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Octopamine enhances phagocytosis in cockroach hemocytesInvolvement of inositol trisphosphate.

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Archives of insect Biochemistry and Physiology 26:249-261 (1 994)
Octopamine Enhances Phagocytosis in
Cockroach Hemocytes: Involvement of
lnositol Trisphosphate
Danica Baines and Roger G.H. D o w n e r
Department of Biology, Universityof Waterloo, Waterloo,Ontario, Canada
Octopamine and 5-hydroxytryptamine (5-HT) were previously shown to affect
phagocytosis in cockroach hemocytes through unidentified receptor-mediated
events. In the present study, we examined the ability of 5-HT and octopamine to
enhance inositol trisphosphate (IP3) production using hemocyte membranes of the
American cockroach, feriplaneta americana. Octopamine enhanced IP3 production with a maximal peak at 100 nM. Similarly, 5-HT enhanced IP3 production
with a maximal effect at 10 nM. The effects of 5-HT and octopamine are not
additive, suggesting that both are working through the same receptor. Phentolamine, a general octopamine antagonist, blocked the effects of octopamine and
5-HT,,while a mammalian 5-HT2 antagonist that blocks 5-HT-sensitive receptors
in insect peripheral tissue, ketanserin, did not. A pharmacological profile indicates
that the receptor is similar to an octopaminel-type.
Octopamine at 1 pM increased phagocytosis in cockroach hemocytes exposed
to Staphylococcus aureus in vitro, and this effect was mimicked by IP3 (1 0 pM).
The octopamine-treated hemocytes were shown to increase IP3 production in the
latter stage of phagocytosis.
Adult cockroaches exposed to an LD50 dose of S. aureus in conjunction with
either 0.1 m M octopamine or the octopaminel agonist, clonidine, had higher
survival rates compared to saline-treated cockroaches. Correspondingly, the octopaminel antagonist, chlorpromazine, partially blocked the octopamine-mediated increase in cockroach survival. o 1994 WiIey-Liss. Inc.
Key words: amine,
iP3,
insect defense, agonist, antagonist
INTRODUCTION
Hemocytes passively moving in the insect hemocoel encounter bacteria and
respond to extracellular signals that facilitate recognition and attachment to
Acknowledgments: This study was supported by the Natural Sciences and Engineering Research
Council of Canada.
Received April 25,1992; accepted September 9, 1993.
Danica Baines is now at Forest Pest Management Institute, Forestry Canada, 1219 Queen Street East,
Sault Ste. Marie, Ontario, Canada P6A 5M7. Address reprint requests there.
0 1994 Wiley-Liss, inc.
250
Baines and Downer
bacteria. Bacterial charge, hydrophobicity, and lipopolysaccharides have been
implicated as agents that affect the antibacterial activities of insect hemocytes
[ll. Attachment of a hemocyte to a bacterium initiates pseudopod extension and
subsequent engulfment of the bacterium in a phagosome 121. From amoeba to
mammalian hemocytes, the initiation of pseudopod extension requires a localized second messenger signal initiated by the binding of a bacterium to a
membrane receptor [31. Several second messengers including CAMP,IP3*, and
diacylglycerol, modulate the reorganization of actin filaments in the cytosol
adjacent to the receptor-bacterium complex that facilitates pseudopod extension. Actin filaments and microtubules have been identified in insect hemocytes
[2,4,5], and it is likely that second messenger systems in these cells coordinate
pseudopod extension and engulfment of bacteria through similar mechanisms.
Amines such as adrenaline and noradrenaline, enhance the activity of mammalian phagocytic cells through an apparent increase in sensitivity to bacterial
factors which facilitate recognition of an infection [6,71. One of the immediate
effects of these amines is the elevation of intracellular calcium [8]. A similar rise
in Ca++occurs in insect hemocytes exposed to the amine, octopamine [91. In
addition, both 5-HT- and octopamine-sensitive receptors were identified as
modulators of the phagocytic activities of cockroach hemocytes in vitro and in
vivo [lo]. In this study, the 5-HT effect was blocked by ketanserin, while the
octopamine effect was blocked by phentolamine. The second messenger, IP3,
regulates the level of intracellular calcium 1111;thus, octopamine or 5-HT could
mediate a change in phagocytosis through an increase in IP3 production.
