The action of iontophoretically applied L-glutamate on an insect visceral muscle

1) lontophoretic application of L-glutamate was employed to study the distribution of glutamate receptors in the superior longitudinal (SL) muscles of the locust (Locusta migratoria) hindgut, in which spontaneous activity was inhibited using normal saline containing 5 mM MgCl_2. 2) Junctional glutamate potentials with a rise time of 50–100 ms (peak) and a decay time of 250–400 ms were recorded at localized sites using ejection pulses in the range 5–10 nC. Most active sites were found in interfiber clefts and were spaced at about 250–300 μm intervals. 3) Desensitization of glutamate receptors occurred using ejection frequencies > 0.2 Hz. Desensitization could be irreversibly blocked using the lectin concanavalin A. 4) Depolarizing (D-) and biphasic depolarizing/hyperpofarizing (DH -) extrajunctional glutamate potentials were observed using ejection pulses > 15 nC. 5) δ-Philanthotoxin (δ-PTX) at concentrations > 0.3 Uml^?1 inhibited junctional glutamate potentials in a dose-dependent manner, 50% inhibition was achieved using 0.45 Uml^?1 δ-PTX. 6) Subthreshold concentrations of proctolin (up to 5 × 10^?10M) had no visible effect on glutamate potentials, suggesting that proctolin possibly does not act by modulating glutamate activity. 7) It is proposed that glutamate plays a transmitter role in SL muscles, while the role of proctolin is still unclear.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Proctolin antagonists bind to [(3)H]proctolin binding sites in the locust hindgut

Proctolin caused dose-dependent (1-200 nM) contraction of the isolated hindgut of S. gregaria which was abolished by [alpha-methyl-L-tyrosine(2)]-proctolin (1 microM). In comparison, cycloproctolin (5 microM) reduced the proctolin maximum response by 41%. Hindgut homogenates contained [(3)H]proctolin binding sites with a K(d) value of 660 nM, a B(max) value of 23.8 pmol/mg protein and a Hill coefficient of 0.934. Cycloproctolin (IC(50,) 220 nM; K(i), 204 nM), unlabeled proctolin (IC(50) 680 nM) and [alpha-methyl-L-tryosine(2)]-proctolin (IC(50) 3.1 microM, K(i), 2.9 microM) but not SchistoFLRFamide (1 nM-10 microM) were capable of displacing bound [(3)H]proctolin.     

Proctolin, its presence in and action on the oviduct of an insect

Proctolin has been isolated from oviduct extracts of Leucophaea maderae by HPLC. Quantitative bioassay with the hindgut of L. maderae demonstrated a proctolin titre of 0.93 +/- 0.15 ng/oviduct. Exposure to proctolin produced three changes in the spontaneous contractile activity of the oviduct: an increase in muscle tonus, an increase in the amplitude and frequency of phasic contractions. The sensitivity of the oviduct to proctolin was compared with the hindgut and foregut organ preparations from the same insect. Oviducts were responsive to proctolin in a calcium-free medium and the peptide also appeared to facilitate the reentry of calcium after depleted preparations were returned to normal levels of external calcium.     

Proctolin: a peptide transmitter candidate in insects.

The slow, striated muscles of the proctodeum (hindgut) of the cockroach, $\text{Periplaneta americana}$ (L.), were examined pharmacologically with reference to the responses evoked by nerve stimulation, glutamate, 5-HT, and proctolin, a myotropic peptide from $\text{Periplaneta}$ recently isolated and identified. The graded contractions evoked by repetitive nerve stimulation were simulated by 5-HT and proctolin at threshold concentrations of about 10⁻⁷ and 10⁻⁹ M respectively; responses to glutamate (∼10⁻⁴ M) were not similarly graded. The 5-HT receptors are distinct from other receptors, including the post-synaptic receptors, since they were specifically blocked by bromolysergic acid diethylamide. Proctolin was fully active on TTX-treated or surgically denervated muscle indicating that the proctolin receptors are located on the muscle fibre membrane. Tyramine, at threshold levels 5×10⁻⁸ M, reversibly antagonized the responses evoked by proctolin and by nerve stimulation but was without effect on the 5-HT and glutamate responses. Neurally evoked responses were potentiated by subthreshold concentrations of proctolin but not by glutamate. Pharmacologically, the proctolin and post-synaptic receptors appear to be identical and distinct from the glutamate and 5-HT receptors. Since proctolin is known to be a constituent of an efferent pathway of the proctodeal nerves, the evidence suggests that it may function as an excitatory transmitter substance. Peptidergic transmission is discussed in relation to the ultrastructural organization of the proctodeal nerve terminals which contain neurosectory granules in addition to electron-lucent, synaptic vesicles.     

