African Snail Achatina Fulica Research Articles

Variation of acharan sulfate and monosaccharide composition and analysis of neutral N-glycans in African giant snail (Achatina fulica).

Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide -->4)-alpha-D-GlcNpAc (1-->4)-alpha-L-IdoAp2S(1-->, analyzed by SAX (strong-anion exchange)-HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex(2-4)-HexNAc(2)), high mannose (Hex(5-9)-HexNAc(2)), and complex (Hex(3)-HexNAc(2-10)) types. None showed core fucosylation.

Ocurrence of nematode larvae in Achatina fulica Bowdich, 1822 (Gastropoda:Achatinidae) snails in vargem Pequena, Rio de Janeiro, Brazil

This paper reports the occurrence of nematode larvae in the giant African snail Achatina fulica inRio de Janeiro City, Brazil. A total of 78 snails was collected in four out of 22 localities in VargemPequena neighborhood between August and December of 2005. In one of the localities (NovoHorizonte residential complex), two forms of nematode larvae were found, which were unsheathedand displayed oxyuroid esophagus. The large and stout form is a third-stage larva with a striatedcuticle and a short undulating tail. The small and slender form exhibited esophago-intestinaland intestinal valves. The prevalence of larval nematodes in A. fulica snails in Vargem Pequenaneighborhood was 4.8%. Additional studies are strongly recommended to identify the nematodespecies found and to determine whether this snail species constitutes a public health concern.

Neurotoxicity of a Novel Local Anesthetic Agent, Ropivacaine: The Possible Roles of Bursts of Potential and Cytoplasmic Second Messenger

Ropivacaine has been shown to induce convulsion following overdose or accidental intravenous injection, but the mechanisms are poorly understood. Using an identifiable central neuron from giant African snail, the authors studied the mechanism of ropivacaine-elicited bursts of potential and explored the possible mechanisms of ropivacaine-induced neurotoxicity. Ropivacaine action on a central neuron (RP4) of the giant African snail (Achatina fulica Ferussac) was recorded by conventional electrophysiologic technique. Interactions between ropivacaine and prazosin, propranolol, atropine, d-tubocurarine, calcium-free solution, H89, U73,122, neomycin, high-magnesium solution, and chelerythrine were also observed. The RP4 neuron showed spontaneous firing of action potentials. Extracellular application of ropivacaine (900 microM) reversibly elicited bursts of potential in the RP4 neuron. The bursts of potential elicited by ropivacaine were not blocked after administration of: (1) prazosin, propranolol, atropine, d-tubocurarine; (2) calcium-free solution; and (3) pretreatment with H89 or chelerythrine. The bursts of potential elicited by ropivacaine were blocked by pretreatment with U73122 (30 microM) or by adding neomycin (3.5 mM) or high-magnesium solution (30 mM). Ropivacaine reversibly elicited bursts of potential in the central snail neuron. The ropivacaine-elicited bursts of potential were associated with phospholipase C activity in the RP4 snail neuron. Our results suggest that ropivacaine-induced neurotoxicity is highly associated with phospholipase C activity and phospholipase C inhibitor may offer a novel therapeutic approach for managing local anesthetic-induced convulsion or other transient neurologic toxicity.

Amphetamine-produced convulsive (bursting) firings in the neuron of the giant African snail Achatina fulica: Effects of inhibitors of phosphodiesterases

Effects of inhibitors of phosphodiesterases (PDE) on bursting activity, produced by d-amphetamine (d-AM) was studied in PPa4 neurons of the giant African snail Achatina fulica F. Action of the following PDE inhibitors was analyzed: vinpocetine (selective to PDE I), erythro-9-(2-hydroxi-3-nonyl) adenine (EHNA; selective to PDE 2), milrinone (selective to PDE 3), rolipram (selective to PDE 4), sildenafil citrate (Viagra@; selective to PDE 5), and caffeine, a non-selective phosphodiesterase inhibitor. Amphetamine at a low concentration (67.5 × 10−5 M) did not produce the bursting firing in the neurons; however, the convulsive activity appeared on addition to this solution of any if the PDE inhibitors except for sildenafil. Forskolin (an adenylyl cyclase activator, 10−4 M) also decreased the neuronal threshold to the d-AM action. The bursting activity produced by d-AM did not develop after a previous administration of the protein kinase A inhibitor KT-5720. The phospholipase C blocker U73122 had no effect on the bursting activity produced by d-AM. It is concluded that the neuronal convulsive activity induced by d-AM is associated with the phosphodiesterase activity.

