| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Anatomy and Histology, Centre for Neuroscience, Flinders University, Adelaide SA 5001, Australia
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
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 ~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 dependant on synaptic vesicle exocytosis, acetylcholine release from small vesicles is highly dependant on the SNARE complex, and neuropeptide release from large vesicles involves SNARE proteins that may interact differently with regulatory factors such as calcium.
pelvic neurons; neuropeptides; nitric oxide; acetylcholine; synaptosomal-associated protein of 25 kDa
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
THE PELVIC VASCULATURE in both males and females receives a dense innervation by autonomic vasodilator neurons that are activated during sexual activity and in response to noxious cutaneous stimulation (9, 15, 46). This vasodilation can be mediated by acetylcholine as well as noncholinergic cotransmitters, but the relative contribution of each cotransmitter varies between species (2, 5, 8, 10, 30, 40, 43). In virgin guinea pigs, the autonomic dilator response has two distinct phases: a fast phase mediated by neuronal nitric oxide (NO), and a slower phase mediated by one or more neuropeptides including vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) (1, 30). Neither phase is endothelium dependent. Acetylcholine is also released from vasodilator neurons, but the sole action of this cotransmitter in the uterine artery from virgin guinea pigs seems to be prejunctional inhibition (Ref. 26, cf Ref. 30). Exogenous application of muscarinic agonists produces endothelium-dependent vasodilation of the uterine artery (30), so the absence of a postjunctional action of neuronally released acetylcholine presumably is a consequence of the long diffusion distance between the perivascular axons and the endothelium in this muscular artery.
Cotransmitters belonging to different chemical classes have different modes of synthesis and storage and act via distinct receptors or intracellular pathways (12, 31). In cranial vasodilator neurons, the selective storage of peptide neurotransmitters in large vesicles is the basis of the preferential release of neuropeptides at higher frequencies of nerve stimulation (17). In contrast, nonpeptide transmitters such as acetylcholine can be stored in both small and large synaptic vesicles (27) and contribute to vasodilator responses over a wide range of stimulation frequencies. NO synthase (NOS), the enzyme responsible for NO synthesis, does not appear to be located inside synaptic vesicles (6, 23, 35), and NO is thought to be synthesized in the cytoplasm on demand in response to an influx of calcium into the nerve terminals (7). However, a NO donor rather than free NO might be the intercellular messenger between nerve terminals and the target cell, and it has been suggested that NO donors are stored in and released from synaptic vesicles (38).
It is now clear that a complex of proteins, soluble NSF attachment protein receptor (SNARE) proteins, is involved in synaptic vesicle exocytosis from many types of neurons (39, 44). Experiments using botulinum or tetanus toxins to cleave specific SNARE proteins demonstrated that release of different chemical classes of neurotransmitters seems to be dependent on SNARE proteins (28, 29). However, the sensitivity of transmitter release from different populations of autonomic neurons to blockade by botulinum toxins can be quite variable (14, 16, 20, 28, 35). One recent study (35) has shown that release of NO from inhibitory motor neurons in the guinea pig colon was not affected by botulinum toxin, whereas release of acetylcholine and substance P from excitatory motor neurons was greatly reduced, indicating differential involvement of SNARE proteins in release of these transmitters. Furthermore, it has been suggested that transmitter release from some large dense-cored vesicles may involve mechanisms different from those involved in release of small synaptic vesicles (21).
Our aim in the current study was to examine the relative role of the SNARE protein complex in release of three different classes of cotransmitters (NO, acetylcholine, and neuropeptides) from the same pelvic vasodilator neurons. We determined the effect of botulinum neurotoxin A (BoNTA), which cleaves and inactivates synaptosomal-associated protein of 25 kDa (SNAP-25), on responses of the isolated uterine artery to each cotransmitter released from the vasodilator neurons. We compared this with the effect of BoNTA on responses to noradrenaline released from sympathetic vasoconstrictor neurons in the same preparations (34).
| |
MATERIALS AND METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
In Vitro Preparations and Treatment with BoNTA
Virgin female guinea pigs (Hartley IMVS, 250-350 g body wt) were killed by stunning and exsanguination via the carotid arteries before removal of the main uterine arteries. These experiments were approved by the Flinders University Animal Welfare Committee. Arteries were placed in HEPES-buffered balanced salt solution [composed of (in mM) 146 NaCl, 4.7 KCl, 0.6 MgSO4, 1.6 NaHCO3, 0.13 NaH2PO4, 2.5 CaCl2, 7.8 glucose, 20 HEPES, and 0.1 ascorbic acid] adjusted to pH 7.3 with NaOH. The entire length of each main uterine artery was dissected free from the adjacent uterine vein and adipose tissue. Both arteries from each animal were then incubated for 2 h at 36°C in a sealed vial containing 250 µl of HEPES-buffered solution with or without 50 nM BoNTA (toxicity, 17 × 106 mouse LD50/mg; Sigma; Castle Hill, New South Wales, Australia). Arteries were washed three times (5 min each) in HEPES solution without BoNTA. A ring segment 4-5 mm long was dissected from the caudal end of one uterine artery from each animal and was mounted in a myograph for measurement of isometric tension (see below). The remaining segments of uterine arteries from each animal were returned to the sealed vial for incubation at 36°C until conclusion of the pharmacological experiments.Arterial segments were stretched between two nichrome wires (diameter,
25 µm) until the wires were separated by 1-1.25 times the
resting internal circumference. Arteries were superfused with HEPES-buffered solution bubbled with 100% O2 at 36°C and
were left to equilibrate for 1 h. Perivascular axons were
stimulated via two platinum wires lying either side of and parallel to
the long axis of the arterial segment. Trains of 200 pulses of 0.3-ms duration at 1 or 10 Hz were delivered every 20-30 min by a Grass S44 stimulator connected to a low-impedance interface (Biomedical Engineering, Flinders Medical Centre). Changes in isometric tension were detected with a Gould-Statham UTC2 transducer and recorded digitally using Chart v3.6 or v4.0 on a PowerLab 4S (AD Instruments; Castle Hill, New South Wales, Australia) connected to a Power Macintosh
7600/200 (Apple Computer). Changes in tension were calibrated in grams,
and the amplitude or area of constrictor and dilator responses were
determined using Chart v4.0 and NIH Image v1.62. The integrated
vasodilator response was calculated as the area of the relaxation below
the level of prostaglandin F2
(PGF) constriction
expressed as a percentage of the total area down to the baseline
without PGF constriction. Group data were analyzed using
repeated-measures ANOVA (SPSS for Windows v9, SPSS; Chicago, IL) or
t-tests (Microsoft Excel v5.0).
