1100 J. Yuan et al. Insect Biochemistry and Molecular Biology 30 2000 1099–1106
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 PGE
2
-specific receptor has been identified in the salivary glands of the female lone star tick Amblyomma
americanum, suggesting a functional role for PGE
2
in tick salivary gland physiology Qian et al., 1997. The
PGE
2
receptor’s binding affinities for various prostano- ids are PGE
2
.PGF
2 α
.PGD
2
.U-46619 thromboxane A
2
analog Qian et al., 1997. PGE
2
stimulates release of anticoagulant protein from dispersed salivary gland
acini, suggesting a linkage between PGE
2
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 PGE
2
receptor and the ability of prostanoids to stimulate secretion of anticoagulant pro-
tein. Since the selective PGE
2
EP1 receptor antagonist AH-6809 affects the stimulatory effect of PGE
2
on anti- coagulant release Qian et al., 1998, we tested the effect
of 17-phenyl trinor PGE
2
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 PGE
2
receptor. We also determined whether endogenous PGE
2
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 PGE
2
Qian et al., 1998. PGE
2
does not affect adenylate cyclase activity in membrane preparations of tick salivary glands Qian et al., 1997,
but does increase IP
3
in dispersed salivary gland acini and the efflux of intracellular Ca
2 +
in whole glands Qian et al., 1998. Qian et al. 1998 demonstrated that PGE
2
mobilizes intracellular Ca
2 +
. 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 IP
3
in response to PGE
2
. We sought to determine if PGE
2
directly acti- vates a salivary gland PLC and the relative importance
of intracellular and extracellular sources of Ca
2 +
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-diethylaminooctyl
ester TMB-8, GTP
γ S and PGE
2
antiserum were from Sigma Chemical Company St. Louis, MO, USA. 6-Isopro-
poxy-9-xanthone-2-carboxylic acid AH-6809 and 1- [
β -[3-4-methoxyphenylpropoxy-4-methoxyphenethyl]-
1H-imidazole HCl] SKF 96365 were from Biomol Plymouth Meeting, PA, USA; PGE
2
, PGF
2 α
, U-46619 and 17-phenyl trinor PGE
2
were from Cayman Chemical Ann Arbor, MI, USA. l-
α -[myo-2-
3
HN]-Phosphatid- ylinositol 11 Cimmol and [5,6,8,11,12,14,15-
3
HN]- prostaglandin E
2
200 Cimmol 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 light10 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 mm
2
. 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 Salivary glands were incubated with Medium-199
containing 20 mM MOPS buffer pH 7.0 containing various drugs of differing concentrations treatment or
the same medium plus the solvent used to dissolve the drugs control. All incubations were performed for
1101 J. Yuan et al. Insect Biochemistry and Molecular Biology 30 2000 1099–1106
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 20
µ l 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 50
µ l was incubated with 20
µ l samples in a
96-well plate at room temperature for 30 min. The coagulation assay was initiated by addition of 100
µ l
APTT reagent:20 mM
CaCl
2
1:1, vv
and the
absorbance monitored at 405 nm. Time to initiation of coagulation V
max
, 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 PGE
2
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 PGE
2
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 PGE
2
antiserum for 30 min. A quantity 0.1 ml of buffer containing 5 pg
of
3
H-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 800 µ
l 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 chromatographymass spectrometry Extracted saliva and salivary gland samples were pre-
pared for
gas chromatographymass
spectrometry GCMS 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 20
µ l 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, 0.25
µ m film thick-
ness, HP-5MS capillary column with oven parameters set at 200
° C for 5 min, ramping to 290
° C at 10
° Cmin
and holding for 8 min. Analysis utilized both total ion scans and selective ion monitoring for the identification
and quantification of PGE
2
using HP Chem Station software and comparison with the same parameters for
authentic PGE
2
standard. Varying quantities of authentic PGE
2
were derivatized and used to construct linear regression analysis for the natural samples. The R
2
were typically between 0.94 and 0.99. Authentic PGE
2
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 PGE
2
to check procedural efficiency. Separate derivatization and
analysis of authentic PGE
2
were 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 200 µ
M and [
3
H]-PI 1
µ Ci in 100 mM N-[2-hydroxyethyl]piperazine-N
9-[2- ethanesulfonic acid] HEPES buffer containing 2 mgml
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 100
µ l reaction mixture were: 50 mM
HEPES buffer pH 7.0, 100 mM KCl, 2 mM CaCl
2
, 1 mgml DOC, 100
µ M PI and 10,000 dpm of [
3
H]-PI. In some assays the pH was adjusted from 6.5 to 7.5. The
mixtures were preincubated at 37 °
C for 2 min before adding 20
µ g enzyme preparation to initiate the reaction.
In some assays the amount of enzyme protein was varied from 5 to 30
µ g. The membrane fraction enzyme
source was prepared from six pairs of salivary glands dissected in 20 mM Tris–HCl pH 7.0, 5 mM of ethyl-
ene glycol bis β
-aminoethyl ether-N,NN 9,N9-tetraacetic
acid EGTA, 1 mM of ethylenediamine-tetraacetic acid EDTA and 1 mM Dithiothreitol DTT, and held on
ice for 5 min. The preparation was sonicated with 30 s bursts six times and then centrifuged at 1310g at 4
° C
for 4 min. The supernatant was centrifuged at 100,000g for 30 min each and the pellet resuspended in 200–
300 µ
l of the above buffer to adjust the protein concen- tration to 2.5
µ g
µ l. Typically, the reaction was conduc-
ted for
15 min at
37 °
C, stopped
by 0.5 ml
chloroformmethanolconcentrated HCl
100:100:0.6 and vortexed for 40 s. An additional 200
µ l of 1 M
HCl5 mM EDTA was added and vortexed for 40 s. After centrifugation at 2000g for 5 min at room tempera-
1102 J. Yuan et al. Insect Biochemistry and Molecular Biology 30 2000 1099–1106
ture, a 200 µ
l aliquot of the upper methanolic phase con- taining inositol phosphates was counted for radioactivity
by a Beckman LS 6000SC counter using 10 ml ScintiSafe
Econo 2 scintillation cocktail. PLC activity was
expressed as
nmol of
inositol phosphate
releasedminmg 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
