Structure of the pentapeptide proctolin, a proposed neurotransmitter in insects.

Proctolin, a myotropic substance with potent activity on the proctodeal (hindgut) muscles of the cockroach, $\text{Periplaneta americana}$ (L.), has been identified by the use of Edman degradation and dansylation techniques as Arg-Tyr-Leu-Pro-Thr. Synthesis of the pentapeptide having this sequence and exhibiting the properties of natural proctolin confirmed the structure. Threshold activity on proctodeal muscle occurs at about 10⁻⁹ M proctolin.     

Experimental studies upon a bundle of tonic fibres in the locust extensor tibialis muscle

The bundle of tonic fibres situated at the proximal end of the locust metathoracic extensor tibialis muscle is innervated by the dorsal unpaired median neurone (DUMETi) as well as by the slow excitatory (SETi)) and common inhibitor (CI) neurones. It is not innervated by the fast excitatory neurone (FETi).These fibres contract spontaneously and rhythmically. The myogenic rhythm can be modified by neural stimulation.Spontaneous slow depolarizing potentials resembling the pacemaker potentials of insect cardiac muscle were demonstrated in these fibres.The actions of glutamate on the tonic muscle fibres are not compatible with its being a specific excitatory transmitter. Glutamate can stimulate weak contractions of the muscle, but this action is inhibited when chloride ions are removed from the saline.10^?6 M Octapamine hyperpolarizes the tonic fibre membrane. Octopamine, GABA and glutamate all inhibit the myogenic contractions and reduce the force of the neurally evoked contractions.The tonic muscle is very responsive to proctolin. At 5 × 10^?11 M proctolin enhances the force and increases the frequency of myogenic contractions. At 10^?9 M it depolarizes the muscle membrane potential, and at that and higher concentrations it causes the muscle to contract. At 2 × 10^?7 M proctolin induces contractures which resemble those evoked by sustained high-frequency neural stimulation. Iontophoretic experiments show that proctolin receptors occur at localized sites on the tonic fibre membrane.     

Some pharmacological properties of neuromuscular transmission in the oviduct of the locust,Locusta migratoria

The effects of various pharmacological agents on neurally evoked contractions of the visceral muscles of the oviduct of Locusta migratoria have been examined. The pentapeptide, proctolin, at low concentrations (10^?11 M?10^?10 M), induced an increase in the amplitude of neurally evoked contractions and basal tonus, and induced the appearance and increased the frequency of myogenic contractions. Glutamate, at 10^?4 M, produced a small transient contraction which in some preparations was accompanied by a reduction in amplitude of neurally evoked contractions. Octopamine, at 10^?6 M, reduced the amplitude of neurally evoked contractions and also resulted in a relaxation of the muscles. The octopaminergic effects were inhibited by the α-aminergic antagonist phentolamine. Neurally evoked contractions were unaffected by dopamine, 5-HT or the acetylcholine receptor antagonists atropine and hexamethonium. Acetylcholine increased the amplitude of neurally evoked contractions, but only at the high concentration of 10^?3 M. The possible role of proctolin and glutamate as excitatory neuro-transmitters and the inhibitory action of octopamine is discussed.     

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.     

5-HT receptors on identified Lymnaea neurones in culture. Pharmacological characterization of 5-HT1A receptors

The ability of the selective 5-HT_1A receptor agonist R(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) to bind with 5-HT receptor(s) on cultured, identified neurones in Lymnaea stagnalis was examined. The identified neurones studied were from the buccal ganglia and the serotonin-containing cerebral giant cells (CGCs). 5-HT and its agonists were applied from puffer pipettes, whilst 5-HT antagonists were applied in the bathing medium. At 10⁻³ M, the 5-HT_1A agonist, always produced paroxysmal depolarizing shifts (PDS) while at a lower concentration (10⁻⁴ M), it always mimicked the effects of 10⁻³ M 5-HT. 8-OH-DPAT (10⁻⁴ M) and 5-HT 10⁻³ M produced dose-dependent increases in the responses they evoked. At 10⁻⁴ M, the 5-HT₃ receptor agonist 1-(m-chlorophenyl)-biguanide hydrochloride (m-CPBG), failed to hyperpolarize most of the neurones hyperpolarized by 5-HT. At 10⁻⁴ M, the antagonists ketanserin (5-HT₂), MDL 72222 (5-HT₃), and pindobind-5-HT_1A (5-HT_1A) consistently abolished spike generation ii spontaneously active neurones. Both ketanserin and MDL 72222 failed to block the actions of 8-OH-DPAT and only partially blocked those of 5-HT, but pindobind-5-HT_1A completely, but reversibly,blocked the 8-OH-DPAT effects while greatly reducing those of 5-HT. These results suggest that 5-HT_1A receptor subtypes might be involved in the hyperpolarizing responses of the CGCs and their follower motor neurones in the buccal ganglia of Lymnaea stagnalis to 5-HT. The presence of 5-HT_1A receptors on these neurones can be considered to correspond with those found in mammals because their pharmacological responses resemble those of mammalian 5-HT_1A receptors.     

