Autoregulation in PC12 cells via P2Y receptors: Evidence for non-exocytotic nucleotide release from neuroendocrine cells

Nucleotides are released not only from neurons, but also from various other types of cells including fibroblasts, epithelial, endothelial and glial cells. While ATP release from non-neural cells is frequently Ca2+ independent and mostly non-vesicular, neuronal ATP release is generally believed to occur via exocytosis. To evaluate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component, the autocrine/paracrine activation of P2Y12 receptors was used as a biosensor for nucleotide release from PC12 cells. Expression of a plasmid coding for the botulinum toxin C1 light chain led to a decrease in syntaxin 1 detected in immunoblots of PC12 membranes. In parallel, spontaneous as well as depolarization-evoked release of previously incorporated [3H]noradrenaline from transfected cells was significantly reduced in comparison with the release from untransfected cells, thus indicating that exocytosis was impaired. In PC12 cells expressing the botulinum toxin C1 light chain, ADP reduced cyclic AMP synthesis to the same extent as in non-transfected cells. Likewise, the enhancement of cyclic AMP synthesis either due to the blockade of P2Y12 receptors or due to the degradation of extracellular neucleotides by apyrase was not different between non-transfected and botulinum toxin C1 light chain expressing cells. However, the inhibition of cyclic AMP synthesis caused by depolarization-evoked release of endogenous nucleotides was either abolished or greatly reduced in cells expressing the botulinum toxin C1 light chain. Together, these results show that spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis, whereas depolarization-evoked nucleotide release relies predominantly on exocytotic mechanisms.
Introduction
Adenine and uridine nucleotides are present in and released from all types of cells [2] and exert effects via P2X and/or P2Y receptors: P2X receptors are ATP-gated cation channels [25]; P2Y receptors are G protein-coupled receptors, and eight different types have been identified (P2Y1, 2, 4, 6, 11, 12, 13, 14; [1]). P2Y1, 11, 12 and 13 are activated by adenine nucleotides, whereas P2Y6 is activated by uridine nucleotides. P2Y2 and 4 are sensitive to adenine and uridine nucleotides, and P2Y14 is activated by UDP-glucose. In a large number of non-neural tissues, e.g. liver, kidney, bones and blood vessels, released nucleotides subserve autocrine/paracrine functions by activating certain P2Y receptors [32, 16, 38, 12, 30]. In a given cell, this autocrine/paracrine regulation depends on the release of nucleotides, the presence of P2Y receptors and occasionally on the enzymatic activities of nucleotidases: in hepatoma cells [16], for instance, released ATP is degraded to ADP to stimulate P2Y1 receptors; in epithelial [32] and osteoblastic [38] cells, in contrast, ATP itself activates P2Y11 and P2Y2 receptors, respectively.
Although ATP has first been described to be released from neurons [15] and to act as a neurotransmitter [6], available information about neuronal autoregulation via released nucleotides and P2Y receptors is limited. Early examples were the feedback inhibition of sympathetic transmitter release via presynaptic P2Y receptors (see [4]) and the ATP-mediated autocrine inhibition of voltage-activated Ca2+ channels in bovine chromaffin cells [9]. More recently, autoregulation via P2Y receptors has been described for the phaeochromocytoma cell line PC12 [24]. There, endogenous P2Y12 receptors were activated by spontaneous nucleotide release to achieve half-maximal inhibition of cyclic AMP (cAMP) synthesis. Depolarization of PC12 cells by 100 mM K+ further enhanced nucleotide release and thereby led to full inhibition of cAMP generation. This latter effect was prevented when voltage-activated Ca2+ channels were blocked by Cd2+ or when extracellular nucleotides were degraded by apyrase. Thus, the inhibition of cAMP synthesis in PC12 cells via P2Y receptors can be viewed as a biosensor for nucleotide release [24].
Nucleotides are released from various cells including fibroblast-like, epithelial, endothelial, glial and neuronal cells [36, 28, 21, 26, 35]. ATP release from non-neural cells can be elicited by different stimuli, such as hypotonic solutions [36], mechanical stimulation [28] or exchange of culture media [21, 26] and is frequently Ca2+ independent and therefore most probably non-vesicular. The mechanisms underlying this non-vesicular ATP release remained largely elusive, but ATP binding cassette transporters, stretch-activated cation channels as well as connexin hemichannels have been implicated [22]. In addition, evidence has been presented that indicates that nucleotide release from non-neural cells may to some extent be vesicular (e.g. [17, 8]).
In neurons and neuroendocrine cells, ATP is stored in vesicles either alone or together with classic neurotransmitters and is thus released by exocytosis [27]. Vesicle exocytosis occurs spontaneously at a slow rate and is largely accelerated when Ca2+ entry is triggered by depolarization-induced opening of voltage-activated Ca2+ channels [29]. Accordingly, depolarization-evoked ATP release from neurons and neuroendocrine cells is entirely Ca2+ dependent [35, 13], but it remained unknown whether all of the spontaneous nucleotide release from neuronal sources is also vesicular.
