Modulation of gamma-aminobutyric acid (GABA) receptors and the feeding response by neurosteroids in Hydra vulgaris.

Gamma-Aminobutyric acid (GABA) receptors are present in membrane preparations from Hydra vulgaris, one of the most primitive organisms with a nervous system. These receptors are sensitive to muscimol and benzodiazepines and appear to be important in the regulation of the feeding response. The effects of neurosteroids, general anaesthetics, and GABA antagonists on GABA(A) receptors in membranes prepared from Hydra and on the feeding response have now been investigated. The neurosteroids tetrahydroprogesterone and tetrahydrodeoxycorticosterone increased [3H]GABA binding to hydra membranes with nanomolar potency (EC50, 141+/-11 and 623+/-36 nM, respectively) and high efficacy (maximal increase 79+/-6.5 and 62+/-4%, respectively), whereas the 3beta-hydroxy epimer of tetrahydroprogesterone was ineffective. The benzodiazepine receptor ligands diazepam (100 microM), clonazepam (100 microM) and abecarnil (30 microM) enhanced [3H]GABA binding to Hydra membranes by 22, 20 and 24%, respectively; effects abolished by the specific benzodiazepine antagonist flumazenil (100 microM). On the contrary, the peripheral benzodiazepine receptor ligand 4'chlorodiazepam failed to affect [3H]GABA binding to Hydra membranes. The general anaesthetics propofol and alphaxalone similarly increased (+38% and +30% respectively) [3H]GABA binding. Moreover, [3H]GABA binding to Hydra membranes was completely inhibited by the GABA(A) receptor antagonist SR 95531, whereas bicuculline was without effect. The modulation of GABA(A) receptors in vitro by these various drugs correlated with their effects on the glutathione-induced feeding response in the living animals. Tetrahydroprogesterone and tetrahydrodeoxy-corticosterone (1 to 10 microM) prolonged, in a dose-dependent manner, the duration of mouth opening induced by 10 microM glutathione, with maximal effects of +33 and +29%, respectively, apparent at 10 microM neurosteroid. Alphaxalone (10 microM) similarly increased (+33%) the effect of glutathione. The effects of steroids on the feeding response were inhibited by SR 95531 in a dose-dependent manner; t-butylbyclophosphorothyonate (1 microM), a specific Cl- channel blocker, which per se, like picrotoxin but not bicuculline, shortened the duration of the response, also counteracted the steroids effects at 1 microM. These results suggest that the modulation of GABA(A) receptors by steroids is an ancient characteristic of the animal kingdom and that the pharmacological properties of these receptors have been highly conserved through evolution.

Biochemical and functional identification of GABA receptors in Hydra vulgaris.

GABA binding sites labelled in vitro with [3H]GABA are present in crude membrane preparations from Hydra vulgaris. [3H]GABA binding is specific (70%), saturable and it is completely displaced by the GABA mimetic muscimol but not by bicuculline or baclofen. Scatchard analysis of saturation data indicates the presence of only one population of binding sites with a Bmax of 4, 75 pmol/mg of protein and a KD of 76 nM. In the living animal GABA and benzodiazepines increase the duration of mouth opening during the glutathione-induced feeding response. Bicuculline prevents the GABA-induced increase of the feeding response. Diazepam activity is enhanced by simultaneous GABA administration and it is suppressed by the specific antagonist flumazenil. In contrast, no [3H]-flunitrazepam binding is detected in membrane preparations. We conclude that a population of GABA receptors is present in Hydra tissues, where they are involved in the modulation of response to chemical stimulation.

[Effects of a new excitotoxic amino acid, dysiherbaine, on cultured Müller cells].

PURPOSE: To determine pharmacological response of dysiherbaine on cultured Müller cells considering the glutamate receptor functions. Dysiherbaine is a new excitotoxic amino acid, which was recently isolated from the water extract of a certain sponge. SUBJECTS AND METHOD: Retinas of adult rabbits were used to prepare the Müller cells. Intracellular calcium ion concentration ([Ca2+]i) analysis was done by fluorophotometry with calcium indicator, Fura-2 AM. RESULTS: A transient increase of [Ca2+]i was observed following the administration of dysiherbaine (2.5 microM -2.5 mM), but it was not observed in the extracellular calcium-free solution. This increase was blocked by the non NMDA glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). A dysiherbaine-induced increase in [Ca2+]i following preincubation of the NMDA glutamate receptor antagonist, (5 R, 10 S)-(+)-5-methyl-10, 11-dihydro-5 H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK 801) was seen in the same number of Müller cells as without the antagonist. CONCLUSION: Dysiherbaine appears to act primarily as a non NMDA glutamate receptor agonist having a secondary action as a NMDA glutamate receptor agonist.

