Possible Regulation of Feeding Behavior in Cockroach Nymphs by the Neurotransmitter Octopamine

Insects, including cockroaches, have the ability to select a proper diet from different nutrient choices. We have showed previously that various neurotransmitters and neuromodulators appear to regulate certain aspects of feeding in the cockroach, Rhyparobia madera. In the current study, we examined the role of octopamine in feeding behavior of cockroach nymphs. By either injection or direct incorporation into the diet blocks, an octopamine agonist (octopamine or synephrine) or antagonist (phentolamine) was effective in altering feeding in R. madera nymphs. Both octopamine and synephrine increased feeding slightly, while phentolamine decreased feeding dramatically. Phentolamine was able to decrease feeding, but not motor activity, when injected directly into the nymphs. Octopamine appears to cause increased feeding in the cockroach.     

Up-regulation of circulating hemocyte population in response to bacterial challenge is mediated by octopamine and 5-hydroxytryptamine via Rac1 signal in Spodoptera exigua

Bacterial challenge induced a significant increase in the total hemocyte population within 4 h in the beet armyworm, Spodoptera exigua. Octopamine and 5-hydroxytryptamine (5-HT) are known to play critical roles in mediating insect immune responses. This study analyzed the effects of both biogenic monoamines on mediating up-regulation of circulating hemocyte population in response to bacterial challenge. Injection of either octopamine or 5-HT induced a significant increase in the total hemocyte count in the hemolymph without any bacterial challenge. On the other hand, the monoamine antagonists, phentolamine (an octopamine antagonist) and ketanserin (a 5-HT antagonist) each suppressed the increase of the circulating hemocyte counts in response to bacterial challenge. This rapid change of circulating hemocyte population did not appear to be the result of de novo hemocyte production from the hematopoietic organ because a physical block (“ligation”) of hemolymph circulation between thorax and abdomen did not inhibit the increase of hemocyte counts in the isolated abdomen in response to bacterial challenge. The effects of the two monoamines on hemocyte numbers were not dependent on the mediatory effects of eicosanoids, because dexamethasone, an eicosanoid biosynthesis inhibitor, had no effect on the hemocyte recruitment induced by the monoamines. On the other hand, an adenylate cyclase inhibitor, NKY80, significantly impaired hemocyte mobilization in response to bacterial challenge, implying involvement of cyclic AMP in the control of hemocyte numbers. Also, a Rac1 inhibitor, NSC23766, significantly antagonized the effects of monoamines in increasing circulating hemocyte numbers. Rac1 activity was necessary to form F-actins in the hemocytes of S. exigua, where its activity showed a quantitative correlation with hemocyte-spreading behavior. This study suggests that octopamine and 5-HT mediate a rapid increase of circulating hemocyte population in response to bacterial challenge via Rac1 signal in S. exigua.     

Characterization of insect neuronal octopamine receptors (OA3 receptors)

Octopamine receptors in the nervous tissue of insects were investigated using a ligand-receptor assay with [³H]NC-5Z or [³H]octopamine as the radioligands. Both ligands recognized a homogenous class of binding sites with the properties of an octopamine receptor. This receptor has been characterized pharmacologically. Both high-affinity agonists (e.g. NC 7, K₁=0.3 nM) and antagonists (e.g. maroxepine, K₁=1.02 nM) were investigated. The neuronal octopamine receptor belongs to a receptor class that can easily be distinguished from peripheral octopamine receptors. Initial investigations of the localization of octopamine receptors within the insect nervous tissue show the greatest receptor density in the optic lobes.     