Vesicle exocytosis in neurons and neuroendocrine cells involves the SNARE proteins syntaxin, SNAP-25 and VAMP/synaptobrevin. These proteins are cleaved by various serotypes of clostridial neurotoxins which thereby prevent exocytosis. In particular, botulinum neurotoxin C1 cleaves syntaxin, and in some cells also SNAP-25, and thereby impedes spontaneous as well as Ca2+-evoked vesicle exocytosis [5]. Transfection of PC12 cells with a plasmid coding for the botulinum toxin C1 light chain was reported to largely reduce spontaneous as well as depolarization-evoked release of co-transfected human growth hormone [10]. Here, we used an analogous method to investigate whether in PC12 cells the activation of P2Y12 receptors by endogenously released nucleotides relies on vesicle exocytosis.
Materials and methods
Materials
[2,8−3H]adenine (specific activity 32 Ci mmol−1) and levo-[ring-2,5,6-3H]noradrenaline (specific activity 37 Ci mmol−1) were obtained from NEN (Vienna, Austria). Na-ADP, 4-(3-butoxy-4-methoxybenzyl)imidazoline-2-one (RO 20-1724), 3′,5′-cyclic AMP, apyrase (grade VII, with an approximately 1:1 ratio in ATPase and ADPase activity), 2-methylthio-AMP, and 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido-adenosine (CGS 21680) were purchased from Sigma (Vienna, Austria).
Cell culture and transfection
PC12 cells were maintained and subcultured as previously described in detail [24]. The cells were plated onto collagen-coated (Biomedical Technologies Inc., Stoughton, MA, USA) culture dishes (NUNC, Roskilde, Denmark) and were kept in OptiMEM (InVitrogen, Vienna, Austria) supplemented with 0.2 mM L-glutamine (HyClone, Aalst, Belgium), 25,000 IU l−1 penicillin and 25 mg l−1 streptomycin (Sigma, Vienna, Austria), 5% fetal calf serum and 10% horse serum (both InVitrogen, Vienna, Austria). Once per week, cell cultures were split; the medium was exchanged twice per week. To investigate the outflow of previously incorporated [3H]noradrenaline under continuous superfusion, PC12 cells were plated onto 5-mm discs, for assays of cAMP accumulation they were plated onto 6-well culture dishes and for membrane preparations for Western blots they were plated onto 100-mm culture dishes. All this tissue culture plastic was coated with collagen (as above).
For the generation of PC12 cell clones stably expressing the botulinum toxin C1 light chain (BoNT/C1), 15 μg of a pcDNA3 vector harbouring the botulinum toxin C1 light chain (pBoNT/C1; kindly provided by R.D. Burgoyne, Liverpool, UK) [10] were mixed with 50 μl of the TransFast transfection reagent (Promega, Mannheim, Germany) and added to semiconfluent PC12 cell cultures in serum-free medium. After a 1 h incubation at 37°C, two additional volumes of serum-free medium and the appropriate amount of serum (as above) were added. This medium was exchanged 48 h after transfection for a medium supplemented with 500 μg/ml neomycin (G418) to allow for selection of drug resistance. This selection medium was replaced every 3–4 days until distinct islands of surviving cells were visible. Individual clones of antibiotic-resistant cells were transferred to 24-well plates and grown in medium containing 200 μg/ml G418.
Immunoblotting of SNARE proteins
Preparation of protein extracts of PC12 membranes was performed as described previously [18]. Materials and methodology used for production and analysis of ECL-immunoblots were also essentially as described therein, with the following modifications: protein extracts in SDS sample buffer were incubated at 95°C for 5 min and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on 5% (stacking) and 10% (separating) gels. After electrophoretic transfer of proteins to nitrocellulose, membranes were cut horizontally along appropriate molecular weight markers in order to separate regions harbouring proteins with apparent molecular weights of 10–20 kD, 21–29 kD and 30–50 kD. These membrane strips were incubated in one of the following monoclonal SNARE antibodies: syntaxin 1 antibody clone 78.3, synaptobrevin 2 antibody clone 69.1 (both obtained from Synaptic Systems, Göttingen, Germany) and SNAP-25 antibody MAB331 (purchased from Chemicon, Temecula, CA, USA).
Measurement of [3H]noradrenaline release
[3H]noradrenaline uptake in and superfusion of PC12 cell cultures were performed as described previously [23]. Culture discs with PC12 cells were incubated in 0.1 μM [3H]noradrenaline in culture medium supplemented with 1 mM ascorbic acid at 36°C for 1 h. After labelling, culture discs were transferred to small chambers and superfused with a buffer containing (mM) NaCl (120), KCl (3.0), CaCl2 (2.0), MgCl2 (2.0), glucose (20), hydroxyethylpiperazine ethanesulphonic acid (HEPES) (10), fumaric acid (0.5), Na-pyruvate (5.0), ascorbic acid (0.57) and desipramine (0.001), adjusted to pH 7.4 with NaOH. Superfusion was performed at 25°C at a rate of about 1.0 ml/min. Collection of 4-min superfusate fractions was started after a 60-min washout period to remove excess radioactivity.
