Octopamine enhances phagocytosis in cockroach hemocytesInvolvement of inositol trisphosphate.код для вставкиСкачать
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 . 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 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 LITERATURE CITED 1. Lackie AM (1988): Hemocyte behaviour. Adv Insect Physiol21:85. 2. Ratcliffe NA, Rowley AF (1979): Role of hemocytes in defense against biological agents. In Gupta AP (ed.):Insect Hemocytes. Cambridge, UK: Cambridge University Press, pp 331414. 3. Bershadsky AD, Vasiliev JM (1988): Cytoskeleton. In Siekevitz P (ed):Cellular Organelles. New York: Plenum Press, pp 67-70,182-190. 4. Baerwald RJ, Boush GM (1970):Time-lapse photographic studies of cockroach hemocyte migrations in vitro. Exp Cell Res 63208. 5. Baerwald RJ, Boush GM (1971):Vinblastine-induced disruption of microtubules in cockroach hemocytes. Tissue Cell 3:251. 6. Forehand JR, Pabst MJ, Phillips WA, JohnstonJr RB (1989):Lipopolysaccharide priming of human neutropluls for an enhanced respiratory burst: Role of inhacellular freecalcium. J Clin Invest 83:74. 7. Mueller H, Sklar LA (1989): Coupling of antagonistic signalling pathways in modulation of neutrophil function. J Cell Biochcm 4:287. 8. Hamachi T, Hirata M, Koga T (1984):Effect of CAMP-elevating drugs on Ca2+efflux and actin polymerization in peritoneal macrophages stimulated with n-formyl chemotactic peptide. Biochem Biophys Ada 804:230. 9. Jahagirdar AP, Milton G, Viswanatha T, Downer RGH (1987): Calcium involvement in mediating the action of octopamine and hypertrehalosemic peptides on insect hemocytes. FEBS Lett 21953. 10. Baines D, DeSantis T, Downer IiGH (1992): Octopamine and 5-hydroxytryptamine enhance phagocytic and nodule formation activities of cockroach (Periplaneta americana) haemocytes. J Insect Physiol38:905. 11. Berridge MJ, Irvine RF (1989): Inositol phosphates and cell signalling. Nature 341:197. 12. Sokal RR, Rohlf FJ (1981):Biometry. New York: WH Freeman, pp 179-207,321-371,691-778. 13. Shapiro M (1979): Changes in hcmocyte populations. In Gupta AP (ed): Insect Hemocytes: Development, Forms, Functions, and Techniques. Cambridge, UK: Cambridge University Press, pp 475-523. 14. Downer RGH (1990): Octopnmine, dopamine and 5-hydroxytryptamine in the cockroach nervous system. In: Huber I, Master El’, Roo BR (eds): Cockroaches as Model Systems for Neurobiology: Applications in Biomedical Research. Boca Raton, FL: pp 103-124. 15. Berridge MJ, Heslop JP (1981):Separate 5-hydroxytryptamine receptors on the salivary gland of the blowfly are linked to the generation of either cyclic adenosine 3’,5’-monophosphate or calcium signals. Br J Pharmacol73:729. 16. Banner SE, Osbourne RH, Cattell KJ (1987):The pharmacology of the isolated foregut of the locust, Schistocerca gregaria-11. Characterization of a 5-HT2 like receptor. Comp Biochem Physiol [C] 88:131. 17. Baines RA, Tyrer NM, Downer KCH (1990):Serotonergicinnervation of the locust mandibular closer muscle modulates contractions through the elevation of cyclic adenosine monophosphate. J Comp Neurol294:623. Role of an Octopamine Receptor in Hemocytes 261 18. Baines I U , Downer RGH (1991):Pharmacologicalcharacterization of a 5- hydroxytryptaminesensitive receptor/adenylate cyclase complex in the mandibular closer muscles of the cricket, GryIlus damestica. J Insect Physiol16:153. 19. Evans PD (1981):Multiple receptor types for octopamine in the locust. J Physiol (Lond) 318:99. 20. Berridge MJ, Dawson RMC, Downes CP, Heslop JP, Irvine RF (1983): Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem J 212:473. 21. Baines RA, Lange AB, Downer RGH (1990):Proctolin in the innervation of the locust mandibu- lar closer muscle modulates contractions through the elevation of inositol trisphosphate. J Comp Neurol294:479. 22. Lange AB (1988): Inositol phospholipid hydrolysis may mediate the action of proctolin on insect visceral muscle. Arch Insect Biochem Physiol10:201. 23. Baines D, Downer RGH (1992): 5-Hydroxytryptamine-sensitive adenylate cyclase affects phagocytosis in cockroach hemocytes. Arch Insect Biochem Physiol21:303. 24. Orchard I, Lange A (1986): Pharmacological profile of octopamine receptors on the lateral oviducts of the locust, Locusta migmtoria. J Insect Physiol32:741. 25. O’Gara BA, Drewes CD (1990): Modulation of tension production by octopamine in the metathoracic dorsal longitudinal muscle of the cricket Teleogryllusoceanicus. J Exp Biol149:161. 26. Roeder T, Gewecke M (1990): Octopamine receptors in locust nervous tissue. Biochem Pharmacol39:1793. 27. Evans PD (1984): The role of cyclic iiucleotides and calcium in the mediation of modulatory effects of octopamine on locust skeletal muscle. J Physiol (Lond) 348:325. 28. Chandra S, Kable EPW, Morrison GH, Webb WW (1991):Calcium sequestration in the golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy. J Cell Sci 100:747. 29. Orr GL, Hollingworth RM (1990):Agonist-induced desensitization of an octopamine receptor. Insect Biochem 20:239.