ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide.

The voltage-gated potassium channel in T lymphocytes, Kv1.3, is an important molecular target for immunosuppressive agents. A structurally defined polypeptide, ShK, from the sea anemone Stichodactyla helianthus inhibited Kv1.3 potently and also blocked Kv1.1, Kv1.4, and Kv1.6 at subnanomolar concentrations. Using mutant cycle analysis in conjunction with complementary mutagenesis of ShK and Kv1.3, and utilizing the structure of ShK, we determined a likely docking configuration for this peptide in the channel. Based upon this topological information, we replaced the critical Lys22 in ShK with the positively charged, non-natural amino acid diaminopropionic acid (ShK-Dap22) and generated a highly selective and potent blocker of the T-lymphocyte channel. ShK-Dap22, at subnanomolar concentrations, suppressed anti-CD3 induced human T-lymphocyte [3H]thymidine incorporation in vitro. Toxicity with this mutant peptide was low in a rodent model, with a median paralytic dose of approximately 200 mg/kg body weight following intravenous administration. The overall structure of ShK-Dap22 in solution, as determined from NMR data, is similar to that of native ShK toxin, but there are some differences in the residues involved in potassium channel binding. Based on these results, we propose that ShK-Dap22 or a structural analogue may have use as an immunosuppressant for the prevention of graft rejection and for the treatment of autoimmune diseases.

Neomycin is an agonist at a polyamine site on the N-methyl-D-aspartate receptor.

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.

Agonist-like character of the (R)-enantiomer of 1-hydroxy-3-amino-pyrrolid-2-one (HA-966).

HA-966 (1-hydroxy-3-amino-pyrrolid-2-one), an antagonist at the strychnine-insensitive glycine site on the N-methyl-D-aspartate (NMDA) receptor complex, only partially inhibits the binding of noncompetitive antagonists to the NMDA receptor but enhances the binding of the NMDA competitive antagonist CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid). Here we report that the IC50 of the active (R)-enantiomer of HA-966 for displacement of [3H]glycine binding is decreased in the presence of spermine, suggesting that spermine increases the affinity of (R)-HA-966 at the [3H]glycine binding site. The IC50 values of the agonist glycine and the partial agonist 1-aminocyclopropane-1-carboxylate are also decreased. The IC50 values of glycine antagonists 6,7-dinitroquinoxalin-2,3-dione and 7-chlorokynurenic acid are not significantly altered. The spermine shift represents the first demonstration of the agonist-like character of the (R)-enantiomer of HA-966 at the glycine site.

Phospholipase A2 (PLA2) activity in undifferentiated U937 cells.

PLA2 activity has been described in U937 cells. The present study characterized PLA2 activity in these undifferentiated cells. Cells were grown in suspension culture, harvested by centrifugation, and washed and homogenized in a neutral buffer containing standard proteinase inhibitors. A low speed supernatant was fractionated either by acid extraction or by sucrose density gradient centrifugation. PLA2 activity was measured using either L-alpha-1-palmitoyl-2-arachidonoyl[1-14C]-phosphatidylcholine or heat-inactivated [3H]oleic acid-labeled E. coli as substrates. Substrate-specific PLA2 activity was found in the acid-extracted and in the 25% sucrose fractions. Standard inhibitors were investigated with these PLA2 activities. Our results suggest undifferentiated U937 cells contain three distinct PLA2 activities. This is the first indication that more than one PLA2 activity is present in undifferentiated U937 cells.