Excitatory neurotransmitters in the tentacle flexor muscles responsible for space positioning of the snail olfactory organ

Recently, three novel flexor muscles (M1, M2 and M3) in the posterior tentacles of the snail have been described, which are responsible for the patterned movements of the tentacles of the snail, Helix pomatia. In this study, we have demonstrated that the muscles received a complex innervation pattern via the peritentacular and olfactory nerves originating from different clusters of motoneurons of the cerebral ganglia. The innervating axons displayed a number of varicosities and established neuromuscular contacts of different ultrastructural forms. Contractions evoked by nerve stimulation could be mimicked by external acetylcholine (ACh) and glutamate (Glu), suggesting that ACh and Glu are excitatory transmitters at the neuromuscular contacts. Choline acetyltransferase and vesicular glutamate transporter immunolabeled axons innervating flexor muscles were demonstrated by immunohistochemistry and in Western blot experiments. Nerve- and transmitter-evoked contractions were similarly attenuated by cholinergic and glutamatergic antagonists supporting the dual excitatory innervation. Dopamine (DA, 10^?5 M) oppositely modulated thin (M1/M2) and thick (M3) muscle responses evoked by stimulation of the olfactory nerve, decreasing the contractions of the M1/M2 and increasing those of M3. In both cases, the modulation site was presynaptic. Serotonin (5-HT) at high concentration (10^?5 M) increased the amplitude of both the nerve- and the ACh-evoked contractions in all muscles. The relaxation rate was facilitated suggesting pre- and postsynaptic site of action. Our data provided evidence for a DAergic and 5-HTergic modulation of cholinergic nerves innervating flexor muscles of the tentacles as well as the muscles itself. These effects of DA and 5-HT may contribute to the regulation of sophisticated movements of tentacle muscles lacking inhibitory innervation.     

Development of cross-tolerance to 5-hydroxytryptamine in organotypic cultures of mouse spinal cord-ganglia during chronic exposure to morphine

Exposure of organotypic explants of mouse spinal cord with attached dorsal root ganglia (DRGs) to low concentrations (～10nM) of 5-hydroxytryptamine (5-HT) markedly depressed sensory-evoked dorsal-horn network responses, resembling the acute effects of opioids in these cultures. Attenuation of cord responses by 5-HT was not prevented by exposure to the 5-HT antagonists, methysergide and cyproheptadiene, nor to the opiate antagonist, naloxone. Explants that had become tolerant to morphine after chronic exposure (1 μM) for > 2 days often developed cross-tolerance to 5-HT. Acute exposure of morphine-tolerant explants to naloxone (1 μM) further attenuated the effects of 5-HT so that the minimum depressant levels of 5-HT were often increased up to 30-fold. Increasing the extra-cellular Ca⁺⁺ concentration (to 5 mM) and/or introduction of 4-aminopyridine markedly antagonized the depressant effects of 5-HT on DRG-evoked cord responses, so that 5-HT levels comparable to those used on morphine-tolerant explants were required to depress naive explants. These depressant effects of 5-HT on cord-DRG explants are consonant with antinociceptive actions of 5-HT administered to dorsal cord $\text{in situ}$. Our data suggest that 5-HT may block neuronal components of dorsal horn networks at similar regions to those that are depressed by opiates, e.g. presynaptic DRG nerve terminals where abundant opiate receptors are located. The marked attenuation of the depressant effects of both 5-HT and opiates on cord-DRG explants by high Ca⁺⁺ raises the possibility that cross-tolerance to 5-HT in morphine-tolerant explants may result from the same neuronal alterations that render dorsal-horn networks tolerant to opiates. Furthermore, the increased degree of cross-tolerance to 5-HT after acute introduction of naloxone in morphine-tolerant cultures may be an expression of opiate dependence.     

