Nitric oxide involvement in Hydra vulgaris very primitive olfactory-like system

Abstract

Hydra feeding response is a very primitive olfactory-like behavior present in a multicellular organism. We investigated the role of nitric oxide (NO) in the induction and control of hydra feeding response. Under basal conditions, hydra specimens produce detectable amounts of nitrite (NO2-), the breakdown product of NO. When hydra were incubated with reduced glutathione (GSH), the typical activator of feeding response, an increase of basal NO production was observed. This effect was inhibited by glutamic or alpha-aminoadipic acids, two GSH antagonists, which block GSH-induced feeding response, and by the NO synthase (NOS) inhibitor L-NAME. Moreover, we found that hydra possess a calcium-dependent (but calmodulin-independent) NOS isoform. By using exogenous NO donors and NOS inhibitors, we demonstrated that NO stimulus can participate both in triggering tentacular movements and in recruiting neighbor tentacles during hydra feeding response. By using dbt2-cGMP, an analog to cGMP, we observed that the NO effect was independent of cGMP pathway. Our results strongly implicate NO involvement in hydra very primitive feeding behavior, thus confirming its preservation throughout evolution.

Fig. 1.

Nitrite (NO₂⁻) release in hydra supernatants. Reduced glutathione (GSH; 2.5 μm) treatment increases basal NO₂⁻levels, as measured by the Griess reaction. A 1 hr pretreatment of hydra by injecting the specific NOS inhibitor l-NAME (L-NAME; 100 μm) into the gastric cavity abolishes GSH-induced NO₂⁻ levels. This effect is reversed by excess l-Arg (L-ARG; 200 μm), whereas the d-isomer of NAME (D-NAME; 100 μm) is inactive. Also, glutamic acid (GLU; 10 μm) or α-aminoadipic acid (AAD; 10 μm) reduces GSH-induced NO₂⁻ production. Finally, nauplia increase basal NO₂⁻ levels, this effect being abolished by l-NAME (100 μm). Results are expressed for NO₂⁻ in nmol · ml⁻¹ · 60 min⁻¹, and each barrepresents mean ± SEM of three experiments. p≤ 0.001 between basal and GSH or nauplia, between GSH and GSH +l-NAME or GSH + GLU, and between nauplia and nauplia +l-NAME; p ≤ 0.01 between GSH and GSH + AAD.

Fig. 2.

Spectroscopic detection of NO release in hydra supernatants. Hemoglobin (25 μm) added to the medium is converted from the oxy (a) to the met form (b) after treatment of hydra with 2.5 μmGSH as monitored by optical spectroscopy. Spectra are measured after a fivefold dilution of the sample. The inset shows the corresponding X-band low-temperature ESR curves atg = ∼2, which reveal the GSH-induced selective formation of a small amount of nitrosyl-hemoglobin (a′, control; b′, GSH-stimulated hydra). Arrowcorresponds to 5 mT.

Fig. 3.

NOS activity in hydra homogenates. InA, a time-dependent [³H]citrulline production is shown. B, Constitutively, hydra express NOS activity in the presence of NADPH, this expression being reduced byl-NAME (100 μm). Excess l-Arg (200 μm) reverses the l-NAME effect, whereasd-NAME (100 μm) is inactive. NOS isoform results in being Ca²⁺-dependent. In fact, when resuspended in EGTA/Ca²⁺-free buffer, NADPH-incubated hydra homogenates show a significant decrease in NOS activity. Data are expressed as the ratio between [³H]citrulline production (cpm × 10⁻³) and mg protein as assayed in the homogenates. InC the isoform of NOS is shown to be CaM-independent. In fact, CaM inhibitors, such as W7 (50 μm) and trifluperazine (TFP; 50 μm), do not alter NOS activity. In contrast, both W7 and TFP are able to strongly inhibit mouse brain Ca²⁺/CaM-dependent [³H]citrulline generation. Results are expressed as percent NOS activity with respect to the basal expression in NADPH-incubated hydra homogenates. Each bar corresponds to mean ± SEM of samples performed in triplicate; *p ≤ 0.001.

Fig. 5.

Hydra feeding behaviors. Hydra at rest (A). GSH (2.5 μm) induces a typical feeding response consisting of tentacle-simultaneous curlings and mouth opening (B). In C a mouth-opening detail (arrowhead) is shown. When hydra are co-incubated with 10 μm SIN-1, tentacle curlings are elicited, but not mouth opening (see arrowhead in D). Nauplia (arrowhead in E) elicit a typical feeding response: the tentacles piercing nauplia are initially activated, and the response successively spreads to the neighboring tentacles (E). When hydra are preincubated withl-NAME (100 μm), the tentacle piercing nauplia is curled (see arrowhead), whereas all nauplia-untouched tentacles are completely at rest (F). Magnification, 10×.

Fig. 4.

cGMP production in hydra. GSH (2.5 μm) induces an increase in hydra levels of cGMP, and the maximal effect is observed after 2 min of treatment. This effect is inhibited by a 24 hr preincubation of hydra with l-NAME (L-NAME; 100 μm), showing that the production of cGMP depends on NO release. Data are expressed as fmol of cGMP per 10 specimens per well. Each point represents mean ± SEM of three experiments performed in triplicate; *p ≤ 0.01.