Depolarization-dependent overflow of previously incorporated [3H]noradrenaline was triggered by inclusion of 30 or 100 mM KCl in the superfusion buffer during two 30-s periods 20 min apart (Fig. 2a). At the end of the experiments, the radioactivity remaining in the cells was extracted by immersion of the discs in 2% (v/v) perchloric acid followed by sonication. Radioactivity in extracts and collected fractions was determined by liquid scintillation counting (Packard Tri-Carb 2100 TR).
The spontaneous (unstimulated) rate of [3H] outflow was obtained by expressing the radioactivity of a collected fraction as percentage of the total radioactivity in the cultures at the beginning of the corresponding collection period. Values obtained during the 4-min period directly preceding the application of 30 mM K+ were used for the comparison of spontaneous (basal) tritium outflow in wild-type and pBoNT/C1 expressing PC12 cells. K+-evoked tritium overflow was calculated as the difference between the total [3H] outflow during and after stimulation and the estimated basal outflow which was assumed to decline linearly throughout the experiments. Therefore, basal outflow during periods of stimulation was assumed to equate the arithmetic mean of the samples preceding and those following stimulation, respectively. The difference between the total and the estimated basal outflow was expressed as a percentage of the total radioactivity in the cultures at the beginning of the respective stimulation (% of total activity).
Determination of cyclic AMP
The accumulation of cAMP in PC12 cell cultures was determined as described before [33]. After labelling of cellular purines with tritiated adenine (2.5 μCi/ml for 12 h), the medium was replaced by a buffer (120 mM NaCl, 3 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 10 mM HEPES, 10 mM LiCl, adjusted to pH 7.4 with NaOH) supplemented with 100 μM of the phosphodiesterase inhibitor Ro-20-1724 [4-(3-butoxy-4-methoxybenzyl) imidazolidin-2-one] and 1 U/ml adenosine deaminase. Dishes were then kept at room temperature for 105 min. During the last 15 min of this incubation period, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido-adenosine (CGS 21680), ADP (10 μM), apyrase (1 U/ml) or 100 mM KCl (NaCl was reduced accordingly) were also included in the medium. When appropriate, the P2Y12 receptor antagonist 2MesAMP (100 μM; [14]) was present for the last 25 min. The incubation was terminated by exchanging the buffer for 1 ml of 2.5% perchloric acid containing 100 μM non-labelled cAMP followed by a 20-min incubation at 4°C. Subsequently, cAMP was separated from the other purines by a chromatographic procedure described before [33]. One tenth of each sample obtained as described above was used for the determination of the total radioactivity. The remaining 900 μl were neutralised by addition of 100 μl 4.2 M KOH and applied to Dowex 50 columns (AG 50W-X4; Bio-Rad, Vienna, Austria), which were then rinsed with 3 ml H2O. The eluate obtained by the subsequent application of 8 ml H2O was directly poured onto alumina columns (Bio-Rad, Vienna, Austria), which were then washed with 6 ml H2O. Finally, cAMP was eluted with 4 ml imidazole buffer (20 mM imidazole in 0.2 M NaCl; pH 7.45). Radioactivity within the samples obtained was determined by liquid scintillation counting. The radioactivity in the fraction of cAMP was expressed as percentage of the total radioactivity incorporated in the cells.
Stimulation of PC12 cells, whether they were clonal cell lines or wild-type cells, with the adenosine A2A receptor agonist CGS 21680 caused reproducible increases in the values of cAMP. However, the extent of basal and stimulated cAMP synthesis may vary between different preparations [33]. Hence, to be able to directly compare changes in CGS 21680-induced cAMP synthesis in different cell clones, values obtained in the presence of ADP, apyrase, 2MesAMP or KCl were expressed as percentage of the values obtained in the very same preparation, but in the absence of these agents (% of control).
Statistics
All data represent arithmetic means±SEM; n represents numbers of culture dishes. Statistical significance of differences between single data points was evaluated by the non-parametric Mann-Whitney test, and p values < 0.05 were accepted as indicators of significance.
Results
To assess the role of vesicle exocytosis in the release of endogenous nucleotides from neuroendocrine cells, this study employed the autocrine/paracrine control of cAMP synthesis in PC12 cells via P2Y12 receptors. To prevent vesicle exocytosis, we generated cell clones stably expressing pBoNT/C1. Depending on the cell types being investigated, this toxin cleaves one or more SNARE proteins and thereby prevents vesicular transmitter release or hormone secretion [5].