Octopamine and 5‐hydroxytryptamine mediate hemocytic phagocytosis and nodule formation via eicosanoids in the beet armyworm,Spodoptera exigua

Octopamine and 5‐hydroxytryptamine (5‐HT) have been known to mediate cellular immune responses, such as hemocytic phagocytosis and nodule formation, during bacterial invasion in some insects. In addition, eicosanoids also mediate these cellular immune reactions in various insects, resulting in clearing the bacteria circulating in the hemolymph. This study investigated a hypothesis on signal cross‐talk between both types of immune mediators in the beet armyworm, Spodoptera exigua, which had been observed in the effect of eicosanoids on mediating the cellular immune responses. In response to bacterial infection, octopamine or 5‐HT markedly enhanced both hemocytic phagocytosis and nodule formation in S. exigua larvae. Their specific antagonists, phentolamine (an octopamine antagonist) or ketanserin (a 5‐HT antagonist) suppressed both cellular immune responses of S. exigua. These effects of biogenic monoamines on the immune mediation were expressed through eicosanoids because the inhibitory effects of both antagonists were rescued by the addition of arachidonic acid (a precursor of eicosanoid biosynthesis). Furthermore, the stimulatory effects of both monoamines on the cellular immune responses were significantly suppressed by different inhibitors acting at their specific levels of eicosanoid biosynthesis. Taken together, this study suggests that octopamine and 5‐HT can mediate hemocytic phagocytosis and nodule formation through a downstream signal pathway relayed by eicosanoids in S. exigua. © 2009 Wiley Periodicals, Inc.     

The effect of a selective octopamine antagonist, epinastine, on pharyngeal pumping in Caenorhabditis elegans

This paper investigates the effect of epinastine, a selective octopamine antagonist in invertebrates, in Caenorhabditis elegans. Specifically, its ability to block the inhibitory action of octopamine on C. elegans-isolated pharynx was assayed. Isolated pharynxes were stimulated to pump by the addition of 500 nM 5-hydroxytryptamine (5-HT) (113 ± 2 per 30 s, n = 15). Octopamine inhibited the 5-HT-induced pumping in a concentration-dependent manner (threshold 1–5 μM) with a 61 ± 11% inhibition with 50 μM (n = 5). Epinastine (0.1 μM) antagonized the inhibitory response to octopamine (P < 0.001; n = 15). Tyramine also inhibited pharyngeal pumping induced by 5-HT but was less potent than octopamine. Tyramine, 50 μM to 1 mM, gave a transient inhibition e.g. of 40 ± 5% at 50 μM (n = 5). A higher (10 μM) concentration of epinastine was required to block the tryamine response compared with octopamine. It is concluded that epinastine selectively antagonizes the effect of octopamine on C. elegans pharynx. Further studies are required to test its selectivity for octopamine in other tissues and other nematodes.     

Agonist-induced desensitization of an octopamine receptor

The octopamine-sensitive adenylate cyclase associated with haemocytes of the American cockroach, Periplaneta americana, has been used as a model system with which to study desensitization of the octopamine receptor. Preincubation of the haemocytes with octopamine results in a large decrease in subsequent maximal stimulation of cyclic AMP production by octopamine with little change in affinity of the receptor for the agonist. This effect of preincubation is dependent upon the concentration of octopamine in the preincubation media and on the duration of exposure. The attenuation appears to be a receptor-mediated event rather than an artifact of the preincubation. Octopamine receptor agonists (octopamine, synephrine, N-demethylchlordimeform) induce desensitization while biogenic amines with poor octopamine receptor affinity (dopamine, serotonin, norepinephrine) are without affect. In contrast, the octopamine receptor antagonist, phentolamine, appears to enhance subsequent stimulation by octopamine. The attenuation of octopamine stimulation of adenylate cyclase is conserved in broken-cell preparations with no alteration of responses to NaF or forskolin. Incubation of the cells with dibutyryl cyclic AMP or forskolin does not induce desensitization. The data indicate that the OA receptors coupled to AC in cockroach haemocytes undergo an homologous desensitization in response to exposure to agonists.     