Analysis of Vasoconstrictor and Vasodilator Responses
Pharmacological experiments were carried out with one of three protocols designed to examine the effects of BoNTA on different cotransmitters or exogenous agonists.Protocol 1a: noradrenergic constrictions. In five control and five BoNTA-treated arteries, two pairs of stimuli were given initially to determine the amplitude of sympathetic vasoconstrictions at 1 and 10 Hz. Guanethidine (1 µM) was then added to abolish neurogenic vasoconstrictions before another pair of stimuli.
Protocol 1b: neurogenic vasodilations mediated by NO and neuropeptides. After the neurogenic constrictions were blocked with guanethidine, the same five control and five BoNTA-treated arteries were preconstricted with PGF (2 µM), and three more pairs of stimulus trains were applied at 1 and 10 Hz to determine the amplitude and area of vasodilator responses. NG-nitro-L-arginine methyl ester (L-NAME) was added to the superfusate between the first and second pairs of stimuli to block the initial component of neurogenic relaxations mediated by neuronal NO (30), leaving the slower relaxation shown previously to be mediated by the neuropeptides VIP and/or CGRP (1, 30). At the conclusion of experiments, all agents were washed out before the determination of the amplitude of contractions produced by KCl.
Protocol 2: prejunctional inhibition of vasodilations by acetylcholine. In 10 control and 10 BoNTA-treated arteries, one pair of stimuli was performed before addition of guanethidine and PGF. Vasodilator responses to two pairs of stimuli at 1 and 10 Hz were then determined. In five control and five BoNTA-treated arteries, hyoscine (1 µM) was added to the superfusate between the first and second pairs of dilator responses to block presynaptic muscarinic receptors. No hyoscine was added in the other 10 experiments.
Protocol 3: vasodilations produced by exogenous agonists. To test for any postjunctional effects of BoNTA, cumulative dose-response curves for the vasodilator agonists VIP (0.1 nM-0.1 µM), carbachol (1 nM-0.1 mM), and sodium nitroprusside (SNP; 1 nM-0.1 mM) were determined after preconstriction with PGF in five control and five BoNTA-treated arteries. A washout period of 1 h was left between each agonist curve. The negative log of the IC50 (pD2) was calculated for each set of curves and was expressed as means ± SE.
Drugs
The drugs used were as follows: carbachol (carbamylcholine chloride, Sigma); guanethidine sulphate (Sigma); hyoscine hydrobromide (Sigma); L-NAME hydrochloride (Sigma); PGF (Sapphire Bioscience; Crows Nest, New South Wales, Australia); SNP (Sigma); and the guinea pig sequence of VIP (Peninsula Laboratories).Immunoblotting for Immunoreactive SNAP-25
After pharmacological experimentation, all segments of uterine arteries from each of 20 animals (10 control and 10 BoNTA treated) were weighed (5-12 mg wet wt/animal), frozen in liquid nitrogen, and stored at
70°C. Proteins were extracted by homogenization in a
solution of 0.1% SDS, 20 mM Tris, 150 mM NaCl, and 1 mM EDTA with a
broad-spectrum protease inhibitor (Roche Diagnostics; Castle Hill, New
South Wales, Australia), followed by centrifugation at 100,000 g for 1 h. The total protein concentration of the
supernatant was determined by Lowry assay (24) using BSA
as the standard. Samples were concentrated to 5 µg/µl by freeze
drying and mixed 1:1 with a reducing buffer containing dithiothreitol
before boiling at 100°C for 5 min. Protein (15 µg) from each sample
(6 µl) was loaded onto 12.5% polyacrylamide gels alongside Bio-Rad
Precision protein standards (Bio-Rad Laboratories; Regents Park, New
South Wales, Australia), and electrophoresis (SDS-PAGE) was performed using a Bio-Rad MiniProtean III.