Effects of FMRFamide-related peptides and morphine on the isolated foregut of the locust schistocerca gregaria

1. Morphine and YAGFMamide were the most effective potentiators of 5-hydroxytryptamine (5-HT)-induced relaxation of the isolated foregut.2. Morphine had no effect on proctolin-induced tissue contraction which was inhibited by YGGF-Mamide and YFMRFamide.3. The differing potency of FaRPs and morphine to potentiate 5-HT effects and reduce proctolin responses suggests that there are two separate FaRP receptor sub types.4. This proposal is supported by the observation that, while naloxone (10⁻⁵ M) is a relatively potent antagonist of FaRP induced inhibition of proctolin contraction, it has less effect on FaRP-induced potentiation of 5-HT-induced relaxation.     

Occurrence of proctolin in six orders of insects

Nine representatives of six orders of insects (Orthoptera, Diptera, Coleoptera, Hemiptera, Hymenoptera, Lepidoptera) were extracted and partially processed by means used in the recent isolation of proctolin, a pentapeptide transmitter candidate in insects. Each insect yielded a substance with pharmacological activity on cockroach proctodeal muscle similar to that of proctolin. Like the responses evoked by proctolin and nerve stimulation, responses to the purified extracts were inhibited by tyramine. All of the active substances behaved as proctolin when subjected to paper chromatography or high voltage paper electrophoresis at pH 6.4 and 3.5. Proctolin appears to be widely and perhaps universally present in the Insecta occurring in most at levels of 2 to 9 μg/kg body weight.     

Isolation of proctolin,a myotropic peptide,from Periplaneta americana

The isolation of a myotropic substance, proctolin, associated with the innervation of the viscera of the cockroach, Periplaneta americana, is reported. Proctolin, previously described as a neurotransmitter or neurohormone, is a small basic peptide with an estimated molecular weight of 500 to 700. The proctodeal (hindgut) muscles of P. americana contract at threshold levels of about 0·5 ng/ml, equivalent to about 10⁻⁹ M proctolin. Approximately 180 μg proctolin were isolated from 125 kg of whole cockroaches. The isolated peptide was homogeneous on paper and thin-layer chromatography and on high-voltage paper electrophoresis at pH 3·5 and 6·4.     

Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.     

Neosurugatoxin blocks nicotinic acetylcholine receptors in the brain

Neosurugatoxin, a neurotoxin isolated from the Japanese ivory mollusc ($\text{Babylonia japonica}$) is a nicotinic antagonist with a specificity towards ganglionic nicotinic receptors. At low concentration (5 × 10^?8 M) neosurugatoxin inhibited the release of [³H]dopamine evoked by 1,1-dimethyl-4-phenylpiperazinium (DMPP) from rat striatal nerve terminals, without affecting the response to K⁺-depolarisation. In contrast, αbungarotoxin did not antagonise the action of DMPP. Neosurugatoxin also inhibited [³H] nicotine binding to rat brain membranes but had no effect on [¹²⁵I]αbungarotoxin binding to the same tissue preparation. These results support the view that functional nicotinic receptors in the CNS resemble ganglionic nicotinic receptors. Neosurugatoxin has considerable potential as a useful probe for such receptors in the brain.     

The pharmacology of the isolated foregut of the locust schistocerca gregaria—I. the effect of a range of putative neurotransmitters

1. Isolated locust foreguts exhibited little or no spontaneous contractile activity.2. Proctolin (10⁻⁹m-10⁻⁶m) caused rapid and powerful contraction of the tissue while the response to similar doses of l-glutamate was much weaker.3. 5-HT (10⁻⁸M-5 × 10⁻⁶M), acting at ketanserin-sensitive receptors, was the most powerful tissue relaxant tested.4. Relaxation caused by octopamine (10⁻⁷M-10⁻⁵M), acting at ketanserin-insensitive receptors was less than 50% of that caused by similar concentrations of 5-HT.5. ACh (10⁻⁵M-10⁻³M) induced relaxation, mimicked by nicotine and antagonised by d-tubocurarine, was seen only in the presence of the anti-cholinesterase neostigmine.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

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.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

Effects of norepinephrine and acetylcholine on 32P incorporation into phospholipids of the rabbit iris muscle following unilateral superior cervical ganglionectomy.

