Anthraquinone polyamines: novel channel blockers to study N-methyl-D-aspartate receptors.

The effects of various anthraquinone polyamines (AQP) were studied at recombinant N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus laevis oocytes. The AQP derivatives had different numbers of methylene groups between the NH(2) (or NH) groups in their spermidine-like tail. Thus, we termed these derivatives AQ33, AQ34, etc. All AQP derivatives inhibited responses of NR1/NR2 receptors in oocytes voltage-clamped at -70 mV, with IC(50) values between 4 and 22 microM. The block was strongly voltage-dependent. AQ34 and AQ33b inhibited responses of NR1/NR2 receptors but did not inhibit responses of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors expressed from GluR1 or GluR2(Q), indicating that AQ34 and AQ33b are preferential NMDA antagonists. Results of experiments using mutant NR1 and NR2 subunits identified residues that influence block by AQ34 and AQ33b. These residues are located in the outer vestibule at the selectivity filter/narrowest constriction of the channel and in the inner vestibule below the level of the selectivity filter. The results with mutant NR1 and NR2 subunits are consistent with the idea that NR1(Asn616) and NR2B(Asn616), but not NR2B(Asn615), make the narrowest constriction of NMDA channel.

Preparation and biological assessment of hydroxycinnamic acid amides of polyamines.

Many plants contain hydroxycinnamic acid conjugates of polyamines that are remarkably similar in general structure to the acylated polyamines found in spider and wasp toxins. In an effort to determine whether these compounds might play a role in the chemical defense of plants against arthropod pests we synthesized a variety of analogues of the coumaric (4-hydroxycinnamic) acid conjugates of di-, tri-, and tetraamines using common protection and acylation strategies. N(1)- and N(8)-coumaroyl spermidine were tested in feeding trials with insect larvae including the European corn borer (Ostrinia nubilalis), the tobacco budworm (Heliothis verescens) and the oblique banded leaf roller (Choristoneura rosaceana). Antifeedant assays with the rice weevil Sitophilus oryzae were also performed. Neither the naturally occurring coumaric acid conjugates of polyamines nor their analogues showed notable toxicity towards insects, despite precautions to maintain these easily oxidized materials in the wet diet. However, more direct bioassays of these compounds on glutamate dependent neuroreceptors including the deep abdominal extensor muscles of crayfish, or mammalian NMDA, delta2, and AMPA receptors, clearly showed that these compounds were inhibitory. N(1)-Coumaoryl spermine, a dodecyl and a cyclohexyl analogue were especially active at NMDA NR1/NR2B receptors. The latter had an IC(50) of 300 microM in the crayfish. N(1)-Coumaroyl spermine had an IC(50) in the crayfish preparation of 70-300 microM and against the mammalian NR1/NR2B receptor of 38 nM. Structure-activity variations show similar trends of length and hydrophobicity as has been seen previously with analogues of spider toxins. We conclude from this work that while the coumaric acid polyamine conjugates are active when directly applied to neuroreceptors, they show no overt toxicity when ingested by insect larvae.

Pharmacology of delta2 glutamate receptors: effects of pentamidine and protons.

The properties of delta2 receptors, which have homology to glutamate receptors but are not gated by glutamate, were studied using the constitutively active Lurcher mutant delta2(A654T) expressed in Xenopus oocytes. The macroscopic current through delta2(A654T) channels in voltage-clamped oocytes was defined as the difference between the holding current measured in the presence of extracellular Na(+) and that in the presence of the large impermeant cation N-methyl-d-glucamine. A-to-T mutations in the delta1 subunit and in NMDA (N-methyl-d-aspartate) receptor subunits, at positions equivalent to delta2(A654T), did not produce constitutively active channels. The current through delta2(A654T) channels was reduced by pentamidine and 9-tetrahydroaminoacridine, antagonists that also inhibit NR1/NR2B NMDA receptors but not AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors. Block of delta2(A654T) currents by these two antagonists was incomplete and weakly voltage-dependent, in contrast to the block of NR1/NR2B receptors, which was complete and strongly voltage-dependent. Pentamidine inhibited a constitutively active NR1(T648A)/NR2B NMDA receptor in a manner similar to its inhibition of a glutamate-gated wild-type NMDA receptor, but different from its inhibition of constitutively active delta2(A654T) receptors. Currents gated by delta2(A654T) were sensitive to the extracellular pH, being smaller at acidic than at alkaline pH, with a pH IC(50) value of 7.47 and a maximum inhibition of 70%. It is concluded that delta2(A654T) channels have some properties in common with NMDA channels but also have characteristics that are different from these receptors. Compounds such as pentamidine may be useful for studies of native delta2 receptors.

Hormonally regulated alpha(4)beta(2)delta GABA(A) receptors are a target for alcohol.

Here we report that low concentrations of alcohol (1-3 mM) increased Cl(-) currents gated by a recombinant GABA(A) receptor, alpha(4)beta(2)delta, by 40-50% in Xenopus laevis oocytes. We also found greater hippocampal expression of receptors containing alpha(4) and delta subunits, using a rat model of premenstrual syndrome (PMS) in which 1-3 mM alcohol preferentially enhanced GABA-gated currents, and low doses of alcohol attenuated anxiety and behavioral reactivity. The alcohol sensitivity of delta-containing receptors may underlie the reinforcing effects of alcohol during PMS, when eye saccade responses to low doses of alcohol are increased.