We propose to utilize the differential sensitivity of the 5-HT and octopamine
receptors to their antagonists 1101 as a method for distinguishing if these same
receptor(s) are linked to enhancing IP3 production in cockroach hemocytes
during phagocytosis. The present study demonstrates that 5-HT and octopamine enhance IP3 production in cockroach hemocytes, but the effect is blocked
by phentolamine and not by ketanserin. This suggests that the octopamine-sensitive receptor previously identified as affecting phagocytosis mediates its effect
through an elevation in IP3 production. Further, a pharmacological profile
establishes the receptor as octopaminel-like.Evidence is provided that suggests
octopamine enhances IP3 production during the latter phase of phagocytosis
when maximal uptake of bacteria occurs.
MATERIALS AND METHODS
Insects
Adult, female cockroaches (Periplaneta ameuicana)were obtained from a labodog chow, sugar, and water.
ratory colony maintained at 28°C and fed PurinaTM
The procedure for collecting hemocytes and cell-free hemolymph from the
cockroaches was described previously [lo].
* Abbreviations used: DCDM = dernethylchlordimeform; DMSO = dimethylsulfoxide; IP3 = inositol
trisphosphate; PBS = phosphate buffered saline; 5-HT = 5- hydroxytryptamine.
Role of an Octoparnine Receptor in Hemocytes
251
IPSAssay-Hemocyte Membranes
The hemocytes of 1.5 cockroaches or approximately 100 pg of protein were
used for each treatment in an assay. After washing, the hemocytes were resuspended in Tris-acetate buffer (10mM, pH 7) containing 1mM dithiothreitol and
sonicated on ice. The resulting membrane preparation was centrifuged at
15,OOOg for 15min at 4°C. The pellet was then resuspended in 1ml of Tris-acetate
+ DTT buffer. My0[2-~H]inositol(10-20 Ci/mmol, 250 pCi in 3 pl) was then
added to the hemocyte membrane suspension and incubated for 2 h at 28°C
with gentle shaking. The sample was placed on ice and resuspended by gently
pipetting the solution.An aliquot (100 pl) of this tissue preparationwas then added
to a tube containing 40 pl of assay cocktail (75 mM Tris-acetate, 0.1 mM GTP, 20
mM ATP, and 30 mM magnesium acetate, pH 7). The samples were shaken
vigorously before a 50 pl aliquot of a neurotransmitter in water, or water alone,
was added. The mixture was shaken and incubated at 30°C for 1min, which was
predetermined as the optimal time period for IP3 production. For the antagonist
studies, the antagonist was added to the membrane preparations 5 min before the
addition of the agonist. The reaction was terminated by the addition of 500 pl of
acidified chloroform/methanol (k2, 5M HC1) and the samples vortexed. To this,
chloroform (500 yl) and water (500 pl) were added and the solutions centrifuged
at 4,000 rpm (HS-4 rotor) for 5 min at 4°C. A sample (400 p1) was removed and
neutralized with 25 pl of 2.5 mM KOH. A 400 p.l sample was assayed for IP3 by
anion exchange chromatographyusing 1ml of Dowex-1 resin (formateform).The
column was sequential1 eluted with the following solutions: 1) 25 ml water
(unincorporatedmy0[2- Hlinositol); 2) 5 ml of 5 mM sodium tetraborate and 60
mM sodium formate (glycerophosphoinositol);3) 7 ml of 100 mM formic acid and
200 mMammonium formate (inositol-l-phosphate);4)7 ml of 100 mM formic acid
and 400 mM ammonium formate (inositol-l,4bisphosphate);and 5) 7 ml of 100
mMformic acid and 800 mMammonium formate (inositol-l,4,5-trisphosphate).
A
3.5 ml aliquot of each fraction was added to 8 ml of EcolumeTM
and counted for 5
min in a Beckman LS 1701scintillationcounter.