Neuromuscular transmission in an insect visceral muscle

The electrical properties of the muscles of locust oviduct have been examined using intracellular recordings. The muscle cells are both dye and electrically coupled. They possess a wide array of spontaneous electrical activity ranging from slow oscillations of membrane potential to action potentials. In addition to possessing spontaneous electrical activity, certain regions of the oviduct are under motor control. The amplitude of evoked excitatory junction potentials (EJPs) increased step wise revealing innervation from a maximum of three motor units. These EJPs underwent summation and facilitation, and reached a critical threshold at which point the membrane revealed an active response. Bath applied glutamate, aspartate, proctolin, and octopamine were tested for their ability to alter resting potential and EJPs. L-glutamate (1.6 X 10(-5) M and above) produced a dose-dependent depolarization of membrane potential accompanied by a reduction in amplitude of EJPs. Although L-aspartate resulted in similar effects, the concentrations required were higher than those for glutamate. Proctolin (6.3 X 10(-11) M-6.0 X 10(-9) M) resulted in a dose-dependent depolarization but had little or no effect on amplitude of EJPs. Application of D, L-octopamine (3.2 X 10(-5) M-1.7 X 10(-4) M) induced a small hyperpolarization and a reduction in amplitude of EJP. It is suggested that contractions of locust oviduct appear to be regulated by a combination of a classical neurotransmitter such as glutamate, along with the neuromodulators octopamine and proctolin.     

Structure function analysis of an arthropod peptide hormone: proctolin and synthetic analogues compared on the cockroach hindgut receptor

Proctolin is a pentapeptide (arg-tyr-leu-pro-thr) found in nervous tissues throughout the phylum Arthropoda. Initially described as a peptidergic neuromuscular transmitter, it now appears that proctolin is a major arthropod neurohormone modulating nervous activity, muscle tonus and contractile force. Structure-function studies with synthetic analogues demonstrate diverse peptides which retain agonistic activity, but few exhibit a high degree of affinity for the cockroach hindgut receptor compared with proctolin (Kdapp = 2 x 10(-8) M). High affinity agonists (Kdapp less than or equal to 10(-7) M) are limited to [phe2]-proctolin, [lys1]-proctolin and specific N-terminal additions. In this regard the hindgut receptor differs in its ligand specificity from that reported for the locust extensor tibia receptor. Using the analogue studies to predict sequences which may act as agonists, we have examined the known vertebrate peptide hormones for proctolin-like sequences. A possible relationship between vasoactive intestinal peptide, proctolin and erythrophore concentrating hormone is critically evaluated.     

Quantification of cockroach allatostatin-like peptide and its myotropic effects in males of the earwig Euborellia annulipes

A monoclonal antibody to allatostatin I of the cockroach Diploptera punctata was used to establish a competitive enzyme‐linked immunosorbent assay for quantification of allatostatin‐like peptides in the hindgut of the adult male earwig, Euborellia annulipes. Hindguts of 0‐day males contained significantly more allatostatin‐positive material than those of 8‐day males fed on catfood. However, males starved for the first 8 days of adult life had significantly higher levels of allatostatin‐positive material than those of either 0‐day or of 8‐day fed males. Hindguts from 0‐day old males exhibited lower spontaneous motility in vitro than those from 8‐day males. Hindguts from males at both ages responded to allostatin with reversible, dosage‐dependent decreases in hindgut motility, and responded to proctolin with reversible, dosage‐dependent increases in hindgut motility. When both allatostatin and proctolin were applied to hindgut preparations simultaneously and in equal concentrations, the response varied with the stage of the male. Starvation enhanced hindgut motility and abolished the response to allatostatin, but not to proctolin. These results indicate the presence of material similar to cockroach allatostatins in male earwigs, and that the levels change with age and physiological stage. Furthermore, such peptides may indeed be regulatory neuropeptides and could modulate hindgut contraction. There was an increase in sensitivity to exogenous allatostatin in the hindgut during development from day 0 to day 8 in feeding males, but a loss in sensitivity in response to starvation; sensitivity to exogenous proctolin also increased with age, but such responsiveness was not diminished by starvation.     