The Three-Dimensional Electrical Field Around the Body of the African Snail Achatina Fulica on Activation of the Giant Neurons

The Three-Dimensional Electrical Field Around the Body of the African Snail Achatina Fulica on Activation of the Giant Neurons

Neuroeffector Connections of Giant Multimodal Neurons in the African Snail Achatina Fulica

A new method of making preparations was used to analyse the neuroeffector connections of the paired giant neurons of the African snail Achatina fulica. These neurons were found to induce postsynaptic potentials in the muscles of the mantle, heart, the wall of the pulmonary cavity, and the muscular elements of the renal complex, the pericardium, the sexual apparatus, the walls of the cerebral arteries, the filaments of the columellar muscles, the wall of the abdomen, and the tentacle retractor muscles. Rhythmic neuron activity led to the development of marked facilitation and long-term potentiation of synaptic potentials. The possible significance of the multiple neuroeffector connections of giant neurons is discussed.

Effects of rolipram on induction of action potential bursts in central snail neurons

Effects of rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of the giant African snail ( Achatina fulica Ferussac). Oscillations of membrane potential bursts were elicited by rolipram and forskolin. The bursts of potential elicited by rolipram were not inhibited after administration with (a) calcium-free solution, (b) high-magnesium solution (30 mM) or (c) U73122. However, the bursts of potential elicited by rolipram were inhibited by pretreatment with KT-5720 (10 μM). Voltage-clamp studies revealed that rolipram decreased the total inward current and steady-state outward currents of the RP4 neuron. The negative slope resistance (NSR) was not detectable in control or rolipram treated RP4 neurons. TEA elicited action potential bursts and an NSR at membrane potential between −50 mV and −30 mV. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters; (2) NSR of steady-state I– V curve; (3) phospholipase activity of the neuron. The rolipram-elicited bursts of potential were dependent on the phosphodiesterases inhibitory activity and the cAMP signaling pathway in the neuron.

Effects of 2,3-Butanedione Monoxime on Induction of Action Potential Bursts in Central Snail Neurons: Direct and Indirect Modulations of Ionic Currents

The effects of 2,3-butanedione monoxime (BDM) on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of giant African snail (Achatina fulica Ferussac). The effect of okadaic acid on the neuron was also tested. The RP4 neuron showed a spontaneous firing of action potential. Okadaic acid (1 µmol/l) did not alter the frequency of spontaneous action potential while BDM (3 mmol/l) reversibly elicited bursts of potential (BoP) of the RP4 neuron. The BoP elicited by BDM (3 mmol/l) were reversed 20 min after incubation with diazoxide (500 µmol/l) while the BoP were not altered in preparations treated with okadaic acid and BDM. The BDM-elicited BoP were not inhibited after administration with (a) hexamethonium (100 µmol/l), (b) atropine (1 mmol/l), (c) d-tubocurarine (100 µmol/l), (d) prazosin (100 µmol/l), (e) propranolol (100 µmol/l), (f) calcium-free solution, (g) high K⁺ (12 mmol/l) or (h) with high Mg²⁺ (30 mmol/l) solutions. The BDM-elicited BoP were inhibited by pretreatment with KT-5720 (10 µmol/l) or H89 (10 µmol/l), the protein kinase A inhibitors. However, the BoP were not affected after application of chelerythrine (10 µmol/l) or Ro 31-8220 (10 µmol/l), the protein kinase C inhibitors. Voltage-clamped studies revealed that BDM elicited a negative slope resistance (NSR) at membrane potentials between –50 and –10 mV. The NSR was not detectable at the same membrane potential in control RP4 neuron. It is suggested that the BoP elicited by BDM were not due to (1) the synaptic effects of neurotransmitters; (2) the activation of cholinergic, adrenergic receptors, or (3) phosphatase activity of the neuron. The BDM-elicited BoP were dependent on the protein kinase A related cAMP in the neuron and the delayed outward K⁺ current may contribute to the BDM-elicited BoP.