Octopamine-Mediated Neuromodulation of Insect Senses

Octopamine functions as a neuromodulator, neurotransmitter, and neurohormone in insect nervous systems. Octopamine has a prominent role in influencing multiple physiological events: (a) as a neuromodulator, it regulates desensitization of sensory inputs, arousal, initiation, and maintenance of various rhythmic behaviors and complex behaviors such as learning and memory; (b) as a neurotransmitter, it regulates endocrine gland activity; and (c) as a neurohormone, it induces mobilization of lipids and carbohydrates. Octopamine exerts its effects by binding to specific proteins that belong to the superfamily of G protein-coupled receptors and share the structural motif of seven transmembrane domains. The activation of octopamine receptors is coupled with different second messenger pathways depending on species, tissue source, receptor type and cell line used for the expression of cloned receptor. The second messengers include adenosine 3′,5′-cyclic monophosphate (cAMP), calcium, diacylglycerol (DAG), and inositol 1,4,5-trisphosphate (IP₃). The cAMP activates protein kinase A, calcium and DAG activate protein kinase C, and IP₃ mobilizes calcium from intracellular stores. Octopamine-mediated generation of these second messengers is associated with changes in cellular response affecting insect behaviors. The main objective of this review is to discuss significance of octopamine-mediated neuromodulation in insect sensory systems.     

The effects of octopamine on juvenile hormone biosynthesis,electrophysiology, and cAMP content of the corpora allata of the cockroach Diploptera punctata

Summary Juvenile hormone production by the corpora allata of the adult female cockroach, Diploptera punctata, can be modulated by treatment with the biogenic amine, octopamine. Endogenous octopamine has been identified within the CA, using HPLC and electrochemical detection. Treatment with octopamine results in a sinusoidal, dose-dependent inhibition of JH biosynthesis by CA from day 2 virgin females, with maximal inhibition occurring at 10^-10 M and 10^-4 M. In day 4 and day 8 mated female corpora allata octopamine inhibited JH biosynthesis at 5·10^-5 M. Although the elevation of either cAMP or cGMP within the CA is known to be associated with an inhibition of JH biosynthesis, treatment with high concentrations of octopamine results in an increase in the level of cAMP but not cGMP. This effect is both dose- and time-dependent.Octopamine treatment also initiates changes in the passive membrane responses of the CA. Superfusion of CA with octopamine results in a pronounced hyperpolarization of CA cells and an increase in the electrotonic potential (indicative of the degree of electrical coupling between CA cells). This effect could be blocked by the octopamine receptor blocker phentolamine. Treatment with octopamine or phentolamine also blocked the hyperpolarization of CA cells normally associated with electrical stimulation of the axon tracts innervating the CA.We hypothesize that octopamine may be a natural neuromodulator of JH production by CA, regulating ion channels in CA cells themselves as well as release of the inhibitory neuropeptide, allatostatin, from the terminals within the CA.Abbreviations 4-AP 4-aminopyridine - CA corpora allata - CC corpora cardiaca - cAMP cyclic adenosine monophosphate - cGMP cyclic guanosine monophosphate - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N2-ethanesulfonic acid - HPLC high pressure liquid chromatography - IBMX 3-isobutyl-1-methylxanthine - JH juvenile hormone - ms millisecond - nA nanoampere - NCA I nervi corporis allati I - OCT octopamine - TEA tetraethyl ammonium     

Pharmacological profile of octopamine and 5HT receptors on the lateral oviducts of the cockroach,Periplaneta americana

The effects of the amines 5HT and octopamine on the myogenic activity of Periplaneta americana (L.) oviducts and the pharmacological profile of octopamine and 5HT receptors on the lateral oviducts have been determined. Application of 5HT to the oviducts resulted in a dose-dependent increase in basal tonus and amplitude of contractions. Antagonist studies revealed that the 5HT receptor on the cockroach oviduct most resembles the mammalian 5HT₂ receptor. Application of octopamine resulted in a decrease in basal tonus and had a biphasic effect on the amplitude of contractions, being stimulatory at low doses and inhibitory at higher ones. The inhibitory effects of octopamine appear to be mediated via cAMP and are blocked by antagonists which indicate that the octopamine receptor is of the octopamine-2 type. © 1995 Wiley-Liss, Inc.     

