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Effects of octopamine on fluid secretion by isolated salivary glands of a feeding ixodid tick.

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Archives of Insect Biochemistry and Physiology 2:217-226 (1985)
Effects of Octopamine on Fluid Secretion by
Isolated Salivary Glands of a Feeding
lxodid Tick
Tom Pannabecker and G l e n R. N e e d h a m
Acarology Laboratory, Department of Entomology, Ohio Stafe university, Columbus
Octopamine elicited a dose-related secretory response by salivary glands
isolated from the feeding female tick Amblyomrna americanum. Half-maximal
stimulation occurred at about 60 pM. Phentolarnine (10 p M ) failed to inhibit
the octoparnine-mediated response; however, thioridazine (50 pM) inhibited
both octopamine (1,000 pM) and dopamine-stimulated (0.1 pM) secretion.
Maximal stimulation by dopamine (1.0 pM) showed no further increase in the
rate of secretion after adding octopamine (1,000 or 0.1 PM).Glands responded
t o octoS:.rnine (100 pM) with rates significantly lower than controls following
exposure to amphetamine (1,000 pM). Octopamine receptors do not appear
t o mediate the secretory response, and octopamine may stimulate secretion
by releasing catecholamines from presynaptic neurons. These results support
the hypothesis that dopamine is the natural transmitter mediating fluid
secretion in the feeding tick salivary gland.
Key words: Amblyomma americanum, dopamine, octopamine, salivary gland, tick
Paired salivary glands of the ixodid tick serve as osmoregulatory organs
during feeding by secreting excess ions and water from the bloodmeal back
into the host [l-41.In addition to this role, the salivary glands of most ixodids
secrete attachment cement, and all ixodids secrete various lesion maintenance factors which allow them to feed on the host for days or weeks [5-71.
*Abbreviations: central nervous system = CNS; dopamine = DA; octopamine = OCT;
percent of the maximum secretory rate of period 1 = % m a ; phentolamine = PHE; thioridazine = THI.
Tom Pannabecker’s present address i s the Department of Zoology, University of Toronto, 25
Harbord St., Toronto, Ontario, M5S IAI, Canada.
Received August 15,1984; accepted November 19,1984.
Address reprint requests to Dr. Glen Needharn, Acarology Laboratory, Department of Entomology, Ohio State University, Columbus, OH 43210.
0 1985 Alan R. Liss, Inc.
Pannabecker and Needham
In nonfeeding ticks the secretory fluid participates in water vapor uptake
although neuronal control of glands in this phase remains virtually unstudied.
Certainly the neuronal control of these numerous secretory processes is
far from being understood, but it is known that dopamine plays a vital
regulatory role 18-16]. A dopamine-sensitive adenylate cyclase has been
identified in the salivary gland of Arnblyomma americanum [14],suggesting
that a specific dopamine receptor is present [13]. In view of the importance
of octopamine in invertebrate nervous systems [lq,its ability to stimulate
fluid secretion in isolated tick salivary glands [10,18], and its presence in both
the brain and salivary glands of the tick (P.D. Evans, personal communication), it is of interest to determine if octopamine may play a role in regulating
tick salivary gland secretory activity during feeding.
It has previously been hypothesized that octopamine receptors are not
present in the gland epithelium because of the relatively high threshold
concentration required for stimulating fluid secretion by isolated glands and
due to the lack of a potent effect by two formamidine compounds which are
reported to be octopamine mimics [18]. Schmidt et a1 [13] found that adenylate cyclase activity did not increase following the exposure of salivary gland
homogenates to 1,000 p M octopamine. This study addresses the question of
how octopamine stimulates fluid secretion in an apparent dopamine-controlled system. Is its action related to an interaction with dopamine receptors?
In this investigation we determined 1)the secretory dose-response curve for
octopamine, 2) the effects of an a-adrenergic and dopaminergic antagonist
on octopamine- and dopamine-induced secretory rates, and 3) the effects of
a maximally effective dopamine concentration with several octopamine doses.
Salivary Gland Assay
The ixodid ticks Amblyumma americanurn (L.) (obtained from the Ohio State
University Acarology Laboratory) were held in glass desiccators over saturated KCl (85% relative humidity) between bloodmeals. Immatures were fed
on hens, and adults were fed on rabbits or sheep. Salivary glands were
dissected from partially fed or fully engorged females (detached less than 6
h), and fluid secretion was monitored as described by Needham and Pannabecker [18]. The gland was alternately bathed in a droplet of saline containing
the drug(s) for 5 min followed by incubation in saline alone for 3 min.