Protein bands were transferred to nitrocellulose and preincubated (1 h) in PBS containing 3% (wt/vol) skim milk powder before exposure for 18 h at 4°C to Sternberger SMI81 mouse monoclonal antibody (dilution 1/2,000, Affiniti Research Products; Mamhead, Exeter, UK) recognizing the NH2-terminus of SNAP-25. Immunoreactive protein bands were detected by incubation with a sheep anti-mouse IgG conjugated to horse radish peroxidase (dilution 1/3,000, AMRAD Biotech; Boronia, Victoria, Australia) for 1 h at room temperature, followed by enhanced chemiluminescence reaction (NEN Renaissance kit, NEL101, NEN Life Science Products; Boston, MA), and were recorded on Kodak X-Omat Blue XB-1 film. Films were scanned and digitized using a Microtek Scanmaker 4, and the optical densities of the immunoreactive bands were determined using NIH Image software. Files were saved in TIFF format and imported into Adobe Photoshop v4.0 (Adobe Systems) for compilation of figures.
Immunohistochemical Detection of Immunoreactive SNAP-25
Uterine arteries were removed from freshly killed guinea pigs, fixed immediately in Zamboni's solution at 4°C for 24-72 h, and then were processed for cryostat sectioning at 10 µm as described previously (32). Transverse sections were triple labeled for immunoreactivity to a combination of markers: 1) tyrosine hydroxylase (TH; antibody AS2-512 raised in a rabbit by Dr. J. Thibault, dilution 1/2,000) to demonstrate axons of catecholamine-synthesizing sympathetic neurons and VIP (antibody FI/III raised in a rat by Dr. R. Murphy, dilution 1/800) to demonstrate vasodilator axons and SNAP-25 (Sternberger mouse monoclonal antibody, dilution 1/2,000); 2) CGRP (Arnel antibody 1780 raised in a goat, dilution 1/1,000) to label sensory nerve fibers (1) together with VIP and SNAP-25 antibodies; and 3) S100 (Sigma antibody S2644 raised in a rabbit, dilution 1/2,400) to label Schwann cells together with VIP and SNAP-25 antibodies. The species-specific secondary antibodies used were donkey anti-rat IgG conjugated to dichlorotriazinylamino fluorescein (DTAF; dilution 1/100), donkey anti-mouse IgG conjugated to Cy3 (dilution 1/1,000), and donkey anti-rabbit or anti-sheep IgG (which recognizes goat IgG) conjugated to Cy5 (dilution 1/50). All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Sections were examined on an Olympus AX70 microscope with a Bio-Rad MRC 1024 laser scanning confocal system using a Kr-Ar laser. Through focus series of optical sections 0.5 µm apart were collected, and Bio-Rad files were saved in TIFF format and processed for brightness and contrast using Adobe Photoshop v4.0.| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Localization of Immunoreactive SNAP-25 in Autonomic Varicosities and Preterminal Axons
Immunoreactive SNAP-25 (SNAP-25-IR) was localized in axon varicosities at the adventitia-medial junction of the uterine artery close to the vascular smooth muscle, as we described previously (33). SNAP-25-IR was present both in vasodilator axons labeled with immunoreactive VIP (VIP-IR) and in adjacent vasoconstrictor axons labeled with immunoreactive TH (TH-IR), albeit at significantly higher levels in the dilator axons than in constrictor axons (Fig. 1) (33). Here, we extend those observations and report SNAP-25-IR in axon bundles distant from transmitter release sites at the adventitia-medial junction. SNAP-25-IR was prominent in axon bundles in the adventitia and appeared to be uniformly distributed throughout the preterminal axons (Fig. 1). Nerve fibers in the outer adventitia with immunoreactive CGRP (CGRP-IR) but not VIP-IR, which have been shown previously to be peptide-containing sensory fibers and not autonomic nerve fibers (1), contained very low levels of SNAP-25-IR. In contrast, SNAP-25-IR in axon bundles sometimes was colocalized with immunoreactivity for S100, a marker for Schwann cells.
|
Effect of BoNTA on SNAP-25-IR
The immunoblotting technique was optimized to demonstrate SNAP-25-IR in extracts of uterine arteries after a single pharmacological experiment. SDS-PAGE followed by immunoblotting demonstrated a single band of SNAP-25-IR at 25 kDa in extracts of uterine arteries incubated at 36°C for 7-8 h without BoNTA (Fig. 2). In contrast, extracts of arteries incubated at 36°C with BoNTA (50 nM) for 2 h and a further 5-6 h after washout of BoNTA showed an immunoreactive band at ~24 kDa in addition to the 25-kDa band (Figs. 2 and 3). The intensity of SNAP-25-IR bands at 25 and 24 kDa was determined in extracts of 10 control and 10 BoNTA-treated arteries run in parallel across four gels. This quantification demonstrated a significant reduction in immunoreactive protein at 25 kDa and a significant increase in immunoreactive protein at 24 kDa after BoNTA treatment (Fig. 3).
|
|
Effect of BoNTA on Constrictions Produced by Sympathetic Nerve Stimulation
At resting tone, electrical stimulation with trains of 200 pulses produced smooth muscle contractions that reached peak amplitude during the stimulation period and were greater in amplitude at 10 Hz than at 1 Hz (Fig. 4). Our previous study (34) demonstrated that this phase of the sympathetic contraction was mediated by norepinephrine. These contractions were abolished by guanethidine, sometimes revealing a small relaxation on stimulation at 10 Hz. BoNTA produced a reduction of borderline statistical significance in the amplitude of contractions produced by the first pair of stimuli at 1 and 10 Hz. However, the amplitude of the second pair of contractions (20-30 min later) was reduced significantly by BoNTA (80% reduction at 1 Hz and 70% reduction at 10 Hz; Fig. 4). Thus BoNTA treatment produced a significant rundown in amplitude of sympathetic constrictions on repeated stimulation at 1 or 10 Hz.