Selective Depletion of Dopamine, Octopamine, and 5-Hydroxytryptamine in the Nervous Tissue of the Cockroach (Periplaneta americana)

High-performance liquid chromatography with electrochemical detection was used to measure the concentrations of 3,4-dihydroxyphenylethylamine (dopamine), 5-hydroxytryptamine (5-HT), p-hydroxyphenylethanolamine (octopamine), alpha-methyl-p-tyrosine, and tryptophan in the cerebral ganglia of cockroaches (Periplaneta americana) after peripheral administration of alpha-methyl-p-tyrosine and alpha-methyltryptophan. In addition, the levels of dopamine, 5-HT, octopamine, alpha-methyl-p-tyrosine, and tryptophan were determined after injection of alpha-methyl-p-tyrosine, 6-hydroxydopamine, or 5,7-dihydroxytryptamine directly into the cerebral ganglia by means of microinjection needles. Peripheral administration of alpha-methyl-p-tyrosine (400-1,600 micrograms/insect) caused a reduction in dopamine and 5-HT concentrations in cockroach cerebral ganglia, although the reduction in dopamine concentrations was more pronounced. Peripheral injections of alpha-methyl-p-tyrosine also reduced octopamine levels in the cerebral ganglia. Peripheral injection of alpha-methyltryptophan (400-1,600 micrograms/insect) caused a marked reduction in 5-HT and tryptophan concentrations in cockroach cerebral ganglia without altering dopamine or octopamine concentrations. Central injections of alpha-methyl-p-tyrosine (80 micrograms/insect) reduced dopamine concentrations in the cerebral ganglia. However, neither 6-hydroxydopamine (20 micrograms/insect) nor 5,7-dihydroxytryptamine (20 micrograms/insect) caused reductions in amine levels when applied near or directly into the cerebral ganglia. The results suggest that specific lesions of aminergic neurons in insects by either 6-hydroxydopamine or 5,7-dihydroxytryptamine are impractical. The specific, long-lasting depletion of 5-HT by alpha-methyltryptophan suggests that this chemical may be useful in elucidating the functions of 5-HT in insects.     

Characterization of a tyramine receptor type 2 from hemocytes of rice stem borer,Chilo suppressalis

Calcium acts as a second messenger in many cell types, including insect hemocytes. Intracellular calcium level has a definite role in innate and adaptive immune signaling. Biogenic amines such as octopamine (OA), tyramine (TA), dopamine (DA) and serotonin (5-HT) play various important physiological roles in insects by activating distinct G-protein-coupled receptors (GPCRs) that share a putative seven transmembrane domain structure. OA and 5-HT have been shown that can mediate insect hemocytic immune reactions to infections and invasions. Here, we showed that TA increase hemocyte spreading in the rice stem borer, Chilo suppressalis. Furthermore, we cloned a cDNA encoding a tyramine receptor type 2 from the hemocytes in the C. suppressalis, viz., CsTA2, which shares high sequence similarity to members of the invertebrate tyramine receptor family. The CsTA2 receptor was stably expressed in human embryonic kidney (HEK) 293 cells, and its ligand response has been examined. Receptor activation with TA induced a dose-dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cells, with an EC₅₀ value of 18.7 ± 5.3 nM, whereas OA, DA, 5-HT and other potential agonists did not have this response. The mRNA is present in various tissues including nerve cord, hemocytes, fat body, midgut, Malpighian tubules, and epidermis in the larval stage. Western blot analysis and immunohistochemistry assay displayed that CsTA2 was detected and presented on hemocytes. We also showed that TA induced Ca²⁺ release from the hemocytes of C. suppressalis.     

Neuromodulation of flight initiation by octopamine in the cockroach Periplaneta americana

We have tested the effect of a known insect neuromodulator, octopamine, on flight initiation in the cockroach. Using minimally dissected animals, we found that octopamine lowered the threshold for windevoked initiation of flight when applied to either of two major synaptic sites in the flight circuitry: 1) the last abdominal ganglion, where wind-sensitive neurons from the cerci excite dorsal giant interneurons, or 2) the metathoracic ganglion, where the dorsal giant interneurons activate interneurons and motoneurons which are involved in producing the rhythmic flight motor pattern in the flight muscles (Fig. 2).Correlated with this change in flight initiation threshold, we found that octopamine applied to the last abdominal ganglion increased the number of action potentials produced by individual dorsal giant interneurons when recruiting the cereal wind-sensitive neurons with wind puffs (Figs. 3, 4, 5) or with extracellular stimulation of their axons (Fig. 6). Octopamine increases the excitability of the giant interneurons (Figs. 7, 8). Also, when we stimulated individual dorsal giant interneurons intracellularly, the number of action potentials needed to initiate flight was reduced when octopamine was applied to the metathoracic ganglion (Fig. 9).Abbreviations EMG electromyogram - dGIs dorsal giant interneurons - GI giant interneuron - A6 sixth abdominal ganglion - T3 third thoracic ganglion - EPSP excitatory postsynaptic potential     

