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

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.     

Nicotinamide adenine dinucleotide is released from sympathetic nerve terminals via a botulinum neurotoxin A-mediated mechanism in canine mesenteric artery

Using high-performance liquid chromatography techniques with fluorescence and electrochemical detection, we found that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in response to electrical field stimulation (4-16 Hz, 0.3 ms, 15 V, 120 s) along with ATP and norepinephrine (NE) in the canine isolated mesenteric arteries. The release of beta-NAD increases with number of pulses/stimulation frequencies. Immunohistochemistry analysis showed dense distribution of tyrosine hydroxylase-like immunoreactivity (TH-LI) and sparse distribution of TH-LI-negative nerve processes, suggesting that these blood vessels are primarily under sympathetic nervous system control with some contribution of other (e.g., sensory) neurons. Exogenous NE (3 micromol/l), alpha,beta-methylene ATP (1 micromol/l), neuropeptide Y (NPY, 0.1 micromol/l), CGRP (0.1 micromol/l), vasoactive intestinal peptide (VIP, 0.1 micromol/l), and substance P (SP, 0.1 micromol/l) had no effect on the basal release of beta-NAD, suggesting that the overflow of beta-NAD is evoked by neither the sympathetic neurotransmitters NE, ATP, and NPY, nor the neuropeptides CGRP, VIP, and SP. Botulinum neurotoxin A (BoNTA, 0.1 micromol/l) abolished the evoked release of NE, ATP, and beta-NAD at 4 Hz, suggesting that at low levels of neural activity, release of these neurotransmitters results from N-ethylmaleimide-sensitive factor attachment protein receptor/synaptosomal-associated protein of 25 kDa-mediated exocytosis. At 16 Hz, however, the evoked release of NE, ATP, and beta-NAD was reduced by BoNTA by approximately 90, 60, and 80%, respectively, suggesting that at higher levels of neural activity, beta-NAD is likely to be released from different populations of synaptic vesicles or different populations of nerve terminals (i.e., sympathetic and sensory terminals).     

Roles of neuropeptide Y and noradrenaline in sympathetic neurotransmission to the thoracic vena cava and aorta of guinea-pigs.

The roles of neuropeptide Y (NPY) and noradrenaline (NA) in sympathetic neurotransmission to large arteries and veins were studied in vitro using the thoracic portions of the aorta and inferior vena cava from guinea-pigs. Both vessels are densely innervated by axons containing NA and NPY. Repetitive transmural stimulation at 2-30 Hz produced contractions of the aorta, which were abolished by prazosin. NPY did not have significant postsynaptic or presynaptic effects on vascular tone of the aorta. Transmural stimulation of the vena cava produced long-lasting contractions which were enhanced by alpha- and beta-adrenoceptor antagonists, and were blocked by guanethidine. Precontracted venae cavae responded to sympathetic stimulation with beta-adrenoceptor-mediated relaxation, followed by contraction. alpha-Adrenoceptor blockade delayed the onset of neurogenic contractions. NPY was a potent contractile agent of the vena cava (EC50 approximately 1.5 x 10(-8) M). A high concentration (3 x 10(-6) M) of NPY, or the specific NPY Y1 receptor agonist, [Leu31, Pro34]NPY, caused parallel, and reversible, desensitization of contractions produced by sympathetic nerve stimulation, and by low concentrations of exogenous NPY. This provides good evidence that NPY is the mediator of the non-adrenergic sympathetic contractions of the vena cava. Furthermore, these results demonstrate that differential location or coupling of postsynaptic receptors for NA and NPY in the aorta and vena cava, leads to differential participation by these substances in sympathetic vasomotor responses. This is likely to be related to the different functions of these two parts of the systemic circulation.     