|
Effects of BoNTA on Contractions Produced by PGF and KCl and Relaxations Produced by Exogenous VIP, Carbachol, and SNP
Treatment with BoNTA did not alter the amplitude of contractions produced by 2 µM PGF [protocol 1: t = 0.16, P = 0.9, df = 8; protocol 2: t = 0.92, P = 0.4, degrees of freedom (df) = 18] or 126 mM KCl (protocol 1: t = 0.14, P = 0.9, df = 8; protocol 2: t = 2.1, P = 0.06, df = 17). VIP, carbachol, and SNP all produced 100% relaxation of arterial segments precontracted with PGF both in control and BoNTA-incubated arteries. Furthermore, treatment with BoNTA did not shift the concentration-response curve for any of the three agonists (VIP: control pD2 = 8.23 ± 0.18, BoNTA pD2 = 8.43 ± 0.12, t = 0.91, P = 0.4; carbachol: control pD2 = 6.15 ± 0.13, BoNTA pD2 = 6.19 ± 0.1, t = 0.25, P = 0.8; SNP: control pD2 = 7.68 ± 0.13, BoNTA pD2 = 7.42 ± 0.1, t = 1.85, P = 0.1).Effect of BoNTA on Autonomic Vasodilation Produced by NO and Neuropeptides
Electrical stimulation of control arteries in the presence of guanethidine and PGF produced a monophasic relaxation of vascular smooth muscle at 1 Hz and a biphasic relaxation at 10 Hz (Fig. 5A). The first phase of the biphasic relaxation reached maximum during the stimulation period and was abolished by L-NAME (30 µM for 30 min). The remaining relaxation at 10 Hz commenced after cessation of stimulation, reached maximum amplitude at ~5 min after stimulation, and lasted for 10-20 min (Fig. 5). Our previous study (30) showed that the response at 1 Hz and the initial phase at 10 Hz is mediated primarily by NO released from autonomic nerve terminals and the response at 10 Hz remaining after L-NAME is mediated by one or more neuropeptides. Thus to quantify the components of neurogenic vasodilatation due to NO and neuropeptides, we determined the amplitude of responses achieved during the stimulation period and the integrated responses (area) both before and after L-NAME treatment.
|
Segments of uterine artery treated with BoNTA showed vasodilator
responses to electrical stimulation at 1 and 10 Hz both before and
after addition of L-NAME (Fig. 5B). The peak
amplitude of smooth muscle relaxation during the stimulation period was
not altered by BoNTA treatment (Fig. 6).
Furthermore, L-NAME produced a significant reduction in
the maximum amplitude of responses at 1 and 10 Hz in both control and
BoNTA-treated arteries (Figs. 5 and 6). Thus BoNTA did not alter the
contribution of neuronal NO to autonomic vasodilation of the uterine
artery. However, BoNTA treatment reduced the duration of vasodilator
responses, particularly after L-NAME treatment (Fig. 5).
This was reflected as a reduction by BoNTA in the area of responses to
both 1- and 10-Hz stimulation (Fig. 7).
Given that the amplitude of NO responses was not affected by BoNTA and
that NO contributes to the area of responses before L-NAME,
these results demonstrate that the peptide component of the autonomic
vasodilations is inhibited partially by BoNTA treatment.
|
|
Effect of BoNTA on Prejunctional Inhibition of Neurogenic Vasodilations by Acetylcholine
In both control and BoNTA-treated arteries, the area of responses to a second pair of stimuli at 1 and 10 Hz was not significantly different from the area of responses to the first pair of stimuli {[F(1,8)] = 0.64, P = 0.5}, where F is the value of ANOVA results with degrees of freedom for effect and error terms, respectively, in parentheses. The addition of hyoscine (1 µM for 30 min) to control arteries between the first and second pairs of stimuli produced a significant enhancement in the duration of vasodilator responses, demonstrated by a significant increase in the area of responses (Fig. 8). Overall, BoNTA significantly reduced the area of vasodilator responses in these experiments (Fig. 8). In particular, BoNTA completely inhibited the enhancement in the area of vasodilations produced by hyoscine (Fig. 8). Thus the predominant effect of neuronally released acetylcholine, a prejunctional inhibition of dilations mediated by NO and neuropeptides, is abolished by this regime of BoNTA treatment.
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Cleavage of SNAP-25 by BoNTA
The mechanisms of action of botulinum toxins in inhibiting neurotransmission are now well understood. The toxins bind to acceptor sites on the neuronal membrane (3), are taken up into the cell by a calcium- and pH-dependent translocation process (18, 42), and cleave specific SNARE proteins (25) in a temperature- and zinc-dependent manner (18, 42). Furthermore, cleavage of the proteins is facilitated by nerve stimulation (42). BoNTA specifically cleaves nine amino acids from the COOH-terminus of SNAP-25 (4). The apparent selectivity of action of botulinum toxins on cholinergic neurons, particularly at the neuromuscular junction, is now known to be due to a concentration of high-affinity toxin acceptor sites on these neurons (3). Although noncholinergic autonomic and central nerve terminals have very few of these high-affinity acceptor sites for BoNTA, higher concentrations of toxins can still be taken up by low-affinity acceptors or by nonspecific uptake mechanisms (3).The regime of BoNTA treatment used in the present study was based on previous functional studies (14, 16, 28) of autonomic neurons showing that BoNTA incubated in the nanomolar range with whole tissues for several hours at 37°C produced substantial reduction or abolition of neurotransmission from some types of neurons. This treatment clearly produced cleavage of the COOH-terminal nine amino acids from SNAP-25 in the uterine artery, leaving a shortened protein of ~24 kDa (4). However, cleavage of SNAP-25 was only partial. Nevertheless, at least one effect of electrical stimulation of perivascular axons innervating the uterine artery, the cholinergic inhibition of the vasodilator response, was abolished by this regime of BoNTA (see below). This is consistent with studies (18, 19, 37) on the neuromuscular junction and cultured spinal cord neurons, where substantial inhibition of neurotransmission occurs at a stage when cleavage of SNAP-25 is minimal.
