The Discharge Of in Situ Nematocysts of the Acontia of Aiptasia Mutabilis is a Ca2+-Induced Response

The ionic events leading to discharge of in situ nematocysts were investigated in acontia excised from Aiptasia mutabilis Gravenhorst. The effect on discharge of various ionic solutions and ion channel blockers was tested. In the absence of Ca2+ in the medium no discharge was elicited, whatever the composition of the medium. In the presence of 10 mmoll−1 Ca2+ total discharge was induced by NaSCN, NaI, choline iodide and KI, whereas KC1 induced both discharge and extrusion of undischarged nematocysts. The latter effect was prevented by La3+ but not by 4-aminopyridine (4-AP) and tetraethylammonium (TEA+). Alcian Blue induced total discharge. NaCl and choline chloride were ineffective. The discharge induced by lyotropic anions depended on Ca2+ concentration and was prevented by the Ca2+ channel blockers La3+, Co2+ and Cd2+, but not by verapamil. It is proposed that the discharge of in situ nematocysts is caused by Ca2+ conductance through the cell membrane of either the nematocyte or the supporting cell. Furthermore, cyclic AMP and cyclic GMP do not seem to be involved as second messengers in the discharge process. The combined effects of the metabolic poisons dinitrophenol (DNP) and monoiodoacetic acid did not affect the discharge process.

Lyotropic anions. Na channel gating and Ca electrode response.

The effects of external anions on gating of Na channels of frog skeletal muscle were studied under voltage clamp. Anions reversibly shift the voltage dependence of peak sodium permeability and of steady state sodium inactivation towards more negative potentials in the sequence: methanesulfonate less than or equal to Cl- less than or equal to acetate less than Br- less than or equal to NO-3 less than or equal to SO2-4 less than benzenesulfonate less than SCN- less than ClO-4; approximately the lyotropic sequence. Voltage shifts are graded with mole fraction in mixtures and are roughly additive to calcium shifts. The peak PNa is not greatly affected. Except for SO2-4, these anions did not change the Ca++ activity of the solutions as measured with the dye murexide. Shifts of gating can be explained as the electrostatic effect of anion adsorption to the Na channel or to nearby lipid. Such adsorption is expected to follow the lyotropic series. Anions also interfere significantly with the response of a Ca-sensitive membrane electrode following the same sequence of effectiveness as the shifts of gating. The lyotropic anions decrease the Ca++ sensitivity and cause anomalously negative responses of the Ca electrode because these anions are somewhat permeant in the hydrophobic detector membrane.

Pasteurization Of Antithrombin III: Stabilization By Heparin And Lyotropic Anions

Pasteurization of Antithrombin III (AT III) for 10 hours at 60°C is necessary to reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 1,8-anilinonaphthalene sulfonate (ANS) which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature as the protein unfolds. The extent of the increase in ANS fluorescence correlated roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure exclusion chromatography. The midpoint, Td, of the thermal denaturation curve increased by 13 and 19°C in the presence of 0.5 M and 1.0 M sodium citrate respectively. Phosphate, sulfate, and EDTA were also strong stabilizers while the chaotropic anions, iodidé and thiocyanate were potent destabilizers. Heparin, at 10 mg/ml, increased Td by 7°, presumbly through a direct binding mechanism. Reducing agents increased ANS fluorescence by an amount similar to that seen with thermally denatured samples, an effect which was inhibited by heparin but not by citrate. Furthermore, incorporation of 14C-iodoacetamide into AT III during thermal titration was coincident with the increase in ANS fluorescence suggesting that disulfide cleavage is the event which triggers the unfolding of the protein. Samples pasteurized for 10 hours at 60°C in the presence of 0.5 M and 1.0 M citrate retained full antithrombin activity but exhibited evidence of minor alterations in the ability to bind heparin.