Cleavage of SNARE proteins by the expression of the BoNT/C1 light chain
The major target of BoNT/C1 is syntaxin 1, which is cleaved by this toxin in all types of preparations [5]. Therefore, several pBoNT/C1 expressing clones were initially tested in immunoblots of harvested membranes for a lack of full-length syntaxin. Two of these pBoNT/C1 expressing clones (BT2 and BT7) were selected for further analysis.
In bovine chromaffin cells, BoNT/C1 was reported to cleave not only syntaxin 1, but also SNAP-25, even though the toxin failed to cleave purified and recombinant SNAP-25 [11]. To evaluate the efficiency of SNARE protein cleavage by the BoNT/C1 light chain expressed in PC12 cells, the immunostaining of syntaxin and SNAP-25 in Western blots was compared with that of synaptobrevin, which is undoubtedly insensitive to BoNT/C1 [11, 5]. As shown in Fig. 1, the stable expression of pBoNT/C1 markedly reduced or even abolished the staining of a protein with an apparent molecular weight of 37 kD by an anti-syntaxin antibody. In contrast, there were no clear-cut reductions in the staining of a 25-kD band by an anti-SNAP-25 antibody, and occasionally the immunostaining of this protein appeared to be even enhanced in pBoNT/C1 expressing clones (Fig. 1a). To quantify these changes in SNARE protein expression, the immunoblots were quantified by densitometry and the values obtained for the anti-syntaxin and anti-SNAP-25 immunoreactive bands were set in relation to those of the anti-synaptobrevin immunoreactive bands (Fig. 1b). This procedure revealed a significant reduction of syntaxin in the pBoNT/C1 expressing clones when compared with wild-type PC12 cells. SNAP-25 immunoreactivity, in contrast, was not significantly altered in these cell clones.
Fig. 1Identification of SNARE proteins in membrane extracts from either wild-type (WT, untransfected) PC12 cells or from two PC12 cell clones stably expressing pBoNT/C1 (BT2 and BT7). SDS-membrane extracts were separated on SDS-PAGE and transferred to nitrocellulose membranes. The membranes were cut horizontally to separate regions harbouring proteins with apparent molecular weights of 1–20 kD, 21–29 kD and 30–50 kD and immunostained with anti-syntaxin 1 antibody clone 78.3 (Stx), anti-SNAP-25 antibody MAB331 (S25) and anti-synaptobrevin 2 antibody clone 69.1 (Sb), respectively. a Representative blot obtained as described above. b The relation between the anti-syntaxin and the anti-synaptobrevin immunoreactivities (Stx/Sb) as determined by densitometric analysis (n = 3). c The relation between the anti-SNAP-25 and the anti-synaptobrevin immunoreactivities (Stx/Sb) as determined by densitometric analysis (n = 3). *Significant differences versus the results obtained with membranes from wild-type cells at p < 0.05
Inhibition of transmitter release by the expression of the BoNT/C1 light chain
Previously, it has been shown that the transient expression of pBoNT/C1 reduced stimulated vesicular catecholamine release from PC12 cells [10]. To evaluate whether catecholamine release was reduced in the pBoNT/C1 expressing clones, clonal and wild-type PC12 cells were labelled with [3H]noradrenaline and the basal and depolarization-dependent outflow of radioactivity was determined. After washout of excess radioactivity during a 1-h washout period, all PC12 cell cultures steadily released small amounts of radioactivity in the superfusion buffer (Fig. 2a). However, this spontaneous outflow of radioactivity was significantly less in the two pBoNT/C1 expressing clones than in untransfected PC12 cells (Fig. 2b).
Fig. 2Basal and depolarization-evoked [3H]noradrenaline release from either wild-type (WT, untransfected) PC12 cells or from two PC12 cell clones stably expressing pBoNT/C1 (BT2 and BT7). PC12 cell cultures were labelled with [3H]noradrenaline and superfused. Subsequent to a 60-min washout period, 4-min fractions of superfusate were collected. Tritium overflow was stimulated twice (S1 after 72 min and S2 after 92 min of superfusion) by the presence of KCl (30 or 100 mM, each for 30 s). a Comparison of the time courses of fractional [3H] outflow from the three different kinds of cell cultures (n = 5 to 6). b The amount of basal [3H] outflow from the three PC12 cell cultures (n = 8 to 9). c The amount of [3H] overflow from the three PC12 cell cultures triggered by 30 mM KCl (n = 8 to 9). d The amount of [3H] overflow from the three PC12 cell cultures triggered by 100 mM KCl (n = 8 to 9). *, **, ***Significant differences versus the results obtained with wild-type cells at p < 0.05, p < 0.01 and p < 0.001, respectively
In wild-type cells, the presence of 30 mM K+ (Na+ was reduced accordingly) for 30 s caused a reproducible increase in the outflow of radioactivity (Fig. 2a), but such an effect was hardly observed in the pBoNT/C1 expressing clones. Accordingly, the overflow of radioactivity triggered by 30 mM K+ was significantly reduced in the pBoNT/C1 expressing clones in comparison with wild-type cells (Fig. 2c). This confirms that the expression of the BoNT/C1 light chain reduced spontaneous noradrenaline release and almost abolished release due to mild depolarizations.