There are several possible explanations for this partial, or slow, cleavage of SNAP-25 after a BoNTA treatment regime that inhibits neurotransmission. First, it is possible that only a small proportion of the total SNAP-25 in the tissue is directly involved in neurotransmission and is accessible to BoNTA. This is likely to be the case in the uterine artery, where we have shown that much of SNAP-25-IR is located in nerve bundles distant from neurotransmitter release sites, including in Schwann cells. Even within nerve terminals, SNAP-25 can be located on recycling synaptic vesicles rather than the terminal membrane (47). This is consistent with our observation that SNAP-25-IR in axon varicosities in the uterine artery is not restricted to the cell membrane but is quite uniformly distributed throughout the varicosities of the vasodilator axons. Furthermore, only a small proportion of the total SNAP-25 at synaptic release sites is likely to be in an uncomplexed form that can be cleaved by BoNTA (13, 36, 37).
A second possibility is that BoNTA treatment inhibits neurotransmission by a mechanism in addition to its inactivation of SNAP-25. It is most unlikely that BoNTA treatment in the current study inactivated another protein involved in neurotransmission, because other studies (4, 19, 36, 37) in vitro using similar or higher concentrations of toxin have found no evidence for nonspecific cleavage of other SNARE proteins by BoNTA. Furthermore, in the present study, BoNTA treatment did not affect receptors or smooth muscle contractile proteins mediating constrictions and dilations by exogenous agonists. It has been recently suggested that the NH2-terminal 197-amino acid cleavage product of SNAP-25 produces a functional antagonism of neurotransmission in cultured neurons from the fetal rat spinal cord (19). Thus inhibition of neurotransmitter release by BoNTA would be the sum effect of a reduction in SNAP-25 able to participate in vesicle exocytosis and production of a cleavage product of SNAP-25 that actively inhibits exocytosis (19). It is not yet known whether such a phenomenon contributes to the functional effects of BoNTA on other neurons. Nevertheless, such an action should be considered a specific effect of BoNTA on the target protein, SNAP-25, rather than a nonspecific effect.
Differential Effect of BoNTA on Different Classes of Cotransmitters
We have shown that 50 nM BoNTA partially inhibits the noradrenergic component of sympathetic vasoconstriction of the uterine artery. In the same preparations, the cholinergic prejunctional inhibition due to stimulation of vasodilator neurons was abolished, and the slow peptide-mediated component of the neurogenic vasodilation was reduced. However, the initial phase of the vasodilation mediated by neurally released NO was not affected. Because BoNTA did not affect postsynaptic mechanisms of vasoconstriction or vasodilation, we conclude that BoNTA has reduced neurotransmission from both vasoconstrictor and vasodilator neurons. Furthermore, because acetylcholine, NO, and neuropeptides are all synthesized by the same pelvic vasodilator neurons (1), we conclude that BoNTA has differential effects on cotransmitters released from the same neurons.The lack of inhibition by BoNTA of the NO-mediated vasodilation of the uterine artery is consistent with previous studies on the bull retractor penis and penile artery (20) and guinea pig taenia coli (28), where BoNTA failed to inhibit noncholinergic inhibitory responses that are now known to be mediated at least partly by neuronal NO (22, 41). In the current study, we can discount the possibility that the neurons utilizing NO as a transmitter do not have acceptor sites for binding BoNTA, because cholinergic transmission from the same neurons was inhibited by BoNTA. Botulinum toxin type B, which cleaves the SNARE protein synaptobrevin, also fails to inhibit release of NO from inhibitory neurons in the guinea pig gastrointestinal tract (35). These studies all support the conclusion that release of NO from autonomic neurons occurs independently of the SNARE complex mediating vesicle exocytosis. They are consistent with the view that NO is synthesized on demand from nonvesicular NOS rather than released from a NO donor stored in synaptic vesicles (cf Ref. 38).
We have shown that two types of cotransmitters released from vesicles in the same vasodilator nerve terminals, acetylcholine and the neuropeptides VIP or CGRP, are both inhibited by BoNTA but to different degrees. Our findings are consistent with the conclusion that release of small vesicles containing acetylcholine alone is blocked completely by BoNTA, whereas release of neuropeptides from large vesicles is only partially blocked (27). This suggests that, although SNARE proteins are involved in exocytosis from large as well as small vesicles, their interactions with other factors regulating transmitter release from different vesicle pools are not identical (21). Further evidence for this proposal has been provided recently by demonstration of the selective role of a calcium-dependent activator protein for secretion (CAPS) in transmitter release from dense core vesicles, but not small synaptic vesicles, in rat brain synaptosomes (45). It is not yet known whether proteins such as CAPS are involved selectively in exocytosis of peptide transmitters from large vesicles in autonomic neurons.