Partial characterization and isolation of earwig `allatostatins'': biological activities in earwigs and cockroaches

Allatostatins are a family of neuropeptides first isolated from the cockroach, Diploptera punctata, that inhibit juvenile hormone production in that species (but do not do so in earwigs), and inhibit hindgut muscle contractions in some insects, including the earwig, Euborellia annulipes. We examined whether material from earwig brains is similar to cockroach allatostatins biochemically, immunologically and physiologically. Brain extracts from adult female earwigs were separated by high performance liquid chromatography (HPLC), followed by radioimmunoassay using antibodies to cockroach allatostatin (Dip-AST). Fractions that co-eluted with cockroach allatostatins were immunoreactive, and at least two peaks of immunoreactivity were detected. Material from each peak at 10 nM Dip-AST equivalents inhibited juvenile hormone biosynthesis in vitro by corpora allata of 2-day virgin D. punctata cockroaches; 1 nM was less effective, and non-immunoreactive fractions failed to inhibit juvenile hormone biosynthesis. Both crude and Sep-Pak (Waters) purified extracts of brains of earwigs containing 1 nM Dip-AST equivalents failed to suppress hindgut contractions in vitro of 2-day earwigs and of brooding female earwigs. In contrast, 1 nM cockroach allostatin 1 (Dip-AST 7) reversibly inhibited hindgut contractions in vitro. These results suggested the presence of another brain factor, such as proctolin, that counteracts the inhibitory effects of Dip-AST. In support of this hypothesis, proctolin stimulated hindgut contractions in vitro at 1 nM; the effects of equal concentrations of allatostatin and proctolin varied with the stage of the female. Furthermore, HPLC-separated fractions that co-eluted with cockroach allatostatin and were immunoreactive with antibodies to Dip-AST suppressed hindgut contractions in vitro of 2-day female earwigs. Finally, crude brain extracts of earwigs suppressed earwig juvenile hormone biosynthesis in vitro in glands of low, but not in glands of high, activity. Thus, earwig brain extract after HPLC separation has Dip-AST-like material that inhibits cockroach corpora allata and suppresses earwig hindgut contractions. Sep-Pak-extracted earwig brain material, however, does not inhibit earwig gut contraction. Although synthetic Dip-AST 7 does not inhibit juvenile hormone synthesis by earwig corpora allata, there is heat-stable material in earwig brain extract that does have this action.     

Comparative actions of the neuropeptides leucopyrokinin and periplanetin CCI on the visceral muscle systems of the cockroaches Leucophaea maderae and Periplaneta americana

1. The effects of two structurally-related peptides, leucopyrokinin (LPK) and periplanetin CC-I (CCI), on contractile activities of visceral muscle systems were compared in the two cockroaches from which these peptides were originally isolated.2. LPK elicited consistent proctolin-like responses on the hindgut, foregut, oviduct and heart of the Madeira cockroach, Leucophaea maderae, with increases in both amplitude and frequency of contraction. CCI, on the other hand, elicited a mostly tonic response on these tissues.3. For the American cockroach, Periplaneta americana, the responses elicited by LPK and CCI were tonic in nature.4. With the exception of the response of the L. maderae hindgut and heart to LPK, threshold levels for either LPK or CCI on all other tissues of both roaches were considerably higher (10–100 times greater) than those for proctolin on the same tissues.5. The maximum response to any concentration of LPK or CCI on the foregut and oviduct of L. maderae and that on the foregut and hindgut of P. americana never reached more than 60% of the maximum contraction achieved with proctolin.     

Comparative studies on the mode of action of proctolin and phorbol-12,13-dibutyrate in their ability to contract the locust mandibular closer muscle.