9
IP3 Production in a Phagocytosis Assay In Vitro
After washing the cells with EDTA-citrate buffer (93 mM sodium chloride,
100 mM glucose,30 mM trisodium citrate, 26 mM citric acid, and 10 mM EDTA
at pH 5),the hemocytes were resuspended in PBS (0.2 M, pH 7) containing a 10
p1 aliquot of my0[2-~H]inositolper 1ml of PBS. The hemocytes were incubated
for 2 h at 28"C, and then 10% cell-free hemolymph [see 101 was added. The
hemocytes were gently resuspended and 100 p1 aliquots or lo6 cells were added
to fresh tubes. An aliquot (20 pl) of octopamine in PBS or PBS alone was then
added to each tube and incubated for 5 min prior to the addition of 100 y1 of
Staphylococcus aweus (lo7 cells, ATCC 6538P). The hemocyte:bacteria ratio was
1:10. The samples were incubated at 28°C for 1h and phagocytosis terminated
at different time periods using the procedure described in the IP3 assay.
Phagocytosis Assay-Stained Bacteria In Vitro
The method used was described previously [lo]. After collecting and washing the hemocytes, they were resuspended in PBS with 10% cell-free hemo-
252
Baines and Downer
lymph. An aliquot (300 pl) of hemocytes (lo4 cells) was added to each of a series
of wells and allowed to stabilize for 30 min at 30°C. Aliquots (30 pl) of either IP3
in 1%DMSO, IP3 alone, inositol in 1%DMSO, or 1%DMSO were added to
different samples and incubated for 5 min at 28°C. Crystal violet-stained S.
aweus (100 pl, l o 5 hemocytes) was then added and the mixture gently shaken
for 1 h at 28°C. The process was terminated by the addition of 50% methanol,
and after several washes with PBS, the hemocytes were assessed for phagocytosis using a compound microscope set at 800x magnification. The data are
expressed as a phagocytic index which is an average of the number of phagocytic cells taking up bacteria per total number of cells observed in an experiment. A minimum of 50 cells were assessed per replicate.
Survival of Cockroaches
The procedure used was described previously 1101. After removing female
cockroaches from the colony and allowing them to sit undisturbed for 2 h, each
insect was injected with 40 pl of S. aureus (108,5cells)resuspended in an agonist
and/or antagonist solution to give a final concentration of 1 mM. The cockroaches were left for 24 h and then assessed for survival. Ten insects were used
in each replicate.
Chemicals
Octopamine, 5-HT, phentolamine, tolazoline, naphazoline, chlorpromazine,
yohimbine, metoclopramide, clonidine, synephrine, DCDM, and buffer salts
were obtained from Sigma Chemical Company (St. Louis, MO). The myo-123Hlinositolwas obtained from Amersham Canada Ltd., Ontario. The Dowex-1
resin was obtained from Bio-Rad (Mississauga, Ontario). EcolumeO was obtained from ICN Biomedicals Inc. (Mississauga,Ontario).
Statistical Analysis
A two-way analysis of variance (ANOVA)with the treatments blocked over
tissue preparations was performed on all data except the cockroach survival
assays. The means were separated with the Neuman-Keuls (SNK) test [121.
Results for IP3 production are presented as mean effects with standard errors
and the percent change relative to control values. The cockroach survival assays
were analyzed using a G-statistic and the means separated using the simultaneous test procedure [121.
RESULTS
IP3 Assays
Membrane preparation for the phospholipase C activity was variable in
different batches of replicate preparations and resulted in variable basal levels
of IP3 (P = 0.05). However, by using the same batch of membrane preparations
for control and experimental studies, the effects [121 of the test chemical on IP3
production can be reliably determined. We were unable to eliminate this
variation among different replicates even after optimization for protein, incubation time, and incubation medium (unpublished data). It is quite possible that
the hemocytes collected from groups of cockroaches could have different pro-
Role of an Octopamine Receptor in Hemocytes
8500
-
7500
-
6500
-
5500
-
4500
I
5500
'
253
oct
1
I
I
I
0
-9
I
I
0
-10
I
I
I
I
-0
-1
-6
-5
I
1
I
I
I
-9
-8
-7
-6
-S
Log Dose (M)
Fig. 1. Effect of varying concentrations of octoparnine (Oct) and 5-HT on the production of IP3 in
hemocyte membrane preparations of the American cockroach ( n = 7, mean k S.E.).
portions of cell types [131. Since the plasmatocyte is the only phagocytic cell in
the hemocyte population, the variation may be a reflection of the proportion of
these cells present in a membrane preparation.