The partial inhibition of noradrenergic neurotransmission from sympathetic vasoconstrictor neurons produced by BoNTA, with the same regime of treatment that completely inhibited cholinergic neurotransmission, is also consistent with previous studies (14, 28) of autonomic neurons. Although neuropeptide Y (NPY) acts as a vasoconstrictor cotransmitter with norepinephrine in the uterine artery, the NPY-mediated component of sympathetic vasoconstriction is slow, occurring after cessation of stimulation, and is only revealed after removal of the vasodilator neurons (34). Thus release of the peptide cotransmitter is unlikely to have contributed to the relative resistance to BoNTA of the early phase of sympathetic neurotransmission examined in the present study. This low sensitivity of noradrenergic neurons to BoNTA may well be due to a low density of high-affinity acceptor sites for the toxin (3). However, we (33) have also shown that the terminals of vasoconstrictor neurons in the uterine artery contain significantly lower levels of immunoreactivity to SNAP-25 than do adjacent vasodilator varicosities (Fig. 1). Thus it is possible that the role of the SNARE complex in exocytosis from small vesicles in sympathetic neurons containing norepinephrine is different from that releasing acetylcholine from small vesicles in the vasodilator neurons in the uterine artery.
Physiological Implications
This study has demonstrated that the release of different classes of cotransmitters from the same autonomic neurons involves regulatory mechanisms with differential sensitivities to clostridial neurotoxins. This further supports the notion that neurons have developed different strategies to match the release of each cotransmitter to different levels of neuronal activation that occur as part of specific reflex pathways (21). Furthermore, endogenous factors or exogenous drugs that interfere selectively with these release mechanisms may have different functional effects on neurotransmission at different levels of neuronal activation. Although botulism is a relatively rare condition, therapeutic use of BoNTA for treatment of muscle spasms, and for cosmetic purposes, is increasing. The doses of toxin injected clinically are very low, but there have been reports of repeated therapeutic doses having autonomic effects, particularly on the cardiovascular system (11). Further studies delineating regional variations in mechanisms of cotransmission from autonomic neurons are likely to have important clinical implications for cardiovascular control.| |
ACKNOWLEDGEMENTS |
|---|
We thank Pat Vilimas for excellent technical assistance.
| |
FOOTNOTES |
|---|
This work was supported by National Health and Medical Research Council of Australia (NHMRC) Project Grant 102114. J. L. Morris is a Principal Research Fellow of the NHMRC.
Address for reprint requests and other correspondence: J. L. Morris, Dept. of Anatomy and Histology, Flinders Univ., GPO Box 2100, Adelaide SA 5001, Australia (E-mail: Judy.Morris{at}flinders.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 May 2001; accepted in final form 23 July 2001.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Anderson, RL,
Gibbins IL,
and
Morris JL.
Five inhibitory transmitters coexist in pelvic autonomic neurons.
Neuroreport
8:
3023-3028,
1997[Web of Science][Medline].
2.
Bell, C.
Transmission from vasoconstrictor and vasodilator nerves to single smooth muscle cells of the guinea-pig uterine artery.
J Physiol (Lond)
205:
695-708,
1969
3.
Black, JD,
and
Dolly JO.
Selective location of acceptors for botulinum neurotoxin A in the central and peripheral nervous systems.
Neuroscience
23:
767-779,
1987[Web of Science][Medline].
4.
Blasi, J,
Chapman ER,
Link E,
Binz T,
Yamasaki S,
De Camilli P,
Südolf TC,
Niemann H,
and
Jahn R.
Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25.
Nature
365:
160-163,
1993[Medline].
5.
Bowman, A,
and
Gillespie JS.
Neurogenic vasodilatation in isolated bovine and canine penile arteries.
J Physiol (Lond)
341:
603-616,
1983
6.
Bredt, DS,
and
Snyder SH.
Isolation of nitric oxide synthase, a calmodulin-requiring enzyme.
Proc Natl Acad Sci USA
87:
682-685,
1990
7.
Bredt, DS,
and
Snyder SH.
Nitric oxide, a novel neuronal messenger.
Neuron
8:
3-11,
1992[Web of Science][Medline].
8.
Creed, KE,
Carati CJ,
and
Keogh EJ.
Autonomic control and vascular changes during penile erection in monkeys.
Br J Urol
61:
510-515,
1988[Web of Science][Medline].
9.
De Groat, WC,
and
Booth AM.
Neural control of penile erection.
In: Nervous Control of the Urogenital System, edited by Maggi CA.. Chur, Switzerland: Harwood, 1993, p. 467-524.
10.
Dorr, LD,
and
Brody MJ.
Hemodynamic mechanisms of erection in the canine penis.
Am J Physiol
213:
1526-1531,
1967.
11.
Girlanda, P,
Vita G,
Nicolosi C,
Milone S,
and
Messina C.
Botulinum toxin therapy: distant effects on neuromuscular transmission and autonomic nervous system.
J Neurol Neurosurg Psychiatry
55:
844-845,
1992
12.
Gibbins, IL,
and
Morris JL.
Pathway specific expression of neuropeptides and autonomic control of the vasculature.
Regul Pept
93:
93-107,
2000[Web of Science][Medline].
13.
Hayashi, T,
McMahon H,
Yamasaki S,
Binz T,
Hata Y,
Südolf TC,
and
Niemann H.
Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.
EMBO J
13:
5051-5061,
1994[Web of Science][Medline].
14.