Espécie invasora em reservas naturais: caracterização da população de Achatina fulica Bowdich, 1822 (Mollusca - Achatinidae) na Ilha Rasa, Guaraqueçaba, Paraná, Brasil

O caramujo africano Achatina fulica é uma das cem espécies invasoras do mundo, causando sérios danos principalmente em ilhas. Objetivou-se caracterizar a população de A. fulica na Ilha Rasa, Guaraqueçaba, PR, Brasil. Coletas sazonais diurnas da A. fulica e fauna associada foram conduzidas na borda e interior de floresta, restinga, mangue e área urbana das comunidades de Almeida e Rasa. Foram coletados 959 caramujos, com uma média de 66,4 animais vivos por terreno vistoriado, estimando-se que haja em torno de 9.000 caramujos na ilha. No entanto a freqüência dos animais esteve condicionada a época do ano, tamanho do animal e comunidade. A pequena e recente população de A. fulica associada ao ambiente antrópico demanda um plano de erradicação urgente e eficaz evitando o seu estabelecimento e a dispersão para as áreas nativas.

Increases in urea synthesis and the ornithine-urea cycle capacity in the giant African snail,Achatina fulica, during fasting or aestivation, or after the injection with ammonium chloride

The objectives of this study are to determine whether a full complement of ornithine-urea cycle (OUC) enzymes is present in the hepatopancreas of the giant African snail Achatina fulica, and to investigate whether the rate of urea synthesis and the OUC capacity can be up-regulated during 23 days of fasting or aestivation, or 24 hr post-injection with NH(4)Cl (10 micromol g(-1) snail) into the foot muscle. A. fulica is ureotelic and a full complement of OUC enzymes, including carbamoyl phosphate synthetase III (CPS III), was detected from its hepatopancreas. There were significant increases in the excretion of NH(4)(+), NH(3) and urea in fasting A. fulica. Fasting had no significant effect on the tissue ammonia contents, but led to a progressive accumulation of urea, which was associated with an 18-fold increase in the rate of urea synthesis. Because fasting took place in the presence of water and because there was no change in water contents in the foot muscle and hepatopancreas, it can be concluded that the function of urea accumulation in fasting A. fulica was unrelated to water retention. Aestivation in arid conditions led to a non-progressive accumulation of urea in A. fulica. During the first 4 days and the last 3 days of the 23-day aestivation period, experimental snails exhibited significantly greater rates of urea synthesis compared with fasted snails. These increases were associated with significant increases in activities of various OUC enzymes, except CPS III, in the hepatopancreas. However, the overall urea accumulation in snails aestivated and snails fasted for 23 days were comparable. Therefore, the classical hypothesis that urea accumulation occurred to prevent water loss through evaporation during aestivation in terrestrial pulmonates may not be valid. Surprisingly, there were no accumulations of ammonia in the foot muscle and hepatopancreas of A. fulica 12 or 24 hr after NH(4)Cl was injected into the foot muscle. In contrast, the urea content in the foot muscle of A. fulica increased 4.5- and 33-fold at hour 12 and hour 24, respectively, and the respective increases in the hepatopancreas were 4.9- and 32-fold. The exogenous ammonia injected into A. fulica was apparently detoxified completely to urea. The urea synthesis rate increased 148-fold within the 24-hr experimental period, which could be the greatest increase known among animals. Simultaneously, there were significant increases in activities of glutamine synthetase (2.5-fold), CPS III (3.1-fold), ornithine transcarbamoylase (2.3-fold), argininosuccinate synthetase+lyase (13.6-fold) and arginase (3.5-fold) in the hepatopancreas 12 hr after the injection of NH(4)Cl. Taken altogether, our results support the view that the primary function of urea synthesis through the OUC in A. fulica is to defend against ammonia toxicity, but suggest that urea may have more than an excretory role in terrestrial pulmonates capable of aestivation.