The effects of varying concentrations of octopamine and 5-HT on the production of IPS in hemocyte membranes of the American cockroach are shown in
Figure 1. Octopamine increases the production of IP3 in a dose-dependent
manner. The threshold dose for this effect is between 1 nM and 10 nM with a
maximal effect at 100 nM. Increasing the concentration above 100 nM results in
a decline in IP3 production; at 10 pM, the effect is significantly higher than
control values ( P = 0.05). The production of IP3 was also increased by 5-HT in
a dose-dependent manner. The threshold dose for this effect is between 0.1 nM
and 1nM, with a maximal effect at 10 nM. Adding more than 10 nM 5-HT caused
a gradual decline in IP3production to control levels. At their maximal response,
octopamine and 5-HT increased IP3 production above the controls by 1,393
254
Baines and Downer
TABLE 1.Additive Effect of 5-HT and Octopamine on IP3 Production by Hemocyte
Membrane Preparations of Periplaneta americana
Treatment
Saline
Octopamine (100 nM)
5-I-IT (10 nM)
Octopamine + 5-€IT
IP3
(DPM/107 cells)
3,044 k 158
3,950 _+ 306"
3,795 _+ 241"
3,938 f 235"
Increase relative to
controla (%)
30
25
29
ahcrease % = (treatment-saline/saline) x 100.
"Significant increase above control values at P = 0.05 ANOVA, SNK test (n = 4,k S.E.).
DPM and 1,022 DPM, respectively. Thus, octopamine produced the largest
increase in IP3 production in hemocyte membrane preparations, but at approximately a tenfold greater concentration than 5-HT.
The effects of 5-HT and octopamine were not additive (Table I), suggesting
that these amines are competing for the same receptor site. Since the two amines
did not greatly differ in their ability to enhance IP3 production, albeit at different
concentrations, it is not apparent which type of receptor is present.
Antagonist Studies
To classify the receptor linked to the phosphoinositide signalling system,
phentolamine, an antagonist of all octopamine receptors identified to date in
insects [141, and ketanserin, an antagonist of all 5-HT receptors identified in
insect peripheral tissues [15-181, were tested for their ability to block the
octopamine and 5-HT-mediated elevation of IPS. Phentolamine blocked the
effect of 5-HT (130%) and octopamine (134%) on IP3 production, while ketanserin did not block either the 5-HT (0%) or octopamine (2%) effect (Table 2).
Phentolamine acts as a partial agonist of the receptor linked to IP3 production
in hemocyte membranes but still effectively blocked the octopamine- and
5-HT-mediated increase in IP3 production.
TABLE 2. Effect of Phentolamine and Ketanserin on the Octopamine- and 5-HT-Mediated
Increase in IP3 Production in Hemocyte Membrane Preparations of Peviplaneta americana
Treatmenta
Saline
5-HT (10 nM)
Octopamine (100 nM)
Ketanserin (K)
Phentolamine (P)
5-HT + K
5-HT + I'
Octopamine + K
Octopamine + I'
IP3
(DPM/107 cells)
1,359
1,599
1,696
1,310
1,587
1,636
1,298
1,690
1,245
80
98*
147"
37
109"
? 135"
Inhibition relative to
agonistb (%I
f
f
k
f
f
k 81**
f 180*
f 51""
0
130
2
134
'Antagonists added at equal concentration to agonist.
%hibition % = [(A - B)/Al x 100, where A = agonist-saline, B = treatment-saline.