Holman, ME,
and
Spitzer NC.
Action of botulinum toxin on transmission from sympathetic nerves to the vas deferens.
Br J Pharmacol
47:
431-433,
1973[Web of Science][Medline].
15.
Hotta, H,
Uchida S,
Shimura M,
and
Suzuki H.
Uterine contractility and blood flow are reflexively regulated by cutaneous afferent stimulation in anesthetized rats.
J Auton Nerv Syst
75:
23-31,
1999[Web of Science][Medline].
16.
Ishikawa, H,
Mitsui Y,
Yoshitomi T,
Mashimo K,
Aoki S,
Mukuno K,
and
Shimizu K.
Presynaptic effects of botulinum toxin type A on the neuronally evoked response of albino and pigmented rabbit iris sphincter and dilator muscles.
Jpn J Ophthalmol
44:
106-109,
2000[Medline].
17.
Johansson, O,
and
Lundberg JM.
Ultrastructural localization of VIP-like immunoreactivity in large dense cored vesicles of "cholinergic type" nerve terminals in cat exocrine glands.
Neuroscience
5:
847-862,
1981.
18.
Kalandakanond, S,
and
Coffield JA.
Cleavage of SNAP-25 by botulinum toxin type A requires receptor-mediated endocytosis, pH-dependent translocation and zinc.
J Pharmacol Exp Ther
296:
980-986,
2001
19.
Keller, JE,
and
Neale EA.
The role of synaptic protein SNAP-25 in the potency of botulinum neurotoxin type A.
J Biol Chem
276:
13476-13482,
2001
20.
Klinge, R,
and
Sjöstrand NO.
Contraction and relaxation of the retractor penis muscle and the penile artery of the bull.
Acta Physiol Scand Suppl
420:
1-88,
1974[Medline].
21.
Langley, K,
and
Grant NJ.
Are exocytosis mechanisms neurotransmitter specific?
Neurochem Int
31:
739-757,
1997[Web of Science][Medline].
22.
Liu, X,
Gillespie JS,
Gibson IF,
and
Martin W.
Effects of NG-substituted analogues of L-arginine on NANC relaxation of the rat anococcygeus and bovine retractor penis muscle and the bovine penile artery.
Br J Pharmacol
104:
53-58,
1991[Web of Science][Medline].
23.
Llewellyn-Smith, IJ,
Song ZM,
Costa M,
Bredt DS,
and
Snyder SH.
Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons.
Brain Res
577:
337-342,
1992[Web of Science][Medline].
24.
Lowry, OH,
Rosenbrough NJ,
Farr AL,
and
Randall RJ.
Protein measurement with the folin reagent.
J Biol Chem
193:
265-275,
1951
25.
Ludger, J,
and
Galli T.
SNAREs drum up!
Eur J Neurosci
10:
415-422,
1998[Medline].
26.
Lundberg, JM,
Änggård A,
and
Fahrenkrug J.
Complementary role of vasoactive intestinal polypeptide (VIP) and acetylcholine for cat submandibular blood flow and secretion. II. Effects of cholinergic antagonists and VIP antiserum.
Acta Physiol Scand
113:
329-336,
1981[Web of Science][Medline].
27.
Lundberg, JM,
Fried G,
Fahrenkrug J,
Hökfelt T,
Lagercrantz H,
Lundgren G,
and
Änggård A.
Subcellular fractionation of cat submandibular gland: comparative studies on the distribution of acetylcholine and vasoactive intestinal polypeptide (VIP).
Neuroscience
6:
1001-1010,
1981[Web of Science][Medline].
28.
MacKenzie, A,
Burnstock G,
and
Dolly JO.
The effects of purified botulinum neurotoxin type A on cholinergic, adrenergic and non-adrenergic, atropine-resistant autonomic neuromuscular transmission.
Neuroscience
7:
997-1006,
1982[Web of Science][Medline].
29.
McMahon, HT,
Foran P,
Dolly JO,
Verhage M,
Wiegert VM,
and
Nicholls DG.
Tetanus toxin and botulinum toxins type A and B inhibit glutamate, gamma-aminobutyric acid, aspartate and met-enkephalin release from synaptosomes.
J Biol Chem
276:
21338-21343,
1992.
30.
Morris, JL.
Co-transmission from autonomic vasodilator neurons supplying the guinea pig uterine artery.
J Auton Nerv Syst
42:
11-22,
1993[Web of Science][Medline].
31.
Morris, JL,
and
Gibbins IL.
Co-transmission and neuromodulation.
In: Autonomic Neuroeffector Mechanisms, edited by Burnstock G,
and Hoyle CHV. Chur, Switzerland: Harwood, 1992, p. 33-119.
32.
Morris, JL,
Kondo M,
and
Gibbins IL.
Selective innervation of different target tissues in guinea-pig cranial exocrine glands by subpopulations of parasympathetic and sympathetic axons.
J Auton Nerv Syst
66:
75-86,
1997[Web of Science][Medline].
33.
Morris, JL,
Lindberg CEY,
and
Gibbins IL.
Different levels of immunoreactivity for synaptosomal-associated protein of 25kDa in vasoconstrictor and vasodilator axons of guinea-pigs.
Neurosci Lett
294:
167-170,
2000[Web of Science][Medline].
34.
Morris, JL,
and
Murphy R.
Evidence that neuropeptide Y released from noradrenergic axons causes prolonged contraction of the guinea-pig uterine artery.