Secreted fluid was collected during the 5-min interval from the main duct
which was secured in liquid paraffin. The collected fluid was placed into
paraffin and its diameter measured to compute its volume.
The support medium consisted of tissue culture medium 199 (Flow Labs,
Rockville, Md) with L-glutamine and Hank’s balanced salts, but without
sodium bicarbonate, and modified according to Kaufman [9] and Needham
and Sauer [19]. The saline was oxygenated with 100% 0 2 for 30 min prior to
use and the pH was adjusted to 7.0 f 0.1 with 2 M NaOH. Osmolarity of
this modified medium 199 was approximately 320 mOsm.
Effects of Octopamine in the Tick Salivary Gland
D,L-dopamine-HC1 and D,L-octopamine-HC1 were purchased from Sigma
Chemical Company, (St Louis, MO). Thioridazine-HC1 was provided by
Sandoz Inc, (East Hanover, NJ), and phentolamine-HCl was a gift from CibaGeigy Corp, (Summitt, NJ). D,L-Amphetamine-sulfate was obtained from
Sumner Chemical Company, New York. All drugs were dissolved directly
into medium 199 and prepared immediately before each experiment.
Experimental Procedure
Three periods of gland exposure to either octopamine or dopamine with
saline washout intervals between each period (Figs. 1,2) served as controls
for the antagonism experiments. The maximum secretory rates achieved
within each of these periods (0-29 min, period 1; 88-125 min, period 2; 152181 min, period 3) were recorded. The greatest rate was always observed in
Fig. 1. Protocol for determining stimulatory effects of dopamine. This experiment served as
a control for tests of potential antagonists. The mean
S.E. is shown (n = 6).
Time (min)
1000 vM
1000 vM
I I I I I Lo
1000 vM
Fig. 2. Protocol for determining stimulatory effects of octopamine. This experiment served
as a control for tests of potential antagonists. The mean f S.E. is shown (n = 7).
Pannabecker and Needham
period 1and was followed by a slight decay during periods 2 and 3. Therefore, the maximum rates of periods 2 and 3 were each evaluated as a OO/ max*
for each gland tested. Inhibitors were tested by exposing the gland to the
potential antagonist alone for 29 min prior to and then during the second
period with the agonist (octopamine or dopamine). Inhibition by the test
drug was indicated if the YO max of the combined agonist and antagonist in
period 2 was less than the YO max of the agonist in the control period 2.
Irreversible or long-term inhibition was indicated when the OO/ max of the
agonist following washout of the test drug in period 3 was less than the
response of the agonist in the control period 3.
The additivity tests were different from above in that the first saline
washout was 32 min rather than 59 min and was followed by simultaneous
gland exposure to octopamine and dopamine (period 2). The procedure for
additivity controls was the same; however, octopamine was not included in
the bathing medium. An additive effect was indicated if the YO max of
dopamine plus octopamine during the second period was greater than the YO
max seen in period 2 of the corresponding control. The significance of the
differences between the treated glands and the controls was determined
using Student’s t-test for each response period with P < 0.05 considered
Octopamine and Dopamine as Agonists
Octopamine stimulated secretion in a dose-dependent manner (Fig. 3). By
extrapolation, the threshold concentration was 10 pM, and 100 pM octopamine stimulated a maximal response. The half-maximal rate was estimated
to occur at 60 pM. Dopamine also stimulated a dose-related secretory response (Fig. 3).
Effects of an a-Adrenergic Antagonist
Phentolamine (10 pM) had no inhibitory effect on the rate of fluid secretion
elicited by octopamine (100 pM), and following the washout period octopamine stimulated a secretory rate that was equal to that of controls (Table 1).
Effects of a Dopamine Antagonist
Thioridazine (50 pM) significantly inhibited 0.1 pM dopamine-stimulated
secretion (Table 1).This antagonism was shown to be irreversible following
the washout period. In two trials it failed to significantly inhibit 1.0 pM
dopamine-stimulated secretion although an apparent reduction was seen.
Following the washout period, these glands were unable to sustain normal
secretion (period 3). Thioridazine at 50 pM significantly inhibited octopamine-stimulated (1,000 pM) secretion. This response was irreversible, as
significant inhibition was apparent following the washout period. Thioridazine at 1.0 pM was an ineffective antagonist of octopamine-stimulated (1,000
pM) secretion (period 2), although following the 35-min washout period the
rates were significantly lower than those of controls (period 3). The molar
Effects of Octopamine in the lick Salivary Gland
6 +4
Agonist Concentration
Fig. 3. Dose-related secretory response for octopamine and dopamine. Rates shown are the
maximum responses achieved during a 29- to 37-min exposure to the agonist (period 1). The
mean f S.E. is shown. The number of experiments is indicated. Open circles = dopamine;
solid circles = octopamine.