Functional and histochemical characterization of a uterine adrenergic denervation process in pregnant rats

The time course of pregnancy-induced changes in the contractile responses of isolated uterine rings and sympathetic innervation pattern were studied using electric field stimulation and histofluorescence techniques, respectively, in intact and 6-hydroxydopamine-treated rats. Neurally mediated contractions elicited by field stimulation (0.6 msec, 1-70 Hz, 40 V) were measured in uterine preparations obtained from nonpregnant, 6-hydroxydopamine-treated and 5-, 10-, 15-, 18-, and 22-day (term) pregnant rats. At all frequencies, the amplitudes of contractions were highest in nonpregnant uteri. Stimulation at 1-2.5 Hz evoked contractions in 10-day pregnant uteri but failed to cause contractions on Day 5 and from Day 15 onward. In uterine preparations obtained from term and from 6-hydroxydopamine-treated rats, contractions could not be evoked by stimulation at 1-20 Hz. Fluorescence histochemistry of uterine adrenergic nerves revealed rich perivascular and myometrial innervation in nonpregnant and in pregnant rats through Day 10. Degeneration and loss of adrenergic nerve fibers was apparent by Day 15, and fluorescent myometrial and perivascular nerves were practically absent by Day 22. These findings demonstrate a progressive, frequency-related reduction of nerve-mediated uterine contractions beginning in midterm pregnancy, in parallel with a gradual loss of adrenergic nerve fibers. Pregnancy-induced nerve degeneration may promote the development of nonsynaptic alpha-adrenergic uterine contractile activity towards term. The reduced responsiveness of uterine smooth muscle to electric field stimulation in early pregnancy appears to be unrelated to alterations in uterine innervation but may be related to changes associated with implantation.     

Inhibitory effects of neuropeptide Y on sympathetic neurotransmission in the rabbit iris-ciliary body

Neuropeptide Y (NPY, 1–300 nM) mediated a concentration-dependent inhibition of field stimulation-evoked [³H]norepinephrine (NE) overflow from the isolated, superfused rabbit iris-ciliary body. At equimolar concentrations (100 nM), the homologous neuropeptide peptide YY (PYY) mimicked the effects of NPY, whereas pancreatic polypeptide (PP) and the C-terminal fragment of NPY_16–36 did not modify [³H]NE release. NPY-induced inhibition of [³H]NE release was unaffected by pretreatment of tissues with atropine (100 nM) plus yohimbine (100 nM) and was nonadditive with the maximal prejunctional effects of carbamycholine or clonidine, indicating that NPY acts independently of prejunctional muscarinic or alpha₂-adrenergic receptor activity to reduce [³H]NE overflow. It is concluded that NPY is a specific, potent modulator of adrenergic neurosecretion in the rabbit iris-ciliary body. These findings confirm the role of NPY as a co-transmitter at ocular sympathetic neuroeffector junctions, either mimicking or augmenting the actions of endogenously released norepinephrine.To whom to address reprint requests.     

Effects of varying impulse number on cotransmitter contributions to sympathetic vasoconstriction in rat tail artery

We examined the contributions of the cotransmitters norepinephrine (NE), ATP, and neuropeptide Y (NPY) to sympathetically evoked vasoconstriction in the rat tail artery in isolated vascular rings by using 1-100 stimulation impulses at 20 Hz. Phentolamine (2 microM), the alpha-adrenoceptor antagonist, markedly reduced responses to all stimuli, although responses to lower impulse numbers were reduced less than responses to longer trains. The purinergic receptor antagonist suramin (100 microM) reduced all responses, but to a much greater extent with few impulse trains. Responses were further reduced or abolished by addition of the second antagonist. Any remaining responses were abolished by the NPY-Y(1) receptor antagonist BIBP-3226 (75 nM). NPY had a direct agonist action and potentiated sympathetically mediated responses. NPY (75 nM) potentiated responses and BIBP-3226 decreased responses to 2- and 20-impulse trains. Both affected responses from 2 impulses to >20 impulses, but there was no preferential effect on purinergic contributions to responses because neurally released NPY potentiated both "pure" NE and ATP responses equally. We conclude that all three cotransmitters contribute significantly to vascular responses and their contribution varies markedly with impulse numbers. There is considerable synergy between cotransmitters, especially with lower impulse numbers where NPY contributions are greater than expected.     

Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.     

SNARE complexes and neuroexocytosis: how many, how close?

Regulated secretion is an essential process in all eukaryotic cells. The release of molecules contained inside exocytic granules and synaptic vesicles is mediated by the assembly of a SNARE complex formed by the coil-coiling of three proteins: SNAP-25, syntaxin and VAMP/synaptobrevin. It seems that SNARE complexes assemble together in rosette-shaped super-complexes but there is controversy on the actual number (N) of copies of SNARE complexes that are necessary to mediate exocytosis. We discuss attempts to determine the value of N and suggest that N varies with the type of exocytic vesicles. In addition, we propose that the N value in neuroexocytosis can be estimated by the comparative use of different types of botulinum neurotoxins.     

In vitro effect of neuropeptide Y on melatonin and norepinephrine release in rat pineal gland

1. To study neuropeptide Y (NPY) effect on melatonin production, rat pineal explants were incubated for 6 hr with 10-1,000 nM NPY in the presence or absence of 10 microM norepinephrine (NE). Melatonin content in the pineal gland and media was measured by radioimmunoassay (RIA). 2. NPY (10-1,000 nM) increased melatonin production and, at 10 or 100 nM concentrations (but not 1,000 nM), enhanced NE stimulation of melatonin production. 3. NPY (1,000 nM) impaired 3H-labeled transmitter release induced by a K+ depolarizing stimulus in rat pineals incubated with 3H-NE. 4. These results suggest that NPY affects both pre- and postsynaptic pineal mechanisms.     

Responses of the uterine circulation to sympathetic nerve stimulation

Pregnant and non-pregnant sheep uteri were perfused in situ with arterial blood at a constant flow rate. Unilateral stimulation (1-2 ma, u ms pulse) of the distal end of the severed sympathetic chain (L3-L4) at frequencies between 5 and 25 Hz produced a graded increase in uterine artery pressure in both horns. At 25 Hz, pressure in the horn ipsilateral to the stimulated sympathetic chain increased by 28 +/ 2% in four pregnant animals and 32 +/ 5% in six non-pregnant ewes. The response of the contralateral horn was significantly smaller than that of the ipsilateral horn (P less than or equal to 0.05). The response was alpha-mediated since it was abolished by local injection of dibenzyline into the middle uterine artery. The responses of the pregnant and non-pregnant animals were similar, indicating that pregnancy did not alter the alpha-adrenergic responses of the uterine vasculature.     

Neuropeptide Y is a cotransmitter with norepinephrine in guinea pig inferior mesenteric vein

Neuropeptide Y (NPY) is a cotransmitter with noradrenaline in guinea pig inferior mesenteric vein. Tyrosine hydroxylase-like immunoreactivity and NPY-like immunoreactivity were colocalized in a dense network of fibers within the adventitial layer of guinea-pig inferior mesenteric vein. Vasoconstrictor responses to electrical field stimulation (0.2-64 Hz, 0.1 ms, 12 V, for 10 s) appear to be mediated primarily by norepinephrine at 0.2 to 4 Hz and by NPY at 8 to 64 Hz. NPY Y1 receptors mediate the contractile responses to both endogenous and exogenous NPY. Norepinephrine and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of norepinephrine and NPY to serve capacitance.     

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca²⁺-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.     

SNARE complex regulation by phosphorylation

SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors) are ubiquitous proteins that direct vesicular trafficking and exocytosis. In neurons, SNAREs act to mediate release of neurotransmitters, which is a carefully regulated process. Calcium influx has long been shown to be the key trigger of release. However, calcium alone cannot regulate the degree of vesicle content release. For example, only a limited number of docked vesicles releases neurotransmitters when calcium entry occurs; this suggests that exocytosis is regulated by other factors besides calcium influx. Regulation of the degree of release is best explained by looking at the many enzymatic proteins that interact with the SNARE complex. These proteins have been hypothesized to regulate the formation, stability, or disassembly of the SNARE complex and therefore may regulate neurotransmitter release. One group of enzymatic regulators is the protein kinases. These proteins phosphorylate sites on both SNARE proteins and proteins that interact with SNARE proteins. Recent research has identified some of the specific effects that phosphorylation (or dephosphorylation) at these sites can produce. Additionally, palmitoylation of SNAP-25, regulates the localization, and hence activity of this key SNARE protein. This review focuses on the location and effects of phosphorylation on SNARE regulation.     