Effects of procaine on a central neuron of the snail, Achatina fulica Ferussac

Effects of procaine on a central neuron (RP1) of the giant African snail ( Achatina fulica Ferussac) were studied pharmacologically. The RP1 neuron showed spontaneous firing of action potential. Extra-cellular application of procaine (10 mM) reversibly elicited bursts of potential. The bursts of potential elicited by procaine were not blocked after administration of (1) prazosin, propranolol, atropine, d-tubocurarine, (2) calcium-free solution, (3) ryanodine (4) pretreatment with KT-5720 or chelerythrine. The bursts of potential elicited by procaine were blocked by adding U73122 (10 μM) and the bursts of potential were decreased if physiological sodium ion was replaced with lithium ion or incubated with either neomycin (3.5 mM) or high magnesium solution (30 mM). Preatment with U73122 (10 μM) blocked the initiation of bursts of potential. Ruthenium red (100 μM) or caffeine (10 mM) facilitated the procaine-elicited bursts of potential. It is concluded that procaine reversibly elicits bursts of potential in the central snail neuron. This effect was not directly related to (1) the extra-cellular calcium ion fluxes, (2) the ryanodine sensitive calcium channels in the neuron, or (3) the PKC or PKA related messenger systems. The procaine-elicited bursts of potential were associated with the phospholipase activity and the calcium mobilization in the neuron.

Nucleolin: acharan sulfate-binding protein on the surface of cancer cells.

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure alpha-D-N-acetylglucosaminyl (1-->4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid-Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 microg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS-PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight approximately 110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells.

Long duration of anticoagulant activity and protective effects of acharan sulfate in vivo

Introduction: We previously reported that a new glycosaminoglycan, acharan sulfate (AS) from the African giant snail Achatina fulica showed anticoagulant activity in vitro, but was much less active when compared to heparin. In the present study, the anticoagulant activity of AS was investigated in vivo. Methods: AS and heparin were administered to mice and rats in various doses and the anticoagulant activities were measured by aPTT assay. Both were also compared in a thrombin-induced lethality animal model. As one of the possible mechanisms, AS-thrombin interaction was studied by using surface plasmon resonance spectroscopy. Results: Intravenous administration of AS to mice prolonged the clotting time (aPTT) in a time and dose-dependent manner. Although the anticoagulant activity was low in rats, it steadily increased over 5 h after administration of AS (30 mg/kg). In contrast, the increase in aPTT induced by 5 mg/kg of heparin was restored to a normal level after 3 h. In a thrombin-induced lethality model in mice, AS (20 mg/kg) protected against lethality by 80%, while heparin (20 mg/kg) did not show any protective activity beyond 3.5 h post-administration. AS could be also detected in plasma even 5 h after i.v. administration to rats. The binding constant ( K D) of AS to thrombin was 7.27×10 -6 M, corresponding to moderate binding affinity. Conclusions: These results show that the longer duration of AS in blood could prolong the clotting time determined by aPTT and offering protection against thrombin-induced lethality. One possible mechanism may result from AS-thrombin interaction.

