF2α had a
stimulatory effect of about 40% of that noted with PGE2
but only at a high concentration (1µM). Somewhat sur-prisingly, low concentrations of PGF2α (1 and 10 nM)
inhibited anticoagulant release 20% (P,0.05) below that observed in the control tissue (Fig. 1). U-46619 (stable thromboxane A2 agonist) was ineffectual at all
concen-trations tested (1 nM–100µM) (data not shown).
3.2. Role of extracellular Ca2+ in anticoagulant
release
Activation of mammalian EP1 receptors leads to an increase in intracellular Ca2+level (Narumiya, 1996). An
increase in intracellular Ca2+ elicited by agonists
typi-cally comes from both intracellular and/or extracellular sources (Zimmermann, 1998). The calcium ionophore A-23187 enhanced anticoagulant release from dispersed salivary glands in a dose-dependent manner (Fig. 2). However, the voltage-gated Ca2+
-channel blocker vera-pamil, the receptor-mediated Ca2+
-entry inhibitor SK&F
Fig. 1. Effects of prostanoids on anticoagulant release from dispersed tick salivary glands. Results are expressed as percentage change (±SEM) of anticoagulant activity in secretion of dispersed salivary glands incubated with the indicated concentration of prostanoids over the solvent controls for 5 min at room temperature. For PGE2, n=7;
PGF2α, n=3; 17-phenyl trinor PGE2, n=4. * indicates a significant
dif-ference from the control (P,0.05).
96365 (Leung et al., 1996; Zimmermann, 1998) and 5 mM EGTA had no inhibitory effect on the 1027M
PGE2-stimulated anticoagulant release from dispersed
isolated salivary gland acini during the course of the 5 min experiments (Table 1). The inability of Ca2+-influx
inhibitors or EGTA to inhibit PGE2-stimulated secretion
of protein during 5 min incubations is consistent with previous results showing that PGE2 stimulates efflux of 45
Ca2+
from dispersed salivary gland acini but has no effect on stimulating an influx of 45
Ca2+
(Qian et al., 1998).
3.3. PLC activity and its activation by PGE2 in tick
salivary glands
Previous studies indicated that PGE2 increases IP3 in
intact salivary glands of female lone star ticks (Qian et al., 1998) but it is unknown whether the increase is a consequence of direct activation of phospholipase C via its G-protein-linked receptor or via an indirect pathway after receptor occupation. Phospholipase C activity (0.3±0.04 nmol IP/min/mg protein) was identified in a crude plasma-membrane-enriched fraction of the sali-vary glands with a pH optimum of|7.0, was shown to be linear with time to 15 min, and increased with amount of membrane protein [Fig. 3(A)–(C)].
Neither 1027M PGE
2 nor 102
5M GTPγS
(non-hydrolyzable GTP analog) stimulated PLC activity in the membrane fraction (Table 2). However, 10210M
AH-6809, a selective PGE2 EP1 receptor antagonist,
signifi-cantly (P,0.05) decreased PLC activity in the mem-brane-enriched fraction to 75% of the basal activity. Tick salivary glands contain an uncommonly high amount of endogenous PGE2, some of which may have been
released into the membrane-enriched fraction during tissue preparation. To account for the findings of the ability of AH-6809 to inhibit PLC activity, yet the inability of exogenous PGE2or GTPγS to stimulate PLC
activity, we hypothesized high levels of endogenous PGE2 in membrane preparations. The amount of
endogenous PGE2was measured in a similarly prepared
plasma-membrane-enriched fraction (n=3) by RIA, and found to be 2.3±0.7×1027M. Furthermore, 1027M
Table 1
Effect of Ca2+channel antagonists and EGTA on PGE
2-stimulatedasecretion of anticoagulant protein
Antagonist Concentration (µM) n Percentage change ±SEM
Verapamil 1 4 22.9 6.6
10 8 +8.2 8.2
100 8 +3.4 7.8
1000 8 20.1 8.4
SK&F 96365 1 5 211.8 6.9
10 4 +14.0 17.8
EGTA 5 18 +8.0 9.6
a[PGE
2]=1027M.
PGE2and 1025M GTPγS completely reversed the
inhi-bition of PLC activity by AH-6809 (Table 2), suggesting that the increase in salivary gland IP3 in response to
PGE2 is caused by direct activation of
membrane-asso-ciated PLC after PGE2 binds to its receptor.
3.4. Effect of IP3 receptor antagonist TMB-8 on
PGE2-stimulated secretion of salivary gland protein
Simultaneous incubation of the IP3 receptor inhibitor
TMB-8 at 1026M abolished the stimulatory effect of
various concentrations of PGE2on anticoagulant release
in dispersed acini, supporting the hypothesis that IP3and
the subsequent intracellular Ca2+ mobilization are
involved in exocytosis of salivary gland proteins (Fig. 4). In the presence of exogenous 1028M PGE
2, various
concentrations of TMB-8 (1028 to 1024M) decreased
the secretion of anticoagulant protein by 14–20% (data not shown). As noted, the salivary glands of female lone star ticks contain an unusually large amount of PGE2.