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.     

Neuropeptide Y cotransmission with norepinephrine in the sympathetic nerve-macrophage interplay

The CNS modulates immune cells by direct synaptic-like contacts in the brain and at peripheral sites, such as lymphoid organs. To study the nerve-macrophage communication, a superfusion method was used to investigate cotransmission of neuropeptide Y (NPY) with norepinephrine (NE), with interleukin (IL)-6 secretion used as the macrophage read-out parameter. Spleen tissue slices spontaneously released NE, NPY, and IL-6 leading to a superfusate concentration at 3-4 h of 1 nM:, 10 pM:, and 120 pg/ml, respectively. Under these conditions, NPY dose-dependently inhibited IL-6 secretion with a maximum effect at 10(-10) M: (p = 0.012) and 10(-9) M: (p < 0.001). Simultaneous addition of NPY at 10(-9) M: and the alpha-2-adrenergic agonist p-aminoclonidine further inhibited IL-6 secretion (p < 0.05). However, simultaneous administration of NPY at 10(-9) M: and the beta-adrenergic agonist isoproterenol at 10(-6) M: or NE at 10(-6) M: significantly increased IL-6 secretion (p < 0.005). To objectify these differential effects of NPY, electrical field stimulation of spleen slices was applied to release endogenous NPY and NE. Electrical field stimulation markedly reduced IL-6 secretion, which was attenuated by the NPY Y1 receptor antagonist BIBP 3226 (10(-7) M, p = 0.039; 10(-8) M, p = 0.035). This indicates that NPY increases the inhibitory effect of endogenous NE, which is mediated at low NE concentrations via alpha-adrenoceptors. Blockade of alpha-adrenoceptors attenuated electrically induced inhibition of IL-6 secretion (p < 0.001), which was dose-dependently abrogated by BIBP 3226. This indicates that under blockade of alpha-adrenoceptors endogenous NPY supports the stimulating effect of endogenous NE via beta-adrenoceptors. These experiments demonstrate the ambiguity of NPY, which functions as a cotransmitter of NE in the nerve-macrophage interplay.     

Munc13 homology domain-1 in CAPS/UNC31 mediates SNARE binding required for priming vesicle exocytosis

Neuropeptide and peptide hormone secretion from neural and endocrine cells occurs by Ca(2+)-triggered dense-core vesicle exocytosis. The membrane fusion machinery consisting of vesicle and plasma membrane SNARE proteins needs to be assembled for Ca(2+)-triggered vesicle exocytosis. The related Munc13 and CAPS/UNC31 proteins that prime vesicle exocytosis are proposed to promote SNARE complex assembly. CAPS binds SNARE proteins and stimulates SNARE complex formation on liposomes, but the relevance of SNARE binding to CAPS function in cells had not been determined. Here we identify a core SNARE-binding domain in CAPS as corresponding to Munc13 homology domain-1 (MHD1). CAPS lacking a single helix in MHD1 was unable to bind SNARE proteins or to support the Ca(2+)-triggered exocytosis of either docked or newly arrived dense-core vesicles. The results show that MHD1 is a SNARE-binding domain and that SNARE protein binding is essential for CAPS function in dense-core vesicle exocytosis.     