Suppression of tumor growth by a new glycosaminoglycan isolated from the African giant snail Achatina fulica

Acharan sulfate is a new type of glycosaminoglycan from the giant African snail, Achatina fulica. Acharan sulfate, which has a primary repeating disaccharide structure of α- d- N-acetylglucosaminyl-2- O-sulfo-α- l-iduronic acid, was studied as a potential antitumor agent in both in vivo and in vitro assays. The antiangiogenic activity of acharan sulfate was evaluated in the chorioallantoic membrane assay and by measuring its effect on the proliferation of calf pulmonary artery endothelial cells. In vivo, a matrigel plug assay showed that acharan sulfate suppressed basic fibroblast growth factor (bFGF)-stimulated angiogenesis and lowered the hemoglobin (Hb) content inside the plug. Acharan sulfate was administered s.c. at two doses for 15 days to C57BL/6 mice implanted with murine Lewis lung carcinoma in the back. It was also administered i.p. to ICR mice bearing sarcoma 180 at a dose of 30 mg/kg. Subcutaneous injection of acharan sulfate at doses of 10 and 30 mg/kg decreased tumor weight and tumor volume by 40% without toxicity or resistance. Intraperitoneal injection of acharan sulfate also decreased tumor weight and volume by 40% in sarcoma 180-bearing mice. These results suggest that the antitumor activity of acharan sulfate may be related to the inhibition of angiogenesis.

Pharmacological activities of a new glycosaminoglycan, acharan sulfate isolated from the giant African snail Achatina fulica.

Acharan sulfate (AS) is a glycosaminoglycan (GAG) prepared from the giant African snail, Achatina fulica. In this study, some biological activities of AS were evaluated on the basis of structural similarities to heparin/heparan sulfate and the biological functions of GAGs. We demonstrated that it exhibited strong immunostimulating activities as measured by carbon clearance test in mice and in vivo phagocytosis. It also exhibited a significant hypoglycemic activity in epinephrine (EP)-induced hyperglycemia as well as antifatigue effects by weight-loaded forced swimming test. And it showed hypolipidemic activities in cholesterol-rich mixture induced hyperlipidemia in rats. The above results indicate that AS has diverse biological activities and suggest therapeutically important target molecules.

Inhibition by acharan sulphate of angiogenesis in experimental inflammation models.

1. The effects of acharan sulphate, a glycosaminoglycan isolated from the giant African snail Achatina fulica, on angiogenesis in the granulation tissue were analysed using an air pouch-type carrageenin-induced inflammation model in rats and a cotton thread-induced inflammation model in mice. 2. In the carrageenin-induced inflammation model in rats, intra-pouch injections of acharan sulphate (5 and 50 micro g) inhibited the pouch fluid accumulation and the granulation tissue formation as well as the angiogenesis in the granulation tissue at day 6 in a dose-dependent manner. 3. The inhibitory effects of acharan sulphate at 50 micro g on the pouch fluid accumulation and the leucocyte infiltration into the pouch fluid was not so effective as that of the cyclo-oxygenase inhibitor indomethacin at 100 micro g, but the inhibitory effects of acharan sulphate at 50 micro g on the granulation tissue formation and angiogenesis in the granulation tissue were almost the same as those of indomethacin at 100 micro g. 4. Acharan sulphate did not affect levels of vascular endothelial growth factor (VEGF) in the granulation tissue and in the pouch fluid at day 6, but indomethacin significantly lowered them. 5. In the cotton thread-induced inflammation model in mice, injections of acharan sulphate (10 micro g) at the site of the cotton thread implantation inhibited the granulation tissue formation and angiogenesis as indomethacin (20 micro g) did. Acharan sulphate (10 micro g) did not affect levels of VEGF in the cotton thread-induced granulation tissue at day 5, but indomethacin (20 micro g) significantly lowered them. 6. In culture of human vascular endothelial cells, acharan sulphate at 10 and 100 micro g ml(-1) inhibited VEGF-induced capillary tube formation. 7. These findings suggest that the inhibitory effect of acharan sulphate on angiogenesis in carrageenin- and cotton thread-induced granulation tissues is not due to the inhibition of VEGF protein induction, but is due to the inhibition of VEGF-induced vascular tube formation.

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica

Acharan sulfate is a glycosaminoglycan (GAG), having the structure →4)-2-acetamido-2-deoxy-α- d-glucopyranose(1→4)-2-sulfo-α- l-idopyranosyluronic acid (1→, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3–5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.