*Indicatesa significant increase above control values, and **indicatesa significant antagonism of
5-HTand octopamine effect at P = 0.05 ANOVA, SNK test (n = 5, fS.E.).
Role of an Octopamine Receptor in Hemocytes
255
TABLE 3. Effect of Octopamine Antagonists on the Octopamine-Mediated Elevation of IP3 in
Hemocyte Membrane Preparations of Periplaizcta americana
Treatment
(100 nM)
Saline
Octopamine
Chlorpromazine (Chl)
Yohimbine (Y)
Metoclopramide (M)
Octopamine + Chl
Octopamine + Y
Octopamine + M
IP3
(DPM/107 cells)
Inhibition relative to
octopaminea (%)
1,250 k 81
1,423
76"
1,199 f 88
1,187 f 108
1,153 f 100
1,208 f 42**
1,083 f 58**
1,322 f 60"
124
196
42
+
aInhibition %
' = [(A- B)/Al x 100, where A = octopamine-saline, B = treatment-saline.
*Indicates a significant increase above control values and **indicatesa significant antagonism of
octopamine effect at P = 0.05 ANOVA, SNK test (n = 4, S.E.).
*
The effect of specific octopamine antagonists [see 191 on the octopaminemediated elevation of IP3 production in cockroach hemocytes was examined
(Table 3). The hierarchy of antagonism was yohimbine (196%) > chforpromazine (124%) > metoclopramide (42%). Yohimbine and chlorpromazine
blocked the effect of octopamine by greater than 1UO%, indicating that the basal
level of IP3 production may be related to the presence of an as yet unidentified
compound.
Agonist Studies
Agonists used to classify octopamine receptors in insects [see 191 were assessed for their effects on IP3production in hemocyte membranes of cockroaches.
The hierarchy of agonism for IP3 production when setting the octopamine effect
to 100% was chlonidine (106%)> tolazoline (77%)> naphazoline (41%) (Table4).
Two general octopamine agonists, synephrine and DCDM, cause an increase in
IP3 production of 127%and 135%,respectively (Table4). Collectively the agonist
and antagonist data support the initial classification of the receptor present on
the hemocytes as primarily octopamine, and more specifically octopaminel-like.
TABLE 4. Effect of Octopamine Agonists on the Elevation of IP3 in Hemocyte Membrane
Preparations of Periplaneia amcricana
Treatment
(100 nM)
Saline
Octopaminc
Synephrine"
DCDMa
Clonidine
Tolazoline
Naphazoiine
IP3
(DPM/107 cells)
Increase relative to
octopamineb (%)
4,243 k 545
_C 665'
399'
5,612 k 215%
5,321 +_ 612*
5,018 f 582*
4,662 k 579
127
135
106
77
41
5,254
5,531
+
"n = 3.
%crease 7%= [(A -B)/AI x 100, where A = octopamine-saline, B = treatment-saline.
*Indicatesa significant increase above control values at P = 0.05 ANOVA, SNK test (n = 6, ? S.E.).
256
Baines and Downer
IPa Production During Phagocytosis In Vitro
The effect of octopamine on IPS production during phagocytosis was investigated. Hemocytes pretreated with either octopamine or saline before exposure
to bacteria caused similar increases in IP3 production in the first 30 min of
phagocytosis (Fig. 2). At 30 min the octopamine- and saline-treated hemocytes
had increased their IP3 production above basal levels (1,040 +_ 60 DPM/107
hemocytes; no bacteria present), and at 60 min, in octopamine-treated hemocytes IP3 production was significantly above that observed for saline-treated
hemocytes ( P = 0.09). The highest level in 11'3 production for octopamine
occurred at 60 min, which corresponds with the inaxiinum uptake of bacteria
during phagocytosis in vitro [lo].
octopamine in vitro (Table
Phagocytosisis enhanced in the presence of 1
5). Exposure of the hemocytes to 10 pM IP3 in 1% DMSO resulted in greater
LO50
0
10
10soI
0
10
I
20
'
20
30
40
I
I
30
40
SO
'
SO
60
'
60
70
'
70
Minutes
Fig. 2. Effect of octopamine (Oct, 100 nM) on the production of IPj during phagocytosis in cockroach
hemocytes (saline = saline-treated cells + bacteria; Oct = octopamine-treated cells + bacteria; n = 5,
mean rf S.E.).