Neuropeptide Y and sympathetic vascular control in man

A parallel increase in systemic plasma levels of neuropeptide Y (NPY)-like immunoreactivity (LI) and noradrenaline (NA) was found during thoracotomy and surgery involving cardiopulmonary bypass in man. Thus, plasma levels of NPY-LI increased from 29 +/- 4 pmol/l before anaesthesia to 59 +/- 10 after thoracotomy and to 87 +/- 8 pmol/l upon cardiopulmonary bypass. The corresponding NA levels increased from 1.3 +/- 0.1 nmol/l before anaesthesia to 3.0 +/- 0.6 and 4.2 +/- 5 nmol/l after thoracotomy and cardiopulmonary bypass, respectively. A significant correlation was found between plasma levels of NPY-LI and NA during the operation but not between NPY-LI and adrenaline. The NPY-LI in human plasma was found to be similar to synthetic porcine NPY on reversed phase high performance liquid chromatography. Human submandibular arteries contained high levels of NPY-LI (24 +/- 3 pmol/g). In in vitro experiments on isolated human submandibular arteries, NPY in low concentrations (1000 pmol/l) was found to potentiate the contractile effects of NA or transmural nerve stimulation and to exert vasoconstrictor activity per se in higher concentrations. The calcium-entry antagonist nifedipine abolished both the NPY-induced contractions and the enhancement of NA-evoked contractions. NPY depressed the nerve stimulation-evoked 3H-NA release from human submandibular arteries via a prejunctional mechanism which was resistant to nifedipine. NPY contracted human mesenteric veins and renal arteries, but not mesenteric arteries. In conclusion, NPY seems to be co-released with NA upon sympathetic activation in man. Furthermore, NPY exerts both pre- and postjunctional effects on sympathetic control of human blood vessels.     

Galanin potentiates electrical stimulation and exogenous norepinephrine-induced contractions in the rat vas deferens

In order to evaluate the mode of action of galanin (GAL) on the neuroeffector mechanism of peripheral sympathetic nerve fibers, the effects of this peptide were tested on the electrical stimulated and the unstimulated preparations of the isolated rat vas deferens in the presence of 10(-7) M atropine. The contractile responses, which were mediated predominantly by activation of postganglionic noradrenergic nerve fibers were dose-dependently potentiated by GAL in concentrations ranging from 1 to 50 nM. The facilitatory action induced by GAL in high concentrations (greater than 10 nM) usually returned to the control level at 2-3 min and were tachyphylactic. The potentiating action of GAL was not modified by pretreatment with 10(-7) M propranolol. Contractions produced by exogenous norepinephrine (NE) in the unstimulated preparations were not affected by pretreatment with low concentrations (less than 5 nM) of GAL. On the other hand, the contractions were dose-dependently potentiated 1 min after pretreatment with higher concentrations (greater than 10 nM) of GAL, which recovered 15 min after constant flow washout. Contractions developed by exogenous 5-hydroxytryptamine were not affected, or slightly inhibited, by GAL (1-50 nM). In some preparations without electrical stimulation, high concentrations of GAL caused a slight contraction, which was not blocked by pretreatment with 10(-6) M phentolamine and 10(-6) M tetrodotoxin. These results suggest that GAL receptors exist presynaptically in the rat vas deferens and that stimulation of the receptors by GAL potentiates the release of NE from the nerve terminals during postganglionic sympathetic nerve stimulation. Other mechanisms for GAL action, such as influence on neuronal uptake and catecholamine metabolism, cannot be ruled out.     

Atrial natriuretic factor attenuates sympathetic neuroeffector responses in hindlimb vasculature of rabbits

To determine whether atrial natriuretic factor (ANF) affects vasoconstrictor responses to electrical stimulation of sympathetic nerves or intra-arterial norepinephrine (NE), changes in perfusion pressure were measured during lumbar sympathetic nerve stimulation (LSNS, 1-8 Hz), or administration of NE (50-200 ng), in an isolated constant flow-perfused hindlimb of chloralose-anesthetized rabbit before and after intra-arterial infusion of ANF (0.5 ng.mL-1.min-1). ANF significantly attenuated responses to LSNS (relative potency, RP = 0.65) and to NE (RP = 0.47). We conclude that ANF attenuates vasoconstrictor responses to both LSNS and NE. Thus ANF alters sympathetic nervous system mediated changes in vascular resistance possibly at the neuroeffector site.     

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.