The role of proctolin has been further investigated in the locust (Locusta migratoria) mandibular closer muscles. Radioactive calcium uptake measurements were made using protease-dissociated muscle cells. Both the phorbol ester, phorbol-12,13-dibutyrate, and proctolin produce tonic contractions which are associated with the influx of extracellular calcium. The thresholds for proctolin and the phorbol ester to contract the muscle were 1-10 nM and 10-100nM, respectively, while their respective thresholds for evoking measurable calcium influx into the muscle cells were 0.1-1 nM for proctolin, and 0.1-1 pM for phorbol-12,13-dibutyrate. The effect of phorbol-12,13-dibutyrate is blocked by a number of protein kinase inhibitors (at a concentration of 0.1 mM), suggesting that an activation of a protein kinase can lead to calcium influx. These inhibitors, however, do not block the effect of proctolin, indicating that these two compounds work through different pathways, possibly converging on the same final target. In light of this finding, a number of other compounds have been tested to try to ascertain how proctolin mediates an increased calcium influx.     

Primary afferent responses of a crustacean mechanoreceptor are modulated by proctolin, octopamine, and serotonin

Modulation of sensory responses recorded intracellularly in primary sensory afferents of a crustacean proprioceptor is described. The neuropeptide proctolin enhances the sensory response, whereas the bioamines octopamine and serotonin depress it. The lobster oval organ of the second maxilla, a simple stretch receptor lacking centrifugal control, provides a useful model for studies on nonsynaptic modulation at peripheral sensory loci. Its three large afferents, X, Y, and Z, were prepared for intracellular recording and tested under five experimental conditions: (1) when fully rested, (2) when adapted to maintained stretch and firing tonically, (3) when showing reduced responses after habituation to repetitive stimulation, (4) not stretched but depolarized with current injections, (5) after TTX blockade. The results, taken together, indicate that conductances contributing to the overall amplitude of the receptor potential are major targets for modulators. Thus proctolin increased receptor potential amplitudes with consequent augmentation of spiking, whereas serotonin and octopamine depressed the receptor potentials, often to subthreshold levels with loss of spiking. Octopamine was a less potent agent than serotonin and failed to act upon fibers under TTX blockade. Fibers Y and Z consistently showed sensitivity to the modulators tested. The largest fiber, X, typically was resistant to proctolin, octopamine, and serotonin. Threshold concentrations of 10(-10)-10(-11) M determined in vitro are well below the circulating levels for serotonin and octopamine found in vivo. Proctolin, however, is usually not detectable in the hemolymph, and it is suggested that a significant site of proctolin release may be the oval organ itself.     

Pharmacological actions of a new class of neuropeptides, the leucokinins I-IV, on the visceral muscles of Leucophaea maderae

1. Leucokinins I-IV did not activate visceral muscles uniformly as a class but rather showed a selective action on the muscles of the hindgut. This organ showed a contractile response to all of the leucokinins at 3 x 10(-10) M that was 5-10% above the mean level of spontaneous activity. The maximum response for each peptide was recorded at 2.1 x 10(-7) M. 2. Both the foregut and the oviduct were 100-1000 fold less sensitive than the hindgut, and each of the former organs required more than 10(-8) M to elicit a detectable excitation. The heart, by comparison, failed to give consistent responses with any of the peptides. 3. The leucokinins caused a protracted excitation of contractile events in the hindgut that lasted for more than 60 min. Moreover, all four peptides evoked contractions from hindguts after membrane depolarization with 158 mM potassium. 4. This result shows that nonsynaptic receptors for the peptides exist in visceral muscle. The leucokinins showed no evidence of facilitating the reentry of calcium into calcium depleted hindgut preparations.     

Comparison of the myotropic activity of position-2 modified analogues of proctolin on the hindgut of Periplaneta americana and the oviduct of Locusta migratoria

The largest series of position-2 modified proctolin analogues to have been examined to date were tested for their ability to mimic the basal contraction induced by proctolin on hindgut of the cockroach, Periplaneta americana, and oviduct of the locust, Locusta migratoria. Twelve analogues of proctolin (Arg-Tyr-Leu-Pro-Thr), differing in the substituent (H, OMe, OEt, OPr, F, Cl, Br, I, NO(2), NH(2), N(3), Me) located at the para-position of the aromatic amino acid, caused dose-dependent contractions of both tissues at concentrations quite similar to proctolin. Seven showed greater or equal potency on the hindgut but, with one exception, they were less active on the oviduct than proctolin. The rank order of potency of the analogues depends on the tissue, lending more support to the notion that insects have more than one type of proctolin receptor. No relationship was observed between myoactivity and lipophilic, steric, electron donating or electron withdrawing properties of the substituents at the para-position of the aromatic amino acid. This may be the result of more than one sub-type of proctolin receptor on the specific tissue with differing structural requirements for optimum activity.     