Role of an Octopamine Receptor in Hemocytes
257
TABLE 5. Effect of Octopamine, Inositol, and Inositol Trisphosphate on Phagocytosis in
Cockroach Hemocytes In Vitro
Treatment
Saline (1% DMSO)
Saline
Octopamine (lym)
Inositol Trisphosphate (10 yM)
Inositol Trisphosphate (1% DMSOb,
Inositol (1%DMSO, 10 FM)
Phagocytic indexa
0.38
0.36
0.50
0.40
0.54
0.40
+ 0.02
i 0.02
? 0.02*
? 0.02
? 0.02%
? 0.02
aEqual to the number of phagocytic cells/total cells counted in a replicate/treatment; minimum
50 cells assessed per replicate with the data presented as the average of 8 replicates.
? h e membrane was permeabilized with DMSO.
*Significantincrease above control values at P = 0.05 ANOVA, SNK test (n = 8,> 800 cells assessed
per treatment, f S.E.).
phagocytosis; however, if IP3 is added without this membrane permeabilizer,
there is no significanteffect on phagocytosis.This indicates that the site of action
of IP3 is intracellular (Table 5). Inositol (+1%DMSO) was unable to mimic the
IP3 effect, again suggesting that the response by hemocytes is specific to P 3 .
Treatment of cockroach hemocytes with 1% DMSO did not affect phagocytosis
compared with saline-treated hemocytes. Octopamine appears to enhance the
level of IP3 production during the uptake phase of phagocytosis.
Survival of Cockroaches
The survival of cockroachesexposed to an LD3o dose of S. aureus in the absence
or presence of agonistsand antagonists of octopaminei receptorsis shown in Figure
3. Octopamine causes significantly higher cockroach survival (85%;P = 0.05)
compared with saline-treated cockroaches receiving bacteria alone (40%). The
octopaminel agonist, clonidine, mimicked the effect of octopamine (75%;P = 0.05).
Further, the octopaminei antagonist, chlorpromazine, reduced (60%)the octopamine-mediated increase in cockroach survival, but not significantly.This information in conjunction with the in vitro phagocytosis data suggests that the benefit
derived from octopamine for artificially infected cockroachesis, at least in part, in
the form of greater phagocytic activity by hemocytes.
DISCUSSION
Calcium oscillations in cells are common cytosolic events that have been
linked to the regulation of cellular activities [lll. Secretion in blowfly salivary
gland and contraction of skeletal and visceral muscle of locusts are affected by
the presence of agonists that promote the elevation of the calcium mobilizing
second messenger, IP3 120-221. The present study suggests that octopamine
increases phagocytosis in cockroach hemocytes through the elevation of IP3
production.
Octopamine and 5-HT were shown previously to enhance phagocytic activities of cockroach hemocytes in vitro and in vivo [lo]. A pharmacological profile
demonstrated the presence of both 5-HT and octopamine receptors affecting
phagocytic activity. The 5-HT-sensitive receptor was subsequently shown to
258
Baines and Downer
50
r
2
3
4
Treatment
Fig. 3 . Survival of female cockroaches exposed to an LDsodose of Staphylococcus aureus (Bacteria)
in conjunction with saline or 1 m M octopamine, clonidine, or octopamine + chlorpromazine (n =
40, 4 replicates).
elevate cAMP production throughout phagocytosis [231. A pharmacological
profile indicated it was a 3-IITFlike receptor. The present study established
that an octopaminei-like receptor increases phagocytic activity through an
increase in IP3 production in the latter phase of phagocytosis. This correlated
with binding, pseudopod extension, and uptake of bacteria. It is likely, then,
that 5-HT and octopamine are coordinating different processes involved with
the phagocytic process that result in a similar increase in phagocytic activity.
Berridge and Heslop [15]observed a similar dual mechanism that increased the
secretionsfrom blowfly salivary gland. In this case, two separate 5-HT receptors
were shown to mediate their effects through the elevation of cAMP and IPS
production.