TABLE 1. Effects of Receptor Antagonists on Dopamine- and Octopamine-Stimulated
Period 2
(PM) Test (n)
29 f 4(2)
2 k 0 (6)
Period 3
(% max f S.E.)
(YOmax k S.E.)
1k 0
30 k 6
P < 0.05
k 13(6)
P < 0.05
57 k 14
P < 0.05
36 i lO(7)
P < 0.05
22 k 9
P < 0.05
Control (n)
46 f 7(4)
k 4 (7)
39 f 10 (4)
k lO(7)
81 f 19(6)
64 f 14(6)
53 f 17 30 f 5
Glands were exposed to OCT or DA alone (control) or to the agonist inhibitor (test). For an
explanation of the protocol see Methods. NSD = test not significantly different from control,
P < 0.05.
Pannabecker and Needham
ratio required to show inhibition was 50011 for thioridazine/dopamine and I/
20 for thioridazineloctopamine.
Effects of Combinations of Octopamine and Dopamine
These experiments were designed to determine if octopamine and dopamine could be acting on the same site. When octopamine (1,000 pM or 0.1
pM) was combined with a maximally effective concentration of dopamine
(1.0 pM) [20], the response was slightly elevated for the higher octopamine
dose, but it was not significantly different from that produced with dopamine
alone (Table 2). Following the washout period, dopamine (1.0 pM) elicited an
amplified rate of secretion by glands previously exposed to 1,000 pM octopamine + 1.0 pM dopamine (78 8% max compared to 51 f 6% for the
control, period 3). Glands exposed to 0.1 pM octopamine 1.0 pM dopamine
showed no change from controls.
Amphetamine- and Octopamine-Stimulated Secretion
A potential mechanism for octopamine stimulation could be a presynaptic
action on neurons which results in the release of endogenous transmitter. To
test for this possibility glands were continuously exposed to amphetamine
until the secretory response subsided, suggesting that a particular pool of
endogenous transmitter had been released.
During a 105-min incubation period in amphetamine (1,000 pM), the secretory response rose to a mean maximum rate of 35 f 11nllmin (n = 4)before
decaying to 3.0 k 0.5 nllmin. Subsequent exposure to 100 pM octopamine
(16-min incubation period) resulted in a mean maximum secretory rate of 14
f 10 nllmin. In contrast, glands treated only with octopamine throughout
the same period secreted at a final mean maximum rate of 88 f 24 nllmin.
15 nllmin after a further 61-min incubation. The
This rate decayed to 49
protocol for glands treated only with octopamine followed that shown in
Figure 2 (periods 1 and 2 only). This response was significantly higher than
the response of the amphetamine-treated glands (P < 0.05).
TABLE 2. The Influence of Octopamine on the Secretory Rate Elicited by a Maximally
Effective Concentration of Dopamine
(pM) Test(n)
Period 2
(% max k S.E.)
Control (n) Significance Test
111 f 19(7) 80 f 7(7) NSD
0.1 88
6 (7)
Period 3
(% max k S.E.)
80 k 7 (7) NSD
78 f 8
51 & 6
P < 0.05
56 k 10
51 k 6
Glands were exposed to DA alone (control) or to dopamine + octopamine (test). For an
explanation of the protocol see Methods. NSD = test not significantly different from control,
P < 0.05.
Effects of Octopamine in the Tick Salivary Gland
At the outset we considered several possible explanations for the ability of
octopamine to stimulate in vitro fluid secretion. These included 1)the presence of octopamine receptors which would be linked to the control of fluid
secretion by the rapidly feeding tick, 2) cross reactivity between catecholamine and octopamine such that octopamine interacts directly with dopamine receptors, and 3) the possibility that octopamine may be effecting
secretion through the dopamine-controlled system but without interacting
directly with dopamine receptors.
The first idea seems unlikely because of the inability of the formamidines
to mimic octopamine in this preparation [18], the high threshold concentration for octopamine, the lack of an antagonistic effect by phentolamine on
octopamine-stimulated secretion, and the inability of octopamine to stimulate
adenylate cyclase in tick gland homogenates [13]. The threshold concentration for stimulation by octopamine was 3 orders of magnitude greater than
for dopamine [20], and 2-4 orders of magnitude greater than the threshold
concentration for octopamine receptors of the locust [21-231. The physiological activity associated with octopamine receptors in the locust neuromuscular
preparation [21] and the locust corpus cardiacum [24] is inhibited by phentolamine at micromolar concentrations. Phentolamine (10 pM) at one-tenth
the agonist concentration clearly did not reduce octopamine-stimulated secretion in the tick salivary gland. Phentolamine may also block dopamine
receptors in vertebrates [25,26] and in invertebrates [27-291. However, in the
tick salivary gland the dopamine-sensitive (2.0 pM) adenylate cyclase is only
slightly inhibited by phentolamine (50 pM) [13]. Dopamine-mediated secretion (1.0 p M ) was unaffected by 100 pM phentolamine but was reversibly
inhibited by 85% at 1,000 p M in another tick species [lo].