Identification of proctolin in the central nervous system of the horseshoe crab, Limulus polyphemus

A proctolin-like peptide was isolated from the prosomal CNS of the chelicerate arthropod, Limulus, and purified using size exclusion, ion exchange and high performance liquid chromatography. Coincident bioassay (cockroach hindgut) and radioimmunoassay were employed to identify fractions which contained proctolin-like material. Proctolin-like activity coeluted with synthetic proctolin with all three chromatographic techniques employed. When applied to either the Limulus heart or hindgut preparations, purified Limulus proctolin produced excitatory responses which were indistinguishable from those produced by the synthetic peptide. Purified samples of the Limulus proctolin-like peptide were subjected to Edman degradation and tandem mass spectrometry and the amino acid sequence of the Limulus peptide was determined to be identical to that of cockroach proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH). The presence of proctolin in the Limulus CNS and its biological action on the isolated heart and hindgut suggest a physiological role for this peptide in the regulation of cardiac output and hindgut motility.     

Pharmacological properties of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate receptors associated with the crayfish hindgut

Pharmacological agents were used to characterize glutamate receptors associated with crayfish hindgut. l-Glutamate reliably increased tonus in isolated hindguts of Procambarus clarkii and suppressed spontaneous hindgut contractions at concentrations of 10 micromol l(-1) or higher. Quisqualate and ibotenate mimicked the effects of L-glutamate. Experiments with strips and rings of hindgut tissue indicate that glutamate acts on both circular and longitudinal muscles. Hindgut contractions were not affected by (+/-)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionoic acid, N-methyl- d-aspartate, domoate or the metabotropic glutamate receptor agonist, (1S,3R)-1-amino-1-carboxycyclopentane-3-acetic acid. Picrotoxin, at 50 micromol l(-1), did not alter the ibotenate-induced reduction in contraction frequency, suggesting that this effect is not produced by inhibitory glutamate receptors. The glutamate-induced increase in tonus was antagonized by Joro spider toxin, JSTX-3. Thus, glutamate receptors associated with crayfish hindgut muscles are of the quisqualate type but are also sensitive to ibotenate. Elevating extracellular potassium concentration mimicked all of the effects of glutamate, suggesting that excessive depolarization may contribute to the suppression of contractions at high agonist concentrations.     

Isolation and partial characterization of five myotropic peptides present in head extracts of the cockroach Leucophaea maderae

Five peptides were isolated by reverse-phase HPLC from head extracts of the cockroach Leucophaea maderae. Four of the peptides were inactivated by aminopeptidase M (APM). The inability of APM to digest the fifth peptide suggests a blocked NH2-terminus. Four of the peptides were inactivated by carboxypeptidase Y (CPY). The activity of the fraction which would have contained proctolin was decreased by about 20%. The complete deactivation of proctolin by CPY indicated that a second peptide, co-eluting with proctolin but refractory to CPY digestion, was responsible for 80% of the biological activity in that fraction. Concentrations of the peptides necessary to produce a threshold response from the isolated cockroach hindgut ranged from 0.009 to 0.083 head equivalents/ml.     