Large intracellular Ca2+ increases have been reported to rescue transmitter release from cells poisoned with BoNT/C1 [7]. Therefore, PC12 cultures were also exposed to 100 mM K+ (Na+ was again reduced) for 30 s. Although this stimulation paradigm caused unequivocal increases in the outflow of radioactivity even from pBoNT/C1 expressing cells (Fig. 2a), the amount of tritium overflow was still reduced when compared with the values obtained in untransfected PC12 cells (Fig. 2d). Thus, the expression of the BoNT/C1 light chain efficiently reduced spontaneous as well as depolarization-evoked vesicular transmitter release.
Changes in the regulation of cAMP synthesis via P2Y receptors due to the expression of the BoNT/C1 light chain
In PC12 cells, activation of endogenous P2Y12 receptors by the exposure to nucleotides causes an inhibition of adenylyl cyclase [33]. Furthermore, spontaneously released nucleotides are also sufficient to partially activate these receptors, and depolarization of the cells fully activates the receptors through enhanced nucleotide release to maximally suppress cAMP synthesis [24]. In accordance with these previous data, exposure of wild-type PC12 cells to 10 μM ADP significantly reduced the amount of cAMP generated due to the activation of A2A adenosine receptor by 1 μM CGS 21680 present for 15 min. In the two pBoNT/C1 expressing clones, the inhibition of cAMP accumulation was as pronounced as in the wild-type cells (Fig. 3a). Thus, the adenylyl cyclase inhibiting P2Y receptors were operating equally well in transfected as well as untransfected PC12 cells.
Fig. 3Changes in cAMP synthesis caused by exogenous ADP or endogenously released nucleotides in either wild-type (WT, untransfected) PC12 cells or in two PC12 cell clones stably expressing pBoNT/C1 (BT2 and BT7). After loading with [3H]adenine, PC12 cells were incubated in RO 20–1724 (100 μM) for 105 min. During the last 15 min of this incubation period, 1 μM CGS 21680 was present either alone or together with 10 μM ADP (a), 1 U/ml apyrase (b), 100 μM MeSAMP (c) or 100 mM KCl (d). The amount of radioactivity retrieved within the fraction of cyclic AMP (cAMP) was calculated as percentage of the total radioactivity extracted from the cell cultures. In order to compare the results of different cell cultures, values obtained in the presence of ADP, apyrase, 2MesAMP or KCl were expressed as percentage of the values obtained in the very same preparation, but in the absence of these agents (% of control). a Results obtained in the presence of 10 μM ADP are expressed as percentage of the results obtained in its absence (% of control; n = 6 to 10). b Results obtained in the presence of 1 U/ml apyrase are expressed as percentage of the results obtained in its absence (% of control; n = 9 to 10). c Results obtained in the presence of 100 μM MeSAMP are expressed as percentage of the results obtained in its absence (n = 6 to 9). d Results obtained in the presence of 100 mM KCl are expressed as percentage of the results obtained in its absence (n = 9; ***p < 0.001 versus the results obtained in wild-type cells; #, ###p < 0.05 and p < 0.001 versus the results obtained in the absence of KCl)
In the presence of apyrase (1 U/ml) to degrade extracellular nucleotides, the cAMP accumulation triggered by CGS 21680 was almost doubled. Again, this effect was not different between wild-type and pBoNT/C1 expressing cells (Fig. 3b). In accordance with these latter results, the blockade of the receptors by the P2Y12 receptor antagonist 2-MeSAMP (100 μM; [14]) also enhanced the stimulation of cAMP about twofold and there were no significant differences between the three types of PC12 cultures (Fig. 3b). Thus, the activation of the P2Y receptors negatively linked to adenylyl cyclase by spontaneously released nucleotides was the same whether pBoNT/C1 was expressed or not.
When wild-type PC12 cells were depolarized by 100 mM K+, the CGS 21680-induced cAMP accumulation was reduced to about the same extent as by 10 μM ADP. However, in one (BT2) of the two pBoNT/C1 expressing clones, the depolarization of PC12 cells failed to cause any alteration in the CGS 21680-induced cAMP accumulation; and in the other clone (BT7), this inhibition was significantly weaker when compared with the values obtained in wild-type PC12 cells (Fig. 3d). Thus, depolarization-dependent inhibition of the cAMP synthesis via P2Y12 receptors was greatly impaired in the pBoNT/C1 expressing cells.
