pp. cGMP pathway. Our results strongly implicate NO involvement in hydra very primitive feeding behavior, thus confirming its preservation throughout evolution. (Colasanti et al., 1995), the most primitive organism possessing a nervous system. Hydra is usually a sessile predator whose tentacles are armed with the typical, characteristic stinging capsules of the coelenterates called nematocysts. When a prey accidentally touches a tentacle, a typical feeding response is activated. Hydra feeding response, which can be considered the most primitive olfactory-like behavior present in a multicellular organism, is usually a complex behavioral phenomenon consisting of tentacle writhing and mouth opening. Loomis (1955) decided that the reduced glutathione (GSH) outflow from the prey when pierced by tentacle nematocysts is the physiological activator of hydra feeding response. GSH is usually thought to interact with a specific receptor, the presence of which in hydra tissues was exhibited by radioligand binding methods (Venturini, 1987) and has been recently characterized, solubilized, and partially purified (Bellis et al., 1991, 1992, Glucocorticoid receptor agonist 1994). However, no data are available concerning the cellular localization of GSH receptor. Hydra feeding response occurs a few seconds after GSH addition, reaches its peak within few minutes, and gradually disappears in 10 min. Different structural GSH analogs, i.e., glutamic or -aminoadipic acids, which bind to but Glucocorticoid receptor agonist do not activate GSH receptors, are able to competitively inhibit GSH activity in eliciting the feeding response (Lenhoff, 1981). Despite the obvious interest for the study of a primitive chemosensorial system as a simple olfactory-like model, little is known about the molecular mechanisms regulating hydra feeding response. Previous reports have indicated that this cytosolic Ca2+influx (Lenhoff, 1981) and the glutamatergic system (Venturini, 1987) are both involved in the induction of feeding response and that calcium ionophore A23187 enhances this response (Venturini et al., 1988). Recently, it has been reported that this interneuronal messenger NO seems to play a central role in the processing of olfactory information in invertebrates (Gelperin, 1994). In particular, a behavioral role for NO in chemosensory activation of feeding in a mollusk has been exhibited (Elphick et al., 1995). In this paper, we have investigated on NO Glucocorticoid receptor agonist involvement in both induction and control of hydra feeding response. At present, however, no data are available concerning the conversation between GSH receptors and the NO pathway. Therefore, Rabbit Polyclonal to PITX1 we decided to approach this problem using GSH antagonists in a study aimed at clarifying the role of the NOCcGMP pathway in the feeding response induced by either GSH or food using NOS inhibitors, exogenous NO donors, and a cGMP analog. MATERIALS AND METHODS Oxyhemoglobin (oxyHb) was prepared from commercial bovine hemoglobin (Hb; Sigma, Milan, Italy) by reduction with a 10-fold excess sodium hydrosulphite (Aldrich, Milan, Italy), followed by gel filtration on prepacked G-25 columns (Pharmacia, Uppsala, Sweden) and equilibration in air; authentic NO solution (NOsol) was obtained by a 30 min bubbling of distilled and deoxygenated water with 99.5% pure NO gas at 4C. This stock solution is in the low millimolar range (2 mm at 25C). GSH was from Merck Italia (Milan, Italy). Sulfanilamide,(formerly Nitrite (NO2?) was determined by the Griess reaction, according to the method ofTracey (1992). Briefly, 170 l of a solution consisting in 1 mm CaCl2 + 1 mm NaHCO3and made up of 100 hydra either untreated or treated with GSH or nauplia for 60 min (3 pulses every 10 min) was mixed with 10 l of sulfanilamide (1 mm final concentration) and 10 l of HCl (0.1N final concentration). The final reaction step was accomplished by the addition of 10 l of NEDA (1 mm final concentration). After a 10 min incubation at room temperature, the absorbance was measured at 548 nm, and nitrite concentration was decided using sodium nitrite as a standard. Results are expressed for NO2? as nmol ml?1 60 min?1. NO production in hydra supernatants was.