IN-VITRO CHARACTERIZATION OF BOTULINUM TOXIN TYPE-A, TYPE-C AND TYPE-D ACTION ON HUMAN TISSUES - COMBINED ELECTROPHYSIOLOGIC, PHARMACOLOGICAL AND MOLECULAR BIOLOGIC APPROACHES
Ja. Coffield et al., IN-VITRO CHARACTERIZATION OF BOTULINUM TOXIN TYPE-A, TYPE-C AND TYPE-D ACTION ON HUMAN TISSUES - COMBINED ELECTROPHYSIOLOGIC, PHARMACOLOGICAL AND MOLECULAR BIOLOGIC APPROACHES, The Journal of pharmacology and experimental therapeutics, 280(3), 1997, pp. 1489-1498
Human exposure to botulinum toxin typically occurs in two settings: 1)
as an etiologic agent in the disease botulism and 2) as a therapeutic
agent for the treatment of dystonia. Epidemiologic studies on botulis
m suggest that the human nervous system is susceptible to five toxin s
erotypes (A, B, E, F and G) and resistant to two (C and D). In the pas
t, these epidemiologic findings have been used as the basis for select
ing serotypes that should be tested as therapeutic agents for dystonia
. Epidemiologic data have been utilized because there are no studies o
f botulinum neurotoxin action on isolated human nerves. In the present
study, electrophysiologic techniques were used to monitor toxin effec
ts on neuromuscular transmission in surgically excised human pyramidal
is muscles, ligand binding studies were done to detect and characteriz
e toxin receptors in human nerve membrane preparations, and molecular
biologic techniques were used to isolate and sequence a human gene tha
t encodes a substrate for botulinum neurotoxin. The results demonstrat
ed that stable resting membrane potentials (-61.5 mV; S.E.M. +/- 0.7)
were maintained in individual fibers of pyramidalis muscle for up to 6
hr at 33 degrees C. The rate of spontaneous miniature endplate potent
ials was low in physiologic solution (0.14 sec(-1)) but increased in r
esponse to elevations in extracellular potassium concentration. In kee
ping with epidemiologic findings, botulinum toxin type A (10(-8) M) pa
ralyzed transmission in human preparations (ca. 90 min). In contrast t
o epidemiologic findings, serotype C (10(-8) M) also paralyzed human t
issues (ca. 65 min). Iodinated botulinum toxin displayed high-affinity
binding to receptors in human nerve membrane preparations (serotype A
high-affinity site: K-d = 0.3 nM, B-max = 0.78 pmol/mg protein; serot
ype C high-affinity site: K-d = 1.96 nM, B-max = 8.9 pmol/mg protein).
In addition, the human nervous system was found to encode polypeptide
s that are substrates for botulinum neurotoxin types A (synaptosomal-a
ssociated protein of M(r) 25,000) and C (syntaxin 1A). These data have
important implications bearing on: 1) the development and administrat
ion of vaccines against botulism and 2) the testing of toxin serotypes
for the treatment of dystonia.