Although the dispersed tissue was washed five times prior to performing the exocytosis assay, the incubation medium still contained 7.7±1.6 nM PGE2 (n=13) as
determined by both RIA and GC/MS. Without exogen-ous PGE2, TMB-8 (102
8
to 1024
M) also decreased the anticoagulant release by 7–23% (data not shown), sug-gesting the existence of some amount of IP3in the tissue
possibly caused by endogenous PGE2 released into the
incubation medium.
4. Discussion
Qian et al. (1997) identified a G-protein-linked, PGE2
-specific receptor in the plasma-membrane fraction of the salivary glands of partially fed female Amblyomma americanum ticks, and determined that the affinities of the receptor for prostanoids were in the rank order of PGE2.PGF2α.PGD2.U-46619. Previously, we found
that exogenous PGE2 stimulates secretion of
anti-Fig. 3. Phospholipase C activity in a plasma membrane-enriched fraction of tick salivary glands as determined by ability to hydrolyze [3H]-inositol phosphate (IP) from [3H]-phosphatidylinositol (PI) as a
function of (A) incubation buffer pH; (B) incubation time; (C) amount of protein in membrane-enriched fraction. Each experiment was perfor-med at least three times with similar results.
coagulant protein from dispersed salivary glands of A. americanum. The rank order of PG effectiveness in sti-mulating release of anticoagulant protein was similar to that of PG binding affinities to the PGE2 receptor found
in a previous study (Qian et al., 1997). The results sug-gest that stimulated exocytosis of bioactive proteins is not a general phenomenon of prostanoids but is PGE2
-specific. However, low concentrations of PGF2α (1 and
Table 2
Effects of PGE2 and GTPγSaon tick salivary gland,
plasma-mem-brane-enriched fraction phospholipase C activity. Enzyme activity is expressed as relative percentage activity over the control (100%). * indicates significant difference from the control (P,0.05, n=3 except AH-6809, n=6)
Treatment PLC activity ±SEM (% of control)
Control 100.0 0.0
PGE2 102.3 3.5
GTPγS 97.6 6.9
AH-6809 76.8* 2.0
AH-6809+PGE2 97.2 7.2
AH-6809+GTPγS 106.5 5.4
a [PGE
2]=1027M; [GTPγs]=1025M; [AH-6809]=10210M.
Fig. 4. Effect of the selective IP3receptor inhibitor TMB-8 (1026M)
on anticoagulant release from dispersed salivary gland acini at different concentrations of PGE2 (solid line) (n=7). Results are expressed as
percentage change (±SEM) of anticoagulant activity on secretion of dispersed salivary glands incubated with the indicated concentration of PGE2over the solvent control for 5 min at room temperature. The
effects of PGE2 on anticoagulant release in the absence of TMB-8
(dashed line) are re-drawn from Fig. 1 for comparison.
10 nM) inhibited release of anticoagulant to about 80% of control tissue. Possible explanations include a PGF2α
-specific receptor that inhibits salivary gland protein secretion or, alternatively, that PGF2α competitively
antagonizes levels of endogenous PGE2 (approximately
7.5 nM in the dispersed acini preparation). It is also worth noting that low levels of selective mammalian PGE2 EP1 receptor antagonist AH-6809 inhibit
antico-agulant release in the absence of exogenous PGE2. Since
absence of exogenous PGE2), the inhibitory effect of low
concentrations of the PGE2EP1 receptor antagonist
AH-6809 suggests that endogenous PGE2may have
contrib-uted to the release. Indeed, despite the five rinses during the preparation of the dispersed acini tissue, we determ-ined the incubation media to contain 7.7±1.6 nM PGE2
(n=13), supporting the above suggestions that our find-ings should take into account levels of endogenous PGE2 present.
A puzzling aspect of the present results and previous studies (Qian et al. 1997, 1998) is the discrepancy between the low dissociation constant (KD) of the PGE2
receptor for PGE2, the low concentration of PGE2
required to stimulate exocytosis but the high concen-tration of PGE2 in the salivary glands (from 0.2 ng/pair
in unfed ticks to 60 ng/pair in partially fed females, unpublished results). The results suggest that there may be PGE2 compartmentation, and different locations for
the PGE2destined to be exported into saliva during tick
feeding and that which binds to the PGE2 receptor
locally. Moreover, it is likely that these high levels of PGE2 are synthesized rapidly in the process of tissue
disruption during acini preparation that could involve the release of the phospholipase A2activity destined for
sali-vary secretion (Bowman et al., 1997) and would not nor-mally be active in the glands.
