Insect Biochemistry and Molecular Biology 30 2000 1099–1106 www.elsevier.comlocateibmb
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,
a
Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078-3033, USA
b
Department of Zoology, University of Aberdeen, Aberdeen AB24 3TZ, UK
c
Department 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 E
2
PGE
2
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 PGE
2
, as compared with PGF
2 α
or the thromboxane analog U-46619, in accordance with their respective binding affinities for the PGE
2
receptor. Furthermore, the selective PGE
2
EP1 receptor agonist, 17-phenyl trinor PGE
2
, was as effective as PGE
2
in stimulating secretion of anticoagulant protein. Calcium ionophore A-23187 1 to 100
µ M stimulated secretion of anticoagulant
protein in a dose-dependent manner but the voltage-gated Ca
2 +
-channel blocker verapamil 1 to 1000 µ
M and the receptor-mediated Ca
2 +
-entry antagonist, SKF 96365 1 and 10 µ
M, and 5 mM ethylene glycol bis β
-aminoethyl ether-N,NN 9,N9-tetraacetic acid
EGTA had no appreciable effect on inhibiting PGE
2
-stimulated secretion of anticoagulant protein. PGE
2
0.1 µ
M and the non- hydrolyzable analog of guanosine triphosphate GTP, GTP
γ S 10
µ M, directly activated phospholipase C PLC in a membrane-
enriched fraction of the salivary glands after PLC was first incubated with the PGE
2
EP1 receptor antagonist AH-6809, which presumably antagonized endogenous PGE
2
0.3 µ
M in the broken-cell-membrane-enriched fraction. TMB-8, an antagonist of intra- cellular inositol trisphosphate IP
3
receptors, inhibited PGE
2
-stimulated secretion. The results support the hypothesis that PGE
2
stimulates secretion of tick salivary gland protein via a phosphoinositide signaling pathway and mobilization of intracellular Ca
2 +
.
2000 Elsevier Science Ltd. All rights reserved.
Keywords: Tick salivary glands; Exocytosis; Calcium; PGE
2
receptor
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: jrs4864okstate.edu J.R. Sauer.
0965-174800 - see front matter
2000 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 Ca
2 +
or increase or decrease cyclic adenosine monophosphate cAMP
Narumiya, 1995. Although most research to date on
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