Discussion
In non-neural cells, release of nucleotides is mostly non-vesicular and involves, for instance, ATP binding cassette transporters, stretch-activated cation channels or connexin hemichannels [22]. In neurons, however, nucleotide release is generally believed to rely on vesicle exocytosis [27]. Here, we employed the autocrine/paracrine activation of P2Y12 receptors in PC12 cells to investigate whether nucleotide release from neuroendocrine cells might involve a non-vesicular component.
Vesicle exocytosis involves the SNARE proteins syntaxin, SNAP-25 and synaptobrevin, and cleavage of one or more of these proteins by clostridial neurotoxins reduces or even abolishes exocytotic transmitter release [5]. The primary target of BoNT/C1 is syntaxin, and its inhibitory effect on transmitter release has been correlated with the cleavage of this protein [3]. However, in cultures of bovine chromaffin cells [11], spinal cord [37] or hippocampal neurons [7], BoNT/C1 was reported to cleave not only syntaxin, but also SNAP-25. In contrast, in sensory neurons the toxin was reported to act exclusively on syntaxin [31]. Furthermore, in cell-free assays BoNT/C1 was found to leave either native SNAP-25 after subcellular fractionation or recombinant SNAP-25 unaltered [11, 37]. Thus, the effects of BoNT/C1 on SNARE proteins appear to vary between different preparations. In this study, expression of the BoNT/C1 light chain reduced syntaxin, but not SNAP-25 immunoreactivity detected in PC12 cells. Likewise, in the insulin secreting β-cell line HIT-T15 expression of the BoNT/C1 light chain only affected syntaxin, even though application of recombinant toxin to permeabilized cells did lead to a reduction in SNAP-25 [20]. Hence, heterologous expression of the toxin’s light chain in neuroendocrine cells does not entirely reproduce the effects of toxin application to these cells. Syntaxin was reported to be more sensitive to BoNT/C1 light chain than SNAP-25 [20] and a 1000-fold higher light chain concentration of BoNT/C was required for SNAP-25 cleavage when compared with the other SNAP-25-targeting serotypes BoNT/A or BoNT/E [34]. Thus, the resistance of SNAP-25 to heterologously expressed BoNT/C1 light chain is likely due to the relatively low toxin affinity and insufficient expression levels of the peptide.
Nevertheless, in functional experiments, expression of the BoNT/C1 light chain significantly reduced spontaneous as well as depolarization-evoked release of [3H]noradrenaline. On the one hand, this confirms that the cleavage of syntaxin alone is sufficient to reduce transmitter release [3]. On the other hand, this result corroborates that catecholamines are released from PC12 cells through vesicular mechanisms and these are largely impaired by the expression of the BoNT/C1 light chain [10].
Considering that the heterologous expression of the BoNT/C1 light chain did efficiently reduce vesicle exocytosis, the question arises as to how this manipulation altered the release of endogenous nucleotides. In PC12 cells, endogenously released nucleotides activate P2Y12 receptors, which mediate a decrease in the synthesis of cAMP triggered by the activation of A2A adenosine receptors [24]. In accordance with these previous results, exposure of non-transfected PC12 cells to the P2Y12 receptor antagonist 2-MesAMP enhanced the cAMP synthesis. An equivalent effect was achieved when the cells were incubated in the nucleotide degrading enzyme apyrase, thus indicating that spontaneously released nucleotides continuously activated the P2Y12 receptors.
To be able to evaluate whether the generation of PC12 clones stably expressing the BoNT/C1 light chain might interfere with the above autocrine/paracrine signalling loop only by affecting exocytosis, we had to establish that the function of the P2Y12 receptor was not compromised by the toxin’s light chain or by the clonal selection procedure. Accordingly, 10 μM ADP, a saturating concentration of the nucleotide in the inhibition of cAMP synthesis in PC12 cells [33], were applied to non-transfected cells and to the two chosen cell clones (BT2 and BT7): the inhibition of cAMP accumulation was not different in the three cultures. Thus, the control of cAMP synthesis via the P2Y12 receptor was not altered by pBoNT/C1 expression or by clonal selection. Previously, we had found that the control of voltage-gated Ca2+ channels via this receptor subtype was the same in a number of PC12 cell clones expressing various types of membrane receptors [19].
Knowing that the P2Y12 receptors operated independently of the BoNT/C1 light chain, the autocrine/paracrine activation of the receptors was assessed. In the toxin expressing clones, the degradation of extracellular nucleotides by apyrase and the blockade of P2Y12 receptors by 2-MesAMP had identical effects on cAMP accumulation as in non-transfected cells. This demonstrates that the spontaneous release of nucleotides was not significantly affected by the BoNT/C1 light chain.
Enhancement of nucleotide release by depolarization of PC12 cells fully activates P2Y12 receptors and thereby reduces cAMP synthesis to about the same extent as 10 μM ADP. This phenomenon is based on Ca2+-dependent vesicle exocytosis as it is suppressed when Ca2+ influx is prevented by the blockade of voltage-gated Ca2+ channels or when extracellular nucleotides are degraded by apyrase [24]. In support of this conclusion, the inhibition of cAMP synthesis through a depolarization of PC12 cells by 100 mM K+ was either significantly attenuated or entirely abolished in the pBoNT/C1 expressing clones. Hence, the K+-evoked and thus Ca2+-dependent release of nucleotides was largely reduced by the BoNT/C1 light chain.