J Auton Nerv Syst
24:
241-249,
1988[Web of Science][Medline].
35.
Olgart, C,
Gustafsson LE,
and
Wiklund NP.
Evidence for non-vesicular nitric oxide release evoked by nerve activation.
Eur J Neurosci
12:
1303-1309,
2000[Web of Science][Medline].
36.
Pellegrini, LL,
O'Connor V,
Lottspeich F,
and
Betz H.
Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.
EMBO J
14:
4705-4713,
1995[Web of Science][Medline].
37.
Raciborska, DA,
Trimble WS,
and
Charlton MP.
Presynaptic protein interactions in vivo: evidence from botulinum A, C, D, and E action at frog neuromuscular junction.
Eur J Neurosci
10:
2617-2628,
1998[Web of Science][Medline].
38.
Rand, MJ,
and
Li CG.
Nitric oxide as a neurotransmitter in peripheral nerves: nature of transmitter and mechanism of transmission.
Annu Rev Physiol
57:
659-682,
1995[Web of Science][Medline].
39.
Risinger, C,
Blomqvist AG,
Lundell I,
Lamertsson A,
Nässel D,
Peribone VA,
Brodin L,
and
Larhammer D.
Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones.
J Biol Chem
268:
24408-24414,
1993
40.
Sato, Y,
Hotta H,
Nakayama H,
and
Suzuki H.
Sympathetic and parasympathetic regulation of the uterine blood flow and contraction in the rat.
J Auton Nerv Syst
59:
151-158,
1996[Web of Science][Medline].
41.
Shuttleworth, CW,
Sweeney KM,
and
Sanders KM.
Evidence that nitric oxide acts as an inhibitory neurotransmitter supplying taenia from the guinea-pig caecum.
Br J Pharmacol
127:
1495-1501,
1999[Web of Science][Medline].
42.
Simpson, LL.
Kinetic studies on the interaction between botulinum toxin type A and the cholinergic neuromuscular junction.
J Pharmacol Exp Ther
212:
16-21,
1980
43.
Sjöstrand, NO,
and
Klinge E.
Principal mechanisms controlling penile retraction and protrusion in rabbits.
Acta Physiol Scand
106:
199-214,
1979[Web of Science][Medline].
44.
Söllner, T,
Whiteheart SW,
Brunner M,
Erdjument-Bromage H,
Geromanos S,
Tempst P,
and
Rothman JE.
SNAP receptors implicated in vesicle targeting and fusion.
Nature
362:
318-324,
1993[Medline].
45.
Tandon, A,
Bannykh S,
Kowalchyk JA,
Banerjee A,
Martin TFJ,
and
Balch WE.
Differential regulation of exocytosis by calcium and CAPS in semi-intact synaptosomes.
Neuron
21:
147-154,
1998[Web of Science][Medline].
46.
Traurig, HH,
and
Papka RE.
Autonomic efferent and visceral sensory innervation of the female reproductive system: special reference to the functional roles of nerves in reproductive organs.
In: Nervous Control of the Urogenital System, edited by Maggi CA.. Chur, Switzerland: Harwood, 1993, p. 103-141.
47.
Walch-Solimena, C,
Blasi J,
Edelman L,
Chapman ER,
von Mollard GF,
and
Jahn R.
The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling.
J Cell Biol
128:
637-645,
1995
This article has been cited by other articles:
|
|
 |
|
  D. L. Kellogg Jr., J. L. Zhao, and Y. Wu Roles of nitric oxide synthase isoforms in cutaneous vasodilation induced by local warming of the skin and whole body heat stress in humans J Appl Physiol, November 1, 2009; 107(5): 1438 - 1444. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Kellogg Jr, J. L. Zhao, and Y. Wu Neuronal nitric oxide synthase control mechanisms in the cutaneous vasculature of humans in vivo J. Physiol., February 1, 2008; 586(3): 847 - 857. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. M. Smyth, L. T. Breen, and V. N. Mutafova-Yambolieva Nicotinamide adenine dinucleotide is released from sympathetic nerve terminals via a botulinum neurotoxin A-mediated mechanism in canine mesenteric artery Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1818 - H1825. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Ansiaux, C. Baudelet, G. O. Cron, J. Segers, C. Dessy, P. Martinive, J. De Wever, J. Verrax, V. Wauthier, N. Beghein, et al. Botulinum Toxin Potentiates Cancer Radiotherapy and Chemotherapy Clin. Cancer Res., February 15, 2006; 12(4): 1276 - 1283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. J. Piovesan, H. G. Teive, P. A. Kowacs, M. V. D. Coletta, L. C. Werneck, and S. D. Silberstein An open study of botulinum-A toxin treatment of trigeminal neuralgia Neurology, October 25, 2005; 65(8): 1306 - 1308. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Voller, T. Sycha, B. Gustorff, L. Schmetterer, S. Lehr, H. G. Eichler, E. Auff, and P. Schnider A randomized, double-blind, placebo controlled study on analgesic effects of botulinum toxin A Neurology, October 14, 2003; 61(7): 940 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. N. James, J. P. Ryan, and H. P. Parkman Inhibitory effects of botulinum toxin on pyloric and antral smooth muscle Am J Physiol Gastrointest Liver Physiol, July 7, 2003; 285(2): G291 - G297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Morris, P. Jobling, and I. L. Gibbins Botulinum neurotoxin A attenuates release of norepinephrine but not NPY from vasoconstrictor neurons Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2627 - H2635. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