Octopamine produced the largest overall increase in IP3 production in hemocyte membranes of the cockroach.The effects of both octopamine and 5-HT were
blocked by an octopamine antagonist, phentolamine, but not by the 5-HT
antagonist, ketanserin. Thus, the receptor is tentatively classified as octopaminergic in nature. Exposure of the hemocytes to specific agonists and antagonists
of octopamine receptors confirm that the receptor is octopaminel-like. Clonidine was the most potent agonist in elevating IPS production in hemocyte
membranes of cockroaches. Yohimbine was a better antagonist of the octopamine-sensitive receptor than chlorpromazine in cockroach hemocytes, and
both were substantially better than metoclopramide. The sensitivity to clonidine and yohimbine is similar to a previously described octopaminei receptor
affecting the myogenic rhythm of the locust extensor tibia muscle [191. Oc-
Role of an Octopamine Receptor in Hemocytes
259
topamine2 receptors in insect skeletal and visceral muscle [24-263 have a
different hierarchy where metoclopramide is the most potent antagonist,
while naphazoline and tolazoline are the most potent agonists. Significantly, the octopaminel receptor in the locust extensor tibia muscle is not
linked to CAMP production, and it is believed to mediate its effects
through the elevation of intracellular calcium [271. Thus, the occurrence
of dual pathways for coordinating similar cellular activities may be common in insect tissues.
Exposure of hemocytes to 10 ,uM octopamine was previously reported to
enhance phagocytosis in cockroach hemocytes 1101. This study demonstrates
that IP3 mimics the effect of octopamine on phagocytosis. In addition, an
octopaminel agonist and an octopaminei antagonist were able to mimic and
partially block the octopamine effect, respectively. Further, octopaminetreated hemocytes enhance IP3 production above control levels during the
bacterial-uptake stage of phagocytosis. Cockroach survival assays also supported the identification of an octopaminel receptor that influenced the fate
of cockroaches exposed to an LD50 dose of S. u w e u s . This study has identified
a receptor-second messenger system that facilitates phagocytosis, but further research is necessary to specifically identify the cellular activities affected by IPS.
The present study did not address the probable intracellular mechanism that
leads to the enhanced phagocytic activity of octopamine-treated hemocytes. In
another study, the calcium concentration in a hemocyte cell line of the forest
tent caterpillar increases upon exposure to 1 pM octopamine 191, and this is
mediated by both a release from intracellular stores and uptake from extracellular medium. For the hemocyte preparation, a similar concentration of octopamine increases the level of IP3 production. Calcium could, therefore, be
released from intracellular stores in cockroach hemocytes through the binding
of IP3 to receptors located in calcium sequestering organelles such as the
calciosome, endoplasmic reticulum, and Golgi apparatus [20,281. The phosphorylation of IP3 to IP4 can also directly open membrane calcium channels allowing
extracellular Ca++into the cell 1201. Our results imply that the resulting Ca++
fluxes could enhance cytoskeletal rearrangements necessary to promote
pseudopod extension and uptake of bacteria by hemocytes.
Phentolamine, when applied alone to cockroach hemocytes, acts as an
agonist for IP3 production, but incubation of the tissue with this agent
before the addition of octopamine completely blocks the octopamine
effect. This phenomenon has also been reported in another study that
examined the desensitization of octopamine receptors in cockroach hemocytes [291. For phentolamine to have both agonist and antagonist qualities,
it might alter the properties of membrane components that affect IP3
production by perhaps binding to a different site than that normally
occupied by octopamine.
On the strength of the data from the whole insect assays, in vitro phagocytic
assays, and the IP3 production assays, the receptor linked to IP3 production in
cockroach hemocytes and associated with the enhancement of phagocytosis by
these cells has been tentatively classified as octopaminel-like.
260
Baines and Downer
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New York: Plenum Press, pp 67-70,182-190.
4. Baerwald RJ, Boush GM (1970):Time-lapse photographic studies of cockroach hemocyte
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