Spontaneous activity of foregut and hindgut visceral muscle of the locust,Locusta migratoria—II. The effect of biogenic amines

1. At 10⁻⁸ M, 5-HT increased both the frequency and amplitude of contractions of isolated locust foregut. At 10⁻⁴ M the foregut general tonus was increased.2. Both spontaneously active and quiescent hindguts were less sensitive to 5-HT, showing only an increase in amplitude of contraction at 10⁻⁶−10⁻⁵ M.3. The Hill plot suggested that although the 5-HT receptor populations in these two gut divisions differed in affinities, they were essentially homogeneous.4. Octopamine (10⁻⁵−10⁻⁴ M) increased foregut contraction frequency but diminished amplitude.5. Octopamine action on the hindgut was varied. At 10⁻⁸ M it slightly increased tonus, while at 10⁻⁵ M it increased contraction amplitude without affecting frequency.6. At 10⁻⁴ M octopamine suppressed activity of spontaneously active preparations and lowered the tonus of quiescent preparations.7. Tyramine induced dose-dependent inhibition of foregut responses to 5-HT. The hindgut was exceptionally sensitive to tyramine, at only 10⁻⁸ M it suppressed 5-HT responses.8. Octopamine inhibited fore- and hindgut responses to 5-HT, but was less effective than tyramine.9. Locust fore- and hindgut have remarkably different pharmacological properties reflecting differences in innervation and in extrajunctional monoamine receptor affinities.     

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α₁-adrenoceptors in EPI action, at least in some fish species. The role of the α₁- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist ³H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors (K_d0.36 nM, B_max 8.61 fmol · mg⁻¹ protein). We provide the first demonstration of α₁-adrenoceptors in these same membranes; analysis of binding data with the α₁-adrenergic antagonist ³H-prazosin demonstrated a single class of binding sites with a K_dof 15.4 nM and a B_max of 75.2 fmol · mg⁻¹ protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP₃) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP₃ (K_d 6.03 nM, B_max 90.2 fmol · mg⁻¹ protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α₁-adrenergic agonists are able to increase intracellular concentrations of IP₃ and Ca²⁺ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α₁-binding sites affinity and of α₁-adrenoceptor/IP₃/Ca²⁺ transduction systems.     

Cockroach oviducts: The presence and release of octopamine and proctolin

The oviducts of the cockroach, Periplaneta americana, contain octopamine (determined by radioenzymatic assay) and proctolin (determined from proctolin-like bioactivity following separation on reversed-phase high performance liquid chromatography). Octopamine content is 5–10-fold greater than proctolin content. Both substances are released in a calcium-dependent manner following depolarisation with high potassium saline. This method resulted in the release of 14% of the total store of octopamine, but only 0.3% of the total store of proctolin. The results are discussed in relation to the recent demonstration of octopamine-containing and proctolin-containing neurones associated with locust oviducts.     

