Effect of 3,4-diaminopyridine on rat extensor digitorum longus muscle paralyzed by local injection of botulinum neurotoxin

Adler, Michaël; MacDonald, Douglas; Sellin, Lawrence C.; Parker, Gerald W.

doi:10.1016/0041-0101(95)00127-1

Cited by 48 publications

(28 citation statements)

References 30 publications

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“…The ionomycin/Ca 2ϩ combination could not produce a full recovery, however. Similar observations were obtained with other neuronal preparations (28,45) and are consistent with observations in vivo that the minimal amount of the K ϩ channel blocker 3,4-diaminopyridine that stimulates a full recovery from BoNT/A does not elicit recovery from BoNT/E (14).…”

Section: Discussionsupporting

confidence: 78%

“…Furthermore, increasing extracellular Ca 2ϩ above physiological concentrations does not overcome the block (Fig. 3), similar to findings with neuromuscular preparations (14).…”

Section: Concentration-dependent Effects Of Bont/e On Snap-25supporting

confidence: 71%

“…In the present work, Ca 2ϩ stimulates release of neurotransmitter with a Hill coefficient of approximately 2 in the absence of toxin, which is consistent with electrophysiological estimates (54,67). Calcium stimulates release following exposure to BoNT/A and to a lesser extent, BoNT/E, each observation being similar to in vivo experiments with local injections of either toxin into the extensor digitorum longus muscle of rats (14,22,23). Our data demonstrate that the amount of P197 generated by BoNT/A is sufficient to block neuronal activity in a concentration-dependent manner.…”

Section: Discussionsupporting

confidence: 59%

“…In this study, we examined the relationship between blockade of neurotransmitter release and cleavage of SNAP-25 caused by BoNT/A and -E to aid in understanding how BoNT/A seems to derive greater potency than BoNT/E (14,22,26,32). Both BoNTs induce a concentration-dependent block in synaptic vesicle exocytosis and proteolysis of SNAP-25.…”

Section: Discussionmentioning

confidence: 99%

“…BoNT/A and -E both cleave SNAP-25 but at distinct sites (12). Interestingly, paralysis from BoNT/A lasts for many months whereas blockade caused by BoNT/E lasts for relatively brief periods (13,14). Two hypotheses were proposed to account for this difference: (a) BoNT/A remains catalytically active for a longer interval than BoNT/E or, alternatively (b) the catalytic activity of both toxins is very short-term but the major SNAP-25 fragment generated by BoNT/A (P197) persists in nerve terminals and interferes with neurotransmitter release (15).…”

mentioning

confidence: 99%

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The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A

Keller

Neale

2001

Journal of Biological Chemistry

112

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. This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca 2؉ . In this case, vesicular release is restored by increasing intracellular Ca 2؉ . These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca 2؉ .Intoxication with botulinum neurotoxin (BoNT) 1 in vivo leads to flaccid paralysis by blockade of acetylcholine release at the neuromuscular junction (1-3). The proteolytic activity of the toxin responsible for causing paralysis resides within the 50-kDa light chain domain (4, 5) and is directed against three synaptic proteins: synaptosomal-associated protein of 25-kDa (SNAP-25), vesicle-associated membrane protein (VAMP), or syntaxin (6 -11). BoNT/A and -E both cleave SNAP-25 but at distinct sites (12). Interestingly, paralysis from BoNT/A lasts for many months whereas blockade caused by BoNT/E lasts for relatively brief periods (13,14). Two hypotheses were proposed to account for this difference: (a) BoNT/A remains catalytically active for a longer interval than BoNT/E or, alternatively (b) the catalytic activity of both toxins is very short-term but the major SNAP-25 fragment generated by BoNT/A (P197) persists in nerve terminals and interferes with neurotransmitter release (15). The first hypothesis has been substantiated by direct demonstration of BoNT/A persistence in primary mouse spinal cord cultures, electroporated chromaffin cells, and mammalian neuromuscular junction preparations in vivo (16 -18). Evidence for the second hypothesis is indirect. Overexpressed P197 in an insulinoma Hit-T15 cell line inhibits insulin secretion (19). BoNT/A action at the neuromuscular junction produces a strong immunogenic signal for P197 within nerve terminals, indicating that P197 resides for some time in the proper intracellular regions where it could exert an inhibitory effect on neurotransmission (20, 21). However, there has been no quantitative analysis to determine whether the concentrations of P197 generated by toxin activity are able to inhibit neurotransmitter ...

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Section: Discussionsupporting

confidence: 78%

“…Furthermore, increasing extracellular Ca 2ϩ above physiological concentrations does not overcome the block (Fig. 3), similar to findings with neuromuscular preparations (14).…”

Section: Concentration-dependent Effects Of Bont/e On Snap-25supporting

confidence: 71%

Section: Discussionsupporting

confidence: 59%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A

Keller

Neale

2001

Journal of Biological Chemistry

112

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Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists

Sheridan¹,

Deshpande²,

Smith³

1999

J. Appl. Toxicol.

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Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists

Sheridan¹,

Deshpande²,

Smith³

1999

J. Appl. Toxicol.

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Measurements of the efficacy of novel botulinum toxin antagonists can be based on classical bioassays of toxin concentration. However, the relative sensitivities of in vivo and in vitro assays to the effects of antagonists are not necessarily correlated with the sensitivities of the assays to toxin. Comparisons of the sensitivity of an in vitro mouse muscle contraction assay with an in vivo mouse survival assay indicated that the in vivo assay was more sensitive to botulinum toxin serotype A by more than one order of magnitude at equivalent molar concentrations. However, in studies of toxin neutralization with equine antisera, the in vitro muscle assay was more than three times more sensitive to the presence of antisera than the equivalent mouse survival assay. For the development of new drugs to treat botulism, antagonist sensitivity is a primary consideration in determining relative efficacy during structure–activity studies. Thus, in studies of toxin antagonists, the in vitro assay appears to be superior for initial testing.

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Effect of 3,4-diaminopyridine on rat extensor digitorum longus muscle paralyzed by local injection of botulinum neurotoxin

Cited by 48 publications

References 30 publications

The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A

The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A

Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists

Comparison of in vivo and in vitro mouse bioassays for botulinum toxin antagonists

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