Differential inhibition by botulinum neurotoxin A of cotransmitters released from autonomic vasodilator neurons

Abstract

The role of the soluble NSF attachment protein receptor (SNARE) protein complex in release of multiple cotransmitters from autonomic vasodilator neurons was examined in isolated segments of guinea pig uterine arteries treated with botulinum neurotoxin A (BoNTA; 50 nM). Western blotting of protein extracts from uterine arteries demonstrated partial cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) to a NH2-terminal fragment of approximately 24 kDa by BoNTA. BoNTA reduced the amplitude (by 70-80%) of isometric contractions of arteries in response to repeated electrical stimulation of sympathetic axons at 1 or 10 Hz. The amplitude of neurogenic relaxations mediated by neuronal nitric oxide (NO) was not affected by BoNTA, whereas the duration of peptide-mediated neurogenic relaxations to stimulation at 10 Hz was reduced (67% reduction in integrated responses). In contrast, presynaptic cholinergic inhibition of neurogenic relaxations was abolished by BoNTA. These results demonstrate that the SNARE complex has differential involvement in release of cotransmitters from the same autonomic neurons: NO release is not dependent on synaptic vesicle exocytosis, acetylcholine release from small vesicles is highly dependent on the SNARE complex, and neuropeptide release from large vesicles involves SNARE proteins that may interact differently with regulatory factors such as calcium.

Substance P is essential for maintaining gut muscle contractility: a novel role for coneurotransmission revealed by botulinum toxin

“…Smooth muscle contraction depends on both the concentration of free cytosolic calcium as well as the sensitivity of the contractile and regulatory proteins to calcium (28). These mechanisms, which include cAMP/cGMP signaling, CPI-17, MLC kinase, and phosphatase among others, converge on the regulatory MLC 20 , whose state of phosphorylation is the principal determinant of smooth muscle cell contraction. This is reflected in our results, with the levels of p-MLC 20 running parallel to the observed effects on contractility in response to various interventions.…”

“…Tetrodotoxin was purchased from Calbiochem (San Diego, CA). Anti-phospho-myosin light chain (p-MLC 20) antibody was purchase from Cell Signaling (Danvers, MA), and myosin light chain (MLC 20) antibody was from Sigma BoNT/A injection. All experiments were approved by the Institutional Animal Care and Use Committee of Stanford University and Johns Hopkins University.…”

“…Measure of p-MLC 20 by Western blot analysis. The strips were homogenized on ice for 30 min in lysis buffer supplemented with protease inhibitors.…”

“…On the basis of our hypothesis that the effects of BoNT/A on cholinergic responsiveness are due to an inhibition of SP release, we compared the effects of BoNT/A and the NK1 receptor antagonist, CP-96345, on pyloric circular muscle contractility and MLC 20 phosphorylation. BoNT/A, boiled BoNT/A, and saline-treated pyloric muscles were isolated 1 h after in vivo injection as above and the strips were incubated in vitro in Krebs's solution for 30 min.…”

Descriptive study of botulism in an Austrian dairy herd: a case report

“…Stillbirth and abortion of calves have also been described. BoNTs are described to reduce the blood supply and muscular action of the uterus (Morris et al 2001). The calf was probably not affected by possible uterine undersupply due to the short time of two days in which its dam showed clinical signs.…”