Structure and function of spiny lobster ligamental nerve plexuses: Evidence for synthesis,storage, and secretion of biogenic amines

Three pairs of ligaments support not only the heart of spiny lobsters but also ligamental nerve plexuses, the complex terminal aborizations of segmental nerves. Segmental nerves 1–4 project from the thoracic ganglia into the pericardial cavity and ultimately ramify along the strands of the anterior, medial, and posterior ligaments. In each branch, a core of large axons sends fibers to terminate in a surrounding cortex of fine and varicose secretory processes. Electron micrographs reveal at least five distinct populations of granule-filled neuronal profiles, many with vesicles clustered at membrane thickenings adjacent to the epineural sheath. The ligamental nerve plexuses synthesize and accumulate octopamine, dopamine, 5-HT, and acetylcholine. Octopamine and 5-HT are predominant, comprising 33% and 65%, respectively, of the synthetic activity devoted to the four amines. Thus, the anatomy, ultrastructure, and neurochemistry of the ligamental nerve plexuses establishes their homology with the pericardial organs of other Crustacea. Octopamine and 5-HT are released by a Ca⁺⁺-dependent mechanism upon electrical stimulation of preterminal nerve trunks, and, in vivo, would be swept immediately through ostia into the heart. These observations, when considered with known effects of octopamine and 5-HT on crustacean cardiac activity, neuromuscular transmission, muscle tension, and cyclic AMP metabolism provide a strong case for hormonal actions at target sites throughout the animal. Segmental nerve processes in the dorsal nerve trunk ramify into a plexus around the dorsal nerve apparatus, a small muscular bulb that lies recessed in the cardiac surface. The dorsal nerve, carrying excitatory and inhibitory input to the cardiac ganglion directly through the bulb's hollow interior. The apparatus synthesizes and contains acetylcholine and the three amines mentioned above. In situ, it may beat rhythmically out of phase with the heart.     

Pharmacology of octopamine-, dopamine-, and 5-hydroxytryptamine-stimulated cyclic amp accumulation in the corpus cardiacum of the american cockroach,Periplaneta americana L

A variety of potential receptor agonists were incubated with isolated corpora cardiaca from the American cockroach Periplaneta americana to determine their effects on cyclic AMP production in this gland. Octopamine, dopamine, and 5-hydroxytryptamine elevated cyclic AMP levels in a dose-dependent manner with estimated K_a values of 15.8, 1.7, and 1.1 m?M, respectively, and their stimulation was found to be additive. Several vertebrate receptor antagonists were tested against the three amines and a preliminary pharmacological profile developed.     

Electrical responses of salivary gland cells of the gastropod mollusc Philine aperta to putative neurotransmitters—II. Octopamine, 5-HT and other biogenic amines

1. Intracellular recordings were made from salivary gland cells of the mollusc Philine aperta. Salivary cell action potentials were produced in a dose-dependent manner in response to bath-application of octopamine, 5-hydroxytryptamine (5-HT) and certain related biogenic amines.2. Evidence that amines act on presynaptic receptors on nerve terminals rather than directly on gland cell receptors is presented. These receptors may serve to modulate salivary neuroglandular transmission.3. Octopamine and 5-HT action was reversibly blocked or reduced by the antagonists phentolamine, chlorpromazine, cyproheptidene, yohimbine, D-tubocurarine, hexamethonium and atropine.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

Regulation of ion and water transport across the eel intestine: effects of acetylcholine and serotonin

Summary Both acetylcholine (ACh) and serotonin (5-HT) lowered the serosa-negative transepithelial potential difference (PD) and the short-circuit current (I_sc), accompanied by a decrease in NaCl and water absorption across the eel intestine. These inhibitory effects of ACh and 5-HT were blocked by atropine, a muscarinic receptor antagonist, and ICS-205930, a 5-HT₃ receptor antagonist, respectively. Even after blocking the ACh receptor with atropine, 5-HT inhibited the PD and I_sc, and ACh lowered them after blocking the 5-HT receptor with ICS-205930, indicating that ACh and 5-HT act independently. Similar inhibition in the PD and the I_sc was observed after electrical field stimulation (EFS) which is expected to release endogenous regulators. These effects of EFS were reduced by 70% after simultaneous addition of atropine and ICS-205930. Since atropine and ICS-205930 block ACh and 5-HT receptors, respectively, these results suggest that endogenous ACh and 5-HT are released by EFS.Abbreviations ACh acetylcholine - EFS electrical field stimulation - 5-HT serotonin - I _sc short-circuit current - PD transepithelial potential difference - R _t tissue resistance - TTX tetrodotoxin