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1. The effect of electrophoretic ejection of philanthotoxin (the polyamine toxin, from the Egyptian digger wasp) was tested on responses of brainstem and spinal neurones in the pentobarbitone-anaesthetized rat to excitatory amino acids. 2. Philanthotoxin caused a dose-dependent reduction of responses to quisqualate, alpha-amino-3-hydroxy-5-phenyl-4-isoxazolepropionate (AMPA) and kainate with little effect on those to N-methyl-D-aspartate (NMDA). 3. The time-course of this antagonist action was slow. In particular the rate of recovery was dependent on frequency of ejection of the agonist. This agonist-dependent recovery suggests that philanthotoxin has a channel blocking mode of action on mammalian central neurones.

Nitric oxide ((*)NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of (*)NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is closely related to glutamate N-methyl-D-aspartate (NMDA) receptors, following a rise in intracellular calcium levels. However, that dependent on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors remains elusive and controversial, despite reports describing a role for these receptors in other brain regions, largely because of lack of quantitative and dynamic measurements of (*)NO. Using a (*)NO-selective microsensor inserted in the diffusional spread of (*)NO in the CA1 region of rat hippocampal slices, we measured its real-time endogenous production, following activation of ionotropic glutamate receptors and under tissue physiological oxygen tension. Both NMDA and AMPA stimulation resulted in a concentration-dependent (*)NO production but encompassing distinct kinetics for lag phases and slower rates of (*)NO production were observed for AMPA stimulation. Robustness of the results was achieved instrumentally and pharmacologically, by means of nitric oxide synthase (NOS) inhibitors and antagonists of NMDA (D-(-)-2-amino-5-phosphonopentanoic acid, AP5) and AMPA (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide, NBQX) receptors. When using glutamate as a stimulus, (*)NO production was of lower magnitude in the presence of AP5 plus NBQX than with AP5 alone, suggesting that even when NMDA receptors are inhibited Ca(2+) rises to levels to induce a peak of (*)NO from the background. Whereas extracellular Ca(2+) was required for the (*)NO signals, Philanthotoxin-4,3,3 (PhTX-4,3,3) a toxin used to target Ca(2+)-permeable AMPA receptors, attenuated (*)NO production. These observations are interpreted on basis of a distinct coupling between the glutamate receptors and neuronal NOS. A role for Ca(2+)-permeable AMPA receptors in the Ca(2+) activation of neuronal NOS is suggested., ((c) 2008 Wiley-Liss, Inc.)

PhTX-343 and PhTX-12, analogues of the natural polyamine wasp toxin PhTX-433, were synthesised in 40-60% yields as pure enantiomers using solid phase synthesis techniques. Capillary electrophoresis procedures were developed for chiral separation and determination of enantiomeric purity (ee) of the enantiomers of PhTX-343 and PhTX-12. The methods were optimised with respect to chiral selector, buffer pH, and temperature around the capillary. Thus, rac-PhTX-343 was resolved using a separation buffer containing 30 mM heptakis-(2, 6-di-O-methyl)-beta-cyclodextrin in 50 mM 6-aminocarproic acid (pH 4. 0) at 15 degrees C. rac-PhTX-12 was not resolvable in this system, but could be resolved using a separation buffer containing 10% w/v of dextrin 10, a linear maltodextrin, in 50 mM 6-aminocaproic acid (pH 4.0) at 15 degrees C. Using these methods, the optical purity of the synthetic enantiomers was determined to be ee > 99%. The enantiomers were also characterised by chiroptical methods. The antagonist potency of the enantiomers was tested on nicotinic acetylcholine receptors (human muscle-type nAChR) expressed in TE671 cells, ionotropic glutamate receptors in Xenopus laevis oocytes (expressing recombinant GluR1flop receptors), and locust muscle ionotropic glutamate receptors sensitive to quisqualate (qGluR). The potencies of each pair of enantiomers were similar (eudismic ratio close to 1)., (Copyright 2000 Wiley-Liss, Inc.)

1. Block by philanthotoxin-343 (PhTX-343), a neurotoxin from wasps, of ionic currents mediated through neuronal nicotinic acetylcholine (ACh) receptor/channels was characterized in rat phaeochromocytoma PC12 cells, by use of whole cell voltage-clamp techniques. 2. In the cells held at -60 mV, PhTX-343 at 0.1 and 1 microM inhibited an inward current activated by 100 microM ACh. The current inhibition was relieved by depolarizing steps, and augmented at negative potentials, suggesting that PhTX-343 blocks the channel in a voltage-dependent manner. Joro spider toxin-3 (JSTX-3) also exerted voltage-dependent inhibition of ACh-activated currents in a similar concentration range, but argiotoxin636 did not affect the currents. 3. Analysis of the current decay during hyperpolarizing steps indicated that the current inhibition by 100 nM PhTX-343 develops in an order of several hundres of milliseconds. On the other hand, the recovery from the current inhibition during depolarizing steps developed in an order of about 100 ms. 4. The results suggest that PhTX-343 blocks neuronal nicotinic receptor channels in PC12 cells at concentrations lower than those required for channel block in non-mammalian cells, and the block exhibits clear voltage-dependence. Estimated from the voltage-dependence, the binding site of PhTX-343 may be located near the outer mouth of the channel.

Philanthotoxin (PhTX) is a neurotoxic constituent of the paralytic venom of the digger wasp, Philanthus triangulum. PhTX inhibits glutamate receptors of insect muscles mostly as a channel blocker, thereby producing muscle paralysis. Since glutamate receptor blockers may be of value as selective insect control agents, numerous derivatives of PhTX were synthesized and tested for their potencies as inhibitors of insect skeletal muscle glutamate receptors. Structure-activity relationship studies revealed that shortening the polyamine chain length reduced potency, and quaternarization of the nitrogen destroyed it. The potency was increased by a bulky anchoring group with moderate hydrophobicity at the end of the polyamine chain. The conversion of the tryosyl moiety to 3,5-diiodo-tyrosyl also increased potency and so did lengthening the butyryl chain from 4 to 10 carbons. Not only did PhTXs inhibit different subtypes of glutamate receptors, including the mammalian N-methyl-D-aspartate receptor, but also nicotinic receptors of insects and vertebrates. Because of this low selectively, and the hydrophilicity of the derivatives tested, which interferes with their penetration to the target receptor, these compounds cannot be used as insecticides. Nevertheless, the insect skeletal muscle glutamate receptor is a viable target for selective insecticides and major changes in PhTX structure may possibly produce derivatives that can be potential insecticides.

In this brief review on Koji Nakanishi's remarkable career in natural products chemistry, we have highlighted a number of his accomplishments that illustrate the broad diversity of his interests. These include the isolation, structure determination, and biological mechanism of action of many natural products including the triterpenoid pristimerin; the diterpenoid ginkgolides; insect and crustacean molting hormones; phytoalexins; the toxic red tide principle brevetoxin; the vanadium tunicate pigments; philanthotoxin from killer wasps; antisickling agents; mitomycin DNA adducts; insect antifeedants; a mitotic hormone, the small molecule fish attractants from the sea anemone; new isolation and purification technologies; molecular chemistry of vision; age-related macular degeneration; and the development of the exciton circular dichroism (CD) chirality method for microscale determination of absolute configuration of natural products and chirality of other chiral molecules and supramolecular assembly., (© 2020 Wiley Periodicals, Inc.)