A potential mechanism for the octopamine-mediated response may occur
through effects on the dopamine system. Cross reactivity exists between
catecholamine and octopamine receptors in the thoracic ganglion of Periplunetu urnericunu [30,31]. In this tissue the stirnulatory effects of noradrenaline
on adenylate cyclase activity were thought to result from partial activation of
receptor sites for octopamine, dopamine, and possibly serotonin. Noradrenaline elevates dopamine-sensitive adenylate cyclase activity in the tick gland,
In tick saliprobably through interactions with the dopamine receptor [B].
vary gland homogenates octopamine failed to elevate adenylate cyclase above
basal levels [13], and it therefore seems unlikely that octopamine interacts
directly with dopamine receptors.
The effect of octopamine nevertheless resembles that of dopamine-mediated secretion. The phenothiazine drug thioridazine, which is an effective
inhibitor of the dopamine-sensitive adenylate cyclase in the tick salivary
gland [13], inhibits both dopamine- and octopamine-stimulated secretions.
The effect of thioridazine in identified octopamine systems has not been
reported although the related compound chlorpromazine is a poor antagonist
of octopamine receptors in the locust [32] (Pannabecker and Orchard, unpublished observations). Therefore both dopamine- and octopamine-stimulated
secretion appear to involve a common, perhaps dopaminergic, receptor site.
Pannabecker and Needham
The secretory response to a combination of a maximally effective concentration of dopamine with octopamine is not significantly different from the
response to dopamine alone, again suggesting that the response to the two
compounds may involve a similar site. However, if octopamine acts directly
on dopamine receptors, a reduction in the secretory rate by competition
might be anticipated when the gland is exposed to the two drugs simultaneously. The failure of octopamine to inhibit the dopamine-stimulated secretory
response is consistent with the idea that octopamine is a poor competitor for
or does not interact directly with dopamine receptors.
Amphetamine, octopamine, and related amines can induce the release of
dopamine from nerve terminals in the rat CNS 133,341, and endogenous
transmitter released by octopamine could account for octopamine-stimulated
secretion in the tick gland. This idea is supported by the reduction in octopamine-stimulated secretion following depletion of transmitter from the
nerves by amphetamine treatment and by the inability of octopamine (1,000
pM, 10 pM) to elevate adenylate cyclase activity above basal levels in salivary
gland homogenates from the tick A . arnericunurn [13]. It seems likely that the
basal adenylate cyclase activity results from endogenous catecholamine in
the salivary gland nerves [13], and gland homogenization might prevent
adenylate cyclase activation (above basal levels) by exogenous octopamine if
transmitter release is the principal mode of action. The slight elevation in
secretory activity of glands exposed to 1pM dopamine + 1,000 ,uM octopamine, following washout (Table 2, period 3), could result from elevated
endogenous dopamine levels which remain high after octopamine removal.
Based on this evidence octopamine stimulates secretion through interactions with a dopaminergic system rather than with octopamine receptors.
These interactions could involve a dopamine uptake mechanism which inactivates endogenous dopamine. Uptake mechanisms are well documented in
vertebrates [35];however, little has been published on invertebrate uptake
mechanisms. One exception is octopamine uptake in the cockroach ventral
nerve cord [36], and it is significant that dopamine is a potent competitor of
both the Na+-sensitive and -insensitive uptake components of this mechanism. Thus dopamine might act as a substrate for this process. Perhaps
octopamine-stimulated secretion in the tick salivary gland operates in a
similar fashion whereby exogenous octopamine accumulates in dopaminergic nerve terminals and subsequently facilitates release of dopamine which
acts upon specific dopamine receptors to elicit fluid secretion.
In summary, the results presented here provide further evidence for specific dopamine receptors which mediate fluid secretion in the tick salivary
gland. The presynaptic action of octopamine may facilitate the release of
dopamine from nerve terminals that lie in close association with the salivary
gland epithelium.
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octopamine, effect, tick, ixodid, feeding, isolated, gland, salivary, secretion, fluid
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