The effect of diterpenoidic diacylglycerols on tentacle regeneration in Hydra vulgaris.

1. The effect of two ichthyotoxic diterpenoid diacylglycerols, verrucosins A and B, previously isolated from the mantle of a marine nudibranch mollusc, were studied on Hydra vulgaris tentacle regeneration. 2. A potent effect was found for both verrucosins in the low nanomolar range; the rank of potency observed was analogous to that reported for diglycerid activation of protein kinase C and to that found for verrucosin activation of this enzyme. 3. In the high nanomolar range, the two verrucosins were found to be toxic to Hydra vulgaris. 4. Verrucosin B-induced toxicity and tentacle regeneration were found to be dependent on [Ca2+] in the assay medium.

Bud induction in decapitated Hydra attenuata by 5-azacytidine: a morphological study.

The effect of 5-azacytidine (5-azaCR) on head regeneration and budding in hydra are reported. Hydra attenuata were exposed to various doses of 5-azaCR for 48 h and then decapitated and cultured. Head regeneration and bud formation were observed for 12 days after decapitation. Untreated control hydra regenerated heads within 7 to 8 days of decapitation with a budding index of 0.2. Buds invariably arose in the normal budding zone (below the gastric region). In the group treated with 0.8 mM-5-azaCR, 9 days after decapitation head regeneration was seen in only 13% of animals, and an average of two buds per hydra were formed, most of which were in the vicinity of the distal end. Induction of budding was also seen in the animals that regenerated heads. In animals exposed to 1 mM-5-azaCR three main types of responses were observed 9 days after decapitation. 44% of the animals regenerated normal heads; about half of them developed at least one bud and these buds originated in the budding zone. 17.5% of the animals developed abnormal, long hypostome-like structures with single or bifurcated tentacles at their tips. There were at least two buds per animal and they were invariably at abnormal sites. 32% of the animals failed to regenerate heads, although they developed two buds. 87% of these buds originated in abnormal sites of the body column and a large number (72%) did not detach even by the 12th day after decapitation. Both 5 and 10 mM of 5-azaCR were toxic to the animals; the survivors formed large globe-shaped heads. Bud induction was seen in 60% and 28% of animals in the 5 and 10 mM groups, respectively. These observations demonstrate that 5-azaCR induces bud formation in hydra at doses that inhibit head regeneration. This bud induction might be due to a specific expression of gene products responsible for bud formation.

Putative glycine receptors in Hydra: a biochemical and behavioural study.

Glycine acts as an inhibitory transmitter in the lower brain stem and spinal cord of vertebrate species, while very few data are yet available to support a similar role in invertebrate nervous systems. Here we report the identification and characterization of glycine receptors in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) by biochemical and behavioural studies. Saturation experiments revealed the occurrence of one population of binding sites of nanomolar affinity (KD = 33 nm) and low capacity (Bmax = 79 fmol/mg protein) for [(3)H]strychnine. The addition of glycine or taurine (0.1 microm-1 mm) produced a dose-dependent inhibition of [(3)H]strychnine binding. Beta-alanine (0.1-1 mm) did not significantly affect [(3)H]strychnine binding. The pharmacological properties of these receptors compare with those of vertebrate glycine receptors. Stimulation of Hydra polyps by reduced glutathione resulted in a significant increase in the duration of mouth opening in the presence of glycine, taurine or beta-alanine. The enhancement of the response was related both to amino acid (10-100 microm) and to glutathione concentration (1-10 microm). The effects of glycine or its agonists were suppressed by strychnine (1-10 microm). D-serine, a glycine agonist at the vertebrate NMDA receptor, produced opposite effects to those of glycine. The effects of d-serine were suppressed by 5,7-dichlorokynurenic acid but not by strychnine. In vitro, [(3)H]strychnine binding was not displaced by d-serine. These results indicate a dual action of glycine in Hydra tissues. The hypothesis that NMDA receptors may also be present in this elementary nervous system is proposed.