The effect of norepinephrine and acetylcholine on the ³²P incorporation into phospholipids of normal and sympathetically denervated rabbit iris muscle was investigated. (1) In the absence of exogenously added neurotransmitters sympathetic denervation exerted little effect on the incorporation of ³²P into the phospholipids of the excised iris muscle. $\text{In vivo}$ thr iris muscle incorporated ³²P into phosphatidylinositol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin in that order of activity while $\text{in vitro}$ phosphatidylinositol was followed by phosphatidylcholine. (2) Tension responses of iris dilator muscle from denervated irises exhibited supersensitivity to norepinephrine. Furthermore, norepinephrine at concentrations of 3 μM and 30 μM produced 1.6 times and 3 times stimulation of the phosphatidic acid of the denervated muscle respectively. In contrast at 30 μM it stimulated this phospholipid by 1.6 times in the normal muscle. This stimulation was completely blocked by phentolamine. (3) While in the normal muscle acetylcholine stimulated the labelling of phosphatidic acid and phosphatidylinositol by more than 2 times, in the denervated muscle it only stimulated 1.4 to 1.7 times. (4) Similarly when ³²Pi was administered intracamerally, the labelling found in the various phospholipids of the denervated iris was significantly lower than that of the normal. (5) It was concluded that denervation decreases the ³²P labelling in the presence of acetylcholine. (6) The norepinephrine-stimulated ³²P incorporation into phosphatidic acid appears to be post-synaptic.     

Effects of the cilioexcitatory neurohumors dopamine and 5-hydroxytryptamine on cyclic AMP levels in the gill of the mussel Mytilus edulis.

Cyclic 3′, 5′-adenosine monophosphate (cAMP) has been identified in the ciliated gill epithelium of the marine mussel $\text{Mytilus}$$\text{edulis}$. In concentrations which stimulate the rate of particle transport by frontal gill cilia, DA and 5HT stimulate levels of cAMP within the gill. The stimulation occurs in as early as 15 sec and is graded from 10^?6M to 10^?4M. DA plus 5HT is not additive at maximal effective concentrations of both amines. ACH does not mimic the DA or 5HT stimulation of cAMP. Theophylline alone has a weak effect on cAMP levels; however, the effect of theophylline is potentiated in the presence of DA or 5HT. Dibutyryl cAMP produces a gradual stimulation in the rate of particle transport. It is suggested that the dopaminergic and serotonergic excitatory control of particle transport by frontal gill cilia of $\text{Mytilus}$$\text{edulis}$ is mediated through a cAMP second messenger system.     

Sympathetic histamine exerts different pre- and post-synaptic functions according to the frequencies of nerve stimulation in guinea pig vas deferens

Histamine (HA) was found to be present in the sympathetic nerve terminals of guinea pig hearts and vasa deferentia in our previous study; however, little is known about the functions of this neurogenic HA. In this study, we used guinea pig vasa deferentia to investigate the pre- and post-synaptic functions of HA evoked by different frequencies of sympathetic nerve stimulation. We found that sympathetic nerve stimulation could evoke HA release, which was independent to mast cell degranulator compound 48/80 and mast cell stabilizer cromolyn, but was highly sensitive to Na⁺ channel blocker tetrodotoxin and chemical sympathectomy with 6-hydroxydopamine. The neurogenically released HA evoked by 12.5 Hz of nerve stimulation activated only pre-synaptic H₃ receptors and mediated pre-synaptic inhibitory effects, while under 25 or 50 Hz stimulation condition, HA simultaneously activated both pre-synaptic H₃ receptors and post-synaptic H₁ receptors. However, the direct contractile responses evoked by sympathetic HA via H₁ receptors were observed at 50 Hz. HA release and HA-mediated contractile responses upon sympathetic nerve stimulation were significantly inhibited by pre-treatment of histidine decarboxylase inhibitor α-fluoromethylhistidine. Furthermore, application of exogenous HA could mimic these pre- and post-synaptic effects. Our findings indicate that HA in sympathetic neurons acts as a neurotransmitter and its functions vary from pre-synaptic inhibition, to post-synaptic facilitation, to direct post-synaptic contractile responses according to sympathetic nerve stimulation frequencies.