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
3.1. Effects of different prostanoids on releasing anticoagulant protein
Incubation of dispersed salivary glands with 1 nM to 1
µ M PGE
2
significantly stimulated secretion of antico- agulant protein. The selective PGE
2
EP1 receptor agonist 17-phenyl trinor PGE
2
also significantly P ,0.05
stimulated anticoagulant release and was as potent as PGE
2
at low concentrations 10 to 100 nM Fig. 1, strongly suggesting that the exocytosis in tick salivary
glands is via an EP1-like PGE
2
receptor. PGF
2 α
had a stimulatory effect of about 40 of that noted with PGE
2
but only at a high concentration 1 µ
M. Somewhat sur- prisingly, low concentrations of PGF
2 α
1 and 10 nM inhibited anticoagulant release 20 P
,0.05 below that observed in the control tissue Fig. 1. U-46619 stable
thromboxane A
2
agonist was ineffectual at all concen- trations tested 1 nM–100
µ M data not shown.
3.2. Role of extracellular Ca
2 +
in anticoagulant release
Activation of mammalian EP1 receptors leads to an increase in intracellular Ca
2 +
level Narumiya, 1996. An increase in intracellular Ca
2 +
elicited by agonists typi- cally comes from both intracellular andor 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 Ca
2 +
-channel blocker vera- pamil, the receptor-mediated Ca
2 +
-entry inhibitor SKF
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 PGE
2
, n =
7; PGF
2 α
, n =
3; 17-phenyl trinor PGE
2
, n =
4. indicates a significant dif- ference from the control P
,0.05.
Fig. 2. Effect of calcium ionophore A-23187 on anticoagulant release
from dispersed tick salivary glands. Results are expressed as percent- age change
± SEM of anticoagulant activity in secretion of dispersed
salivary glands incubated with the indicated concentration of the drug over the solvent control dimethyl sulfoxide DMSO for 5 min at room
temperature n =
5. indicates a significant difference from the con- trol P
,0.05.
1103 J. Yuan et al. Insect Biochemistry and Molecular Biology 30 2000 1099–1106
96365 Leung et al., 1996; Zimmermann, 1998 and 5 mM EGTA had no inhibitory effect on the 10
27
M PGE
2
-stimulated anticoagulant release from dispersed isolated salivary gland acini during the course of the
5 min experiments Table 1. The inability of Ca
2 +
-influx inhibitors or EGTA to inhibit PGE
2
-stimulated secretion of protein during 5 min incubations is consistent with
previous results showing that PGE
2
stimulates efflux of
45
Ca
2+
from dispersed salivary gland acini but has no effect on stimulating an influx of
45
Ca
2+
Qian et al., 1998.
3.3. PLC activity and its activation by PGE
2
in tick salivary glands
Previous studies indicated that PGE
2
increases IP
3
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 IPminmg 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. 3A–C].
Neither 10
27
M PGE
2
nor 10
25
M GTP γ
S non- hydrolyzable GTP analog stimulated PLC activity in the
membrane fraction Table 2. However, 10
210
M AH- 6809, a selective PGE
2
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 PGE
2
, 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 PGE
2
or GTP γ
S to stimulate PLC activity, we hypothesized high levels of endogenous
PGE
2
in membrane preparations. The amount of endogenous PGE
2
was measured in a similarly prepared plasma-membrane-enriched fraction n
= 3 by RIA, and
found to be 2.3 ±
0.7 ×
10
27
M. Furthermore, 10
27
M
Table 1 Effect of Ca
2 +
channel antagonists and EGTA on PGE
2
-stimulated
a
secretion of anticoagulant protein Antagonist
Concentration µ
M 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
SKF 96365 1
5 211.8
6.9 10
4 +
14.0 17.8
EGTA 5
18 +
8.0 9.6
a
[PGE
2
] =
10
27
M.
PGE
2
and 10
25
M GTP γ
S completely reversed the inhi- bition of PLC activity by AH-6809 Table 2, suggesting
that the increase in salivary gland IP
3
in response to PGE
2
is caused by direct activation of membrane-asso- ciated PLC after PGE
2
binds to its receptor. 3.4. Effect of IP
3
receptor antagonist TMB-8 on PGE
2
-stimulated secretion of salivary gland protein Simultaneous incubation of the IP
3
receptor inhibitor TMB-8 at 10
26
M abolished the stimulatory effect of various concentrations of PGE
2
on anticoagulant release in dispersed acini, supporting the hypothesis that IP
3
and the subsequent intracellular Ca
2 +
mobilization are involved in exocytosis of salivary gland proteins Fig.
4. In the presence of exogenous 10
28
M PGE
2
, various concentrations of TMB-8 10
28
to 10
24
M 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 PGE
2
. Although the dispersed tissue was washed five times
prior to performing the exocytosis assay, the incubation medium still contained 7.7
± 1.6 nM PGE
2
n =
13 as determined by both RIA and GCMS. Without exogen-
ous PGE
2
, TMB-8 10
28
to 10
24
M also decreased the anticoagulant release by 7–23 data not shown, sug-
gesting the existence of some amount of IP
3
in the tissue possibly caused by endogenous PGE
2
released into the incubation medium.
4. Discussion