Effect of Single and Binary Combinations of Plant-Derived Molluscicides on Reproduction and Survival of the Snail Achatina fulica

The effects of sublethal treatments (20% and 60% of LC(50)/24 h) with plant-derived molluscicides on the reproduction of the giant African snail Achatina fulica were studied. Azadirachta indica oil, Cedrus deodara oil, Allium sativum bulb powder, and Nerium indicum bark powder singly and binary combinations on reproduction and survival of A. fulica were investigated. Repeated treatment occurred on day 0, day 15, and day 30. These plant-derived molluscicides significantly reduced fecundity, egg viability, and survival of A. fulica within 15 days. Discontinuation of the treatments after day 30 did not lead to a recovery trend in the next 30 days. Day 0 sublethal treatment of all the molluscicides caused a maximum reduction in protein, amino acid, DNA, RNA, and phospholipid levels and simultaneous increase in lipid peroxidation in the ovotestis of treated A. fulica. It is believed that sublethal exposure of these molluscicides on snail reproduction is a complex process, involving more than one factor in reducing the reproductive capacity of A. fulica.

First Record of Giant African Land Snail Achatina fulica from Rajasthan

First Record of Giant African Land Snail Achatina fulica from Rajasthan

Effects of inhibitors for intracellular signal transduction systems on the inward current produced by GABA in a snail neuron

1. 1. An inward current ( I in) was produced by γ-aminobutyric acid (GABA) and muscimol, but not by baclofen, in an identifiable giant neuron type, v-LCDN (ventral-left cerebral distinct neuron), of an African giant snail ( Achatina fulica Férussac) under voltage clamp. 2. 2. The pharmacological features of the excitatory GABA receptors in this Achatina neuron type, termed the Achatina muscimol II type GABA receptors, were mainly comparable to those of the mammalian GABA c receptors. 3. 3. It was demonstrated in the present study that the following inhibitors for intracellular signal transduction systems showed no significant effect on the I in produced by GABA in this Achatina neuron type: H-7 [1-(5-isoquinolinyl sulfonyl)-2-methylpiperazine], an inhibitor of cyclic AMP-dependent protein kinase (PKA), cyclic GMP-dependent protein kinase (PKG) and protein kinase C (PKC); H-8 ( N-[2-(methylamino)-ethyl]-5-isoquinolinesulfonamide), a PKA and PKG inhibitor; H-9 [ N-(2-aminoethyl)-5-isoquinolinesulfonamide), a PKA inhibitor; staurosporine ((9α,10β,11β,13α)-(+)-2,3,10,11,12,13-hexahydro-10-methoxy-9-methyl-11-(methylamino)-9,13-epoxy-1H-9H-diindolo[ 1,2,3-gh: 3′,2′,1′-lm]pyrrolo[3,4-j] [1,7]benzodiazonin-1-one), a PKA and PKC inhibitor; KT5823 ((8R,9S,11S)-9-methoxy-9-methoxycarbonyl-2N,8-dimethyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b, 11a-triazadibenzo[a,g]cycloocta[c,d,e]-trinden-1-one), a PKG inhibitor; W-7 [ N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide], a calmodulin inhibitor; ML-9 [1-(5-chloronaphthalene-l-sulfonyl-1 H-hexahydro-1,4-diazepine hydrochloride], a myosin light-chain kinase inhibitor; genistein [5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one], a tyrosine protein kinase inhibitor; IBMX (3-isobutyl-l-methylxanthine), a cyclic nucleotide phosphodiesterase (PDE) inhibitor; fluphenazine nitrogen-mustard (2-chloroethyl)-4[3-(2-trifluoromethyl-10-phenothiazinyl)-propyl]piperazine dihydrochloride), a calmodulin-dependent PDE inhibitor; calyculin A, a type 1 protein phosphatase inhibitor; and okadaic acid (9,10-deepithio-9,10-didehydroacanthifolicin), a type 1, 2A and 2B protein phosphatase inhibitor. 4. 4. With these results, it was proposed that the excitatory Achatina muscimol II type GABA receptors in v-LCDN are not metabotropic but ionotropic.