In mammals, PGE2 can induce diverse and opposite
cellular responses through multiple signal transduction pathways, thus PGE2 receptors are classified into four
subtypes: EP1, EP2, EP3 and EP4 (Coleman et al., 1990; Negishi et al., 1993). EP1 receptors are involved in Ca2+
mobilization, while EP2, EP3 and EP4 receptors have been shown to affect intracellular cAMP levels via stimulation or inhibition of adenylate cyclase (Coleman et al., 1994). PGE2 did not affect adenylate cyclase
activity in tick salivary gland membrane preparations (Qian et al., 1997) but did stimulate the formation of IP3
and mobilization of intracellular Ca2+in dispersed tissue
(Qian et al., 1998). That the PGE2EP1 receptor agonist,
17-phenyl trinor PGE2, was as effective as PGE2
sup-ports the hypothesis that the exocytosis observed is via the PGE2receptor EP1-like subtype. This is also
consist-ent with the previous finding that 17-phenyl trinor PGE2
partially reverses the small inhibition of dopamine-stimulated salivary fluid secretion by PLA2inhibitor
ole-yloxyethyl phosphorylcholine (Qian et al., 1997). A phosphatidylinositol phospholipase C (PLC) activity was identified in the salivary glands of A. amer-icanum and the results strongly implicate its involvement in PGE2-stimulated exocytosis. Earlier studies found a
G-protein-linked, PGE2-specific receptor in the
plasma-membrane fraction of tick salivary glands that increases IP3 and mobilizes intracellular Ca2+ in intact glands,
implying activation of salivary gland PLC (Qian et al., 1997). However, it was uncertain if PGE2activated tick
salivary gland PLC directly or indirectly. In this study,
neither 1027M PGE
2 nor 1025M GTPγS affected the
PLC activity in the broken cell tick salivary gland mem-brane fraction, suggesting that PGE2 may activate PLC
indirectly. However, a low concentration (10210M) of
the selective PGE2EP1 receptor antagonist AH-6809 (in
the absence of exogenous PGE2) suppressed PLC
activity in the membrane preparation to |75% of the control, indicating the existence of endogenous PGE2in
the membrane-enriched fraction. This was verified when 1027
M PGE2 and 102 5
M GTPγS completely reversed the PLC activity inhibited by AH-6809, and when 2.3×1027M PGE
2 was found in a similarly prepared
plasma-membrane-enriched fraction. The results provide strong support for the hypothesis that PGE2directly
acti-vates a membrane-associated PLC via a G-protein in tick salivary glands, leading to an increase in intracellular IP3.
Activation of mammalian EP1 receptors leads to an increase in intracellular Ca2+level (Narumiya, 1996). In
our results, the calcium ionophore A-23187 enhanced anticoagulant release in a dose-dependent manner and was as effective as PGE2, suggesting a role for increased
intracellular calcium in controlling exocytosis of antico-agulant proteins. That Ca2+
mobilized by PGE2 is
important in regulating exocytosis of salivary gland pro-teins is consistent with the observation that regulated exocytosis in most secretory cells is caused by a rise in intracellular Ca2+ during cell stimulation (Edwardson et
al., 1997). The voltage-gated Ca2+-channel blocker
vera-pamil, the receptor-mediated Ca2+-uptake inhibitor SK&
F 96365 and the Ca2+chelator EGTA, however, had no
effect on inhibiting anticoagulant release in the presence of 1027M PGE
2, indicating that the increase in
intra-cellular Ca2+ needed by PGE
2 to stimulate exocytosis
under the present experimental conditions came from intracellular sources via an increase in IP3. In mammals,
the PLC signaling pathway, especially the second mess-enger IP3, is thought to be important in secretory cells
(Putney, 1988). The IP3 receptor inhibitor TMB-8
(1026M) effectively inhibited PGE
2-stimulated
antico-agulant release, further supporting this hypothesis. TMB-8 also significantly decreased the secretion of anticoagu-lant protein in the absence of exogenous PGE2, providing
further evidence for the existence of endogenous PGE2
in the prepared salivary gland tissue. However, Ca2+
mobilization in secretory cells via phosphoinositide sig-naling is complex; typically Ca2+ signals are achieved
by IP3-induced release from intracellular stores and,
depending on experimental conditions, subsequent Ca2+
influx from the extracellular space (Zimmermann, 1998). More detailed studies such as the use of Ca2+ imaging
with Ca2+-fluorescent dyes and other experimental
con-ditions are needed to more fully clarify how Ca2+
mobil-ization is regulated and how subsequent Ca2+ entry is
activated in tick salivary glands during or after PGE2
Many bioactive proteins such as anticoagulants, anti-inflammatory factors and immuosuppression factors are secreted in tick saliva (Bowman et al., 1997). These pro-teins may assist pathogen transmission by ticks. Further-more, membrane-bounded parasites such as spirochetes and rickettsiae may exploit the tick’s exocytotic mech-anisms of transport for transmission to the host, since both spirochetes and rickettsiae are believed to enter sali-vary glands via endocytosis and exit via exocytosis (Munderloh and Kurtti, 1995). Continued studies on the regulation and mechanisms of bioactive protein secretion by tick salivary glands may provide valuable insights into how ixodid ticks remain attached and feeding for long periods and how they transmit pathogens to host animals.
Acknowledgements
We thank Drs Robert Barker and Tom Phillips for critically reviewing this manuscript. This article was approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was sup-ported by NSF Grant IBN 9974299 and the Oklahoma Center for the Advancement of Science and Technology (OCAST), grant number HR-98-057.
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