Effects of octopamine on fluid secretion by isolated salivary glands of a feeding ixodid tick.код для вставкиСкачать
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 INTRODUCTION 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. 218 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 ,suggesting that a specific dopamine receptor is present . 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 . Schmidt et a1  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. MATERIALS AND METHODS 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 . 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  and Needham and Sauer . 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 219 Drugs 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 120 80 Secretory Rate (nllmln) 40 0 1-11 DopamiM O.*Y 1111 1-11 Dopamine 0.1,Y Dopamha O.lpN 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). -160 .120 .80 I -= 60 Time (min) I I m40 Octoparnine 1000 vM 6b I II II I I I 160 120 m==m Octopamine 1000 vM 140 I I I I I Lo 160 120 111 Octopamine 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). 220 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 significant. RESULTS 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 221 II I I 6 +4 l I I I ! Agonist Concentration (pM) 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 Secretion Period 2 Drug (PM) Test (n) 29 f 4(2) THI 50 DA THI 50 2 k 0 (6) DA THI 0.1 50 8 + + Period 3 (% max f S.E.) (YOmax k S.E.) Conc Test Control NSD 1k 0 30 k 6 P < 0.05 k 13(6) P < 0.05 7k6 57 k 14 P < 0.05 36 i lO(7) P < 0.05 2k1 22 k 9 P < 0.05 5+3 22+9 P<O.O5 Control (n) 46 f 7(4) Significance Significance 1.0 k 4 (7) 85 k OCT THI + OCT PHE + OCT 1,000 1.0 39 f 10 (4) 36 k lO(7) NSD 1,OOO 10 81 f 19(6) 64 f 14(6) NSD 53 f 17 30 f 5 NSD 100 + 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. 222 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) , 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 Drug OCT + DA OCT + DA Conc (pM) Test(n) 1,OOO Period 2 (% max k S.E.) Control (n) Significance Test 111 f 19(7) 80 f 7(7) NSD 1.0 0.1 88 6 (7) Period 3 (% max k S.E.) 80 k 7 (7) NSD Control Significance 78 f 8 51 & 6 P < 0.05 56 k 10 51 k 6 NSD 1.0 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 223 DISCUSSION 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 , 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 . The threshold concentration for stimulation by octopamine was 3 orders of magnitude greater than for dopamine , 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  and the locust corpus cardiacum  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) . 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 , 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 , 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  (Pannabecker and Orchard, unpublished observations). Therefore both dopamine- and octopamine-stimulated secretion appear to involve a common, perhaps dopaminergic, receptor site. 224 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 . It seems likely that the basal adenylate cyclase activity results from endogenous catecholamine in the salivary gland nerves , 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 ;however, little has been published on invertebrate uptake mechanisms. One exception is octopamine uptake in the cockroach ventral nerve cord , 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. LITERATURE CITED 1. Tatchell RJ: Salivary secretion in the tick as a means of water elimination. Nature 213, 940 (1967). 2. Tatchell RJ: The ionic regulatory role of salivary secretions of the cattle tick, Boophilus microplus. J Insect Physiol 15, 1421 (1969). Effects of Octopamine in the lick Salivary Gland 225 3. 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