title: Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.
abstract: We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.
authors.affiliation: Anesthesia Research Laboratories, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: J
authors.initials: J
authors.lastname: McHugh
authors.forename: W M
authors.initials: WM
authors.lastname: Mok
authors.forename: G K
authors.initials: GK
authors.lastname: Wang
authors.forename: G
authors.initials: G
authors.lastname: Strichartz
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis*; D000779:Anesthetics, Local / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D001496:Batrachotoxins / Q000494:pharmacology*; D001665:Binding Sites / Q000502:physiology; D002045:Bupivacaine / Q000494:pharmacology; D010777:Photochemistry; D011817:Rabbits; D026941:Sodium Channel Blockers; D013574:Synaptosomes / Q000502:physiology; D014466:Ultraviolet Rays
chemical_list: D000345:Affinity Labels; D000779:Anesthetics, Local; D001386:Azides; D001496:Batrachotoxins; C093720:N-(2-di-N-butylaminoethyl)-4-azidobenzene; D026941:Sodium Channel Blockers; C029534:hydroxysuccinimidyl-4-azidobenzoate; C031775:batrachotoxinin A 20-alpha-benzoate; D002045:Bupivacaine