Pharmacological actions of the calcium antagonist propyl-methylenedioxyindene in skinned vascular smooth muscle

Previous studies provided strong evidence that propyl-methylenedioxyindene (pr-MDI) interfered with calcium at an intracellular site. To further characterize the mechanism of action of pr-MDI, its pharmacological actions on chemically skinned vascular smooth muscle were examined. Rat caudal artery strips were chemically skinned with saponin (0.15 mg/mL for 1 h). The efficiency of the skinning was evidenced by a loss of contractile response to 74 mM K+. The intactness of the regulatory and contractile proteins was ascertained by the ability of the skinned tissue to contract in response to Ca2+ (free Ca2+ concentration of 10(-4) or 10(-6)M). Caffeine (25 mM) induced contraction was used as an index of the functional integrity of the sarcoplasmic reticulum in the skinned preparations. Contraction of the skinned artery with a free Ca2+ concentration of 10(-6)M was significantly obtunded by 1 X 10(-4)M trifluoperazine (a calmodulin antagonist) but not by 1 X 10(-4)M pr-MDI. Contraction of the skinned artery evoked by 25 mM caffeine in the absence of extracellular calcium was significantly obtunded by 1 X 10(-4)M pr-MDI but not by 1 X 10(-6)M nifedipine (a calcium channel blocker). The results indicate that pr-MDI acts intracellular to block calcium mobilization from the sarcoplasmic reticulum without directly interfering with the regulatory and contractile proteins.     

Ventral Horn Neuropeptides Modulate the Release of Noradrenaline from Tissue Slices of Rat Brainstem and Ventral Thoracic Spinal Cord

The release of endogenous noradrenaline (NA) from slices of adult rat brainstem and ventral thoracic spinal cord was investigated using a fixed-volume incubation technique and HPLC with electrochemical detection. Incubation with potassium (15-50 mM) produced a dose-related increase in basal NA release that was calcium dependent. The potassium-evoked release of NA from spinal cord or brainstem slices was potentiated according to dose by preincubation with either (a) the selective alpha 2-adrenoceptor antagonist idazoxan (10(-6)-10(-4) M) or (b) the thyrotrophin-releasing hormone (TRH) analogue RX 77368 (pGlu-His-3,3'-dimethyl ProNH2; 10(-5) and 10(-4) M). Incubation of spinal cord slices with the NA uptake inhibitor maprotiline (1 microM) enhanced the effect of idazoxan but inhibited that of RX 77368. The effects of RX 77368 and potassium alone (15 mM) on NA release from both spinal cord and brainstem slices were reduced to basal levels with tetrodotoxin (10(-7) M). Similarly, preincubation of spinal cord, but not brainstem, slices with the insect neuropeptide proctolin (10(-4) M) significantly attenuated the potassium- or RX 77368-induced release of NA, whereas substance P (3 X 10(-5) and 1 X 10(-4) M) had no effect on either tissue. These results suggest that changes in NA release in the spinal cord and brainstem may mediate some of the actions of neuropeptides in ventral spinal cord, although the peptides may not be acting directly on the noradrenergic nerve terminals in these tissues.     

The association of serotonin with the alimentary canal of the African migratory locust, Locusta migratoria: distribution, physiology and pharmacological profile

The association of serotonin with the alimentary canal of Locusta migratoria was investigated using immunohistochemistry and high performance liquid chromatography (HPLC) coupled to electrochemical detection. Serotonin-like immunoreactive processes were differentially distributed between and within three regions of the alimentary canal; the foregut, midgut and hindgut. The midgut possessed the most serotonin-like immunoreactive processes, while the hindgut contained only a few immunoreactive processes. Using HPLC coupled to electrochemical detection the serotonin content was highest in the midgut followed by the foregut and hindgut. The physiological response of the midgut to serotonin as well as to the combination of serotonin and proctolin was also examined. It was found that the application of serotonin to the midgut leads to a dose-dependent reduction in tonus of the circular muscles. Serotonin was also able to inhibit a proctolin-induced contraction of the midgut in a dose-dependent manner. The physiological and pharmacological properties of serotonin agonists and antagonists on the midgut were also investigated. The results indicate that alpha-methyl 5-HT was the most effective agonist leading to a 108% relaxation at 10(-9) M compared to that caused by the same serotonin concentration. Among several serotonin receptor antagonists tested, mianserin was the most potent. The application of mianserin at 10(-5) M in combination with 5x10(-6) M serotonin resulted in a 66% reduction of the serotonin-induced relaxation of midgut muscle. The serotonin antagonist cyproheptadine was less effective leading to a 39% reduction of the 5x10(-6) M serotonin-induced relaxation. Ketanserin was a weak antagonist.