Taken together, the present results show that the expression of the BoNT/C1 light chain impairs vesicular transmitter release from PC12 cells, but does not alter the autocrine/paracrine activation of P2Y receptors by spontaneously released nucleotides. Thus, the spontaneous nucleotide release from neuroendocrine cells may occur independently of vesicle exocytosis. The depolarization-evoked nucleotide release, in contrast, relies predominantly on exocytotic mechanisms.

pc12 cells
p2y receptor
nucleotide release
cyclic amp
vesicle exocytosis


Dilatation tracheoscopy for laryngeal and tracheal stenosis in patients with Wegener’s granulomatosis

Wegener’s granulomatosis (WG) frequently involves the subglottis and trachea and may compromise the upper airway. The objective of this study is to evaluate retrospectively the effect of treatment of subglottic stenosis (SGS) and tracheal stenosis (TS) by dilatation tracheoscopy (DT) in patients with WG. We performed a cohort study on all patients who underwent DT between February 2001 and September 2005 in our institution. From this cohort we identified a total of nine WG patients. In all patients, clinical, serological and histopathological data had been prospectively collected by a standardized protocol from the time point of diagnosis. In the nine patients that were identified with SGS or TS due to WG (eight women and one man), a total of 22 DT’s were performed. Two patients needed a tracheostoma (one temporarily). The mean follow-up after the first DT was 25.4 ± 14.1 months. Two patients did not experience a recurrence of SGS or TS. Six patients required a second DT without recurrence of local disease. The remaining patient underwent 8 DT's in a 4-year period. DT can offer a simple and repeatable solution to SGS and TS due to WG. Seven of the nine patients required more than one dilatation and some patients experience a functional restriction. One patient has a definitive tracheostoma.
Introduction
Wegener’s granulomatosis (WG), first described by Friedrich Wegener in Stuttgart in 1936, is multi-system disease characterized by a necrotizing granulomatous arteritis of the upper and lower respiratory tract and a necrotizing crescentic glomerulonephritis [1]. Vasculitis may also affect other organs such as the eyes, skin, joints, heart and the nervous system [1]. Upper respiratory tract manifestations—particularly sinusitis, ulcerations of the nasal mucosa and epistaxis—are common and debilitating presentations. Subglottic stenosis (SGS) and tracheal stenosis (TS), however, are potentially life threatening presentations of WG. SGS has been found to occur in approximately 16–23% in patients carrying a diagnosis of WG [2, 3]. This narrowing of the upper airway at the level of cricoid cartilage and/or upper tracheal rings presents a management dilemma. Dilatation tracheoscopy (DT) is one of the possible options for treatment of SGS and TS. The long-term effects of DT in patients with WG have never been reported.
DT is a minimally invasive self-standing procedure. It can be used for elective and emergency intervention in patients with subglottic and tracheal stenosis. This has been described elsewhere (G.B. Halmos, F.G. Dikkers, Dilatation tracheoscopy in treatment of subglottic and tracheal stenosis, submitted for publication). Patients suffering from WG present differences (age, sex, response to treatment) compared to other aetiologies, which justifies separate publication. The objective of this study is therefore to evaluate the effect of the treatment of SGS and TS by DT in patients with WG.
Methods
We retrospectively identified all patients from our institution that underwent a DT between February 2001 and September 2005. The total cohort of benign, grade II (Myer-Cotton) subglottic or tracheal stenosis consisted of patients with a history of posttraumatic tracheal stenosis, thermal tracheal stenosis, posttracheotomy cicatricial stenosis, tracheal rupture, postintubation stenosis, and WG, amongst others. As stated before, the cohort is described elsewhere.
From the cohort we identified a total of nine WG patients. These nine patients had been diagnosed with WG between May 1990 and August 2003. During this period a total of 195 patients in our institution were diagnosed as having WG (including the nine patients who underwent DT). From this cohort, one additional patient underwent a DT for SGS in 1992. Three other patients were diagnosed with relatively mild SGS or TS that did not necessitate DT. The reference group for the comparison of demographic and serological data consisted of the total cohort, minus the patients who underwent DT and minus the three patients with mild tracheal stenosis who did not undergo DT. Differences in age between the DT group and the reference group were tested with the Mann Whitney U test; differences in the male/female ratio and antineutrophil cytoplasmatic autoantibodies (ANCA) specificity were tested with chi-square test. Although this was a retrospective study, the clinical, serological and histopathological data of both the DT group and the control group had been prospectively collected by a standardised protocol starting at the time of the diagnosis of Wegener’s granulomatosis. Extrarenal organ involvement was categorized as described previously [4]. ANCA was assayed with indirect immunofluorescence. Sera from all patients were assayed for the presence of antibodies against proteinase 3 (PR3–ANCA), myeloperoxidase (MPO–ANCA) and elastase as described previously [5].
DT was performed when patients complained of progressive dyspnoea in combination with decrease of peak flows. DT is an endoscopically performed intervention. An intubation laryngoscope, a Groningen optical dilatation tracheoscope (Karl Storz 1033R) (Fig. 1), telescope and suction tubes are required for this procedure. The tracheoscope has a length of 30 cm. The proximal end of the tracheoscope is designed in such a way that customary ventilation tubes and a 30 cm Hopkins® straight forward telescope (Karl Storz 27005AA) can be connected. The distal end of the instrument contains numerous lateral tiny openings, which enable air to come through in the centre of the stenosis. The dilatation tracheoscope is available with a diameter of 8 and 12 mm. The appropriate size of the instrument is determined by the size of the patient’s larynx and the healthy portion of the trachea.
Fig. 1Groningen dilatation tracheoscope
The intervention is carried out under general anaesthesia. Following the administration of the anaesthesia, with ventilation taking place via an anaesthesia mask, the dilatation tracheoscope is introduced under endoscopic control. The stenosis is then visible through the vocal cords (Fig. 2). The bevelled design of the tip, which can be advanced forward through the stenosis, ensures that the ventilation is maintained during the process. The conical construction of the tip enables the instrument to be advanced up to the wider section of the tracheoscope (Fig. 3), after which the tracheoscope remains in place for 5 to 10 min.
Fig. 2Patient nr 9 pre dilatation. During laryngotracheoscopy a subglottic stenosis is clearly visible. The vocal cords can be seen bilaterallyFig. 3Patient nr 9 during DT. The dilatation tracheoscope is introduced through the stenosis. Parts of the tissue protrude through the tiny distal openings of the tracheoscope
The constellation of the tracheoscope is suitable for most grade II (Myer-Cotton) subglottic or tracheal stenosis. The intervention can be repeated after any time interval.
Typically, no antibiotics or corticosteroids are used. The use of mitomycin-C was considered in each case, but it was never used, because mitomycin-C should be used in fresh wounds. Patients are dismissed the day after DT. Peakflows were measured three times (Respironics Healthscan Inc., Cedar Groove, NJ, USA) in an upright position. The mean of the three measurements was taken.
Results
Patients
The total cohort of patients who underwent DT because of SGS and TS was 25. Nine of them were identified as patients with WG. Eight were female, one was male. The other 16 patients did not have vasculitis as cause of their stenosis. The causes in these 16 patients were posttraumatic [5], postintubation [4], idiopathic [4], post tracheotomy [2] and post thermal [1] injury. The mean age of the patient group at first presentation of WG was 41.6 ± 11.2 years (Table 1). The interval between the presentation of WG and the first symptoms of laryngeal and tracheal stenosis varied between 0 and 122 months (mean 48.9 ± 44.1). The average age at of the time of presentation of the stenosis was 45.6 ± 11.9 years (Table 1).
Table 1Patient characteristics of nine patients with Wegener’s Granulomatosis (WG) and subglottic stenosis (SGS) or tracheal stenosis (TS). Clinical and laboratory findings. ANCA antineutrophil cytoplasmatic autoantibodies, PR3 proteinase 3, MPO myeloperoxidasePatient No.SexAge at presentation of WGOrgans involved at first presentation of WGANCA specificityNumber of relapses of WGOrgans involved at first relapse of WGFollow-up after diagnosis of WG (years)Age at first presentation of SGS or TSInterval between diagnosis of WG and first treatment of TS or SGS (months)1M44JointsPR312Joints, skin1751922F26Lungs, TracheaPR31Lungs, kidney142603F43TracheaMPO1Lungs124304F57JointsPR32Ear, eye, kidney, joints12671225F26TracheaPR31Kidney, eye1131606F51MastoidPR32Nose (concha inferior), mastoid1058817F42Ear drumAtypical0Lung, ear drum845358F52NosePR31Nose (septum and concha), joints, eye756489F33Nasal vesselsPR30Vessels4332
At diagnosis, all WG patients had additional organ involvement outside the otorhinolaryngologic area. The kidneys, lungs and eyes were affected three times. The joints were affected two times. The skin was affected once (Table 1).
As shown in Table 1, the number of relapses of WG varied between 0 and 12. There was no relation between the number of relapses and the occurrence of TS or SGS. In only one of the DT procedures there was endoscopic and/or histopathological evidence of vasculitis activity. The TS or SGS occurred in 95% in periods where the disease appeared to be less active.
In Table 2 the patient characteristics of the 9 patients who underwent DT for SGS or TS are compared with those patients who were diagnosed with WG in the same time frame but who did not have evidence of SGS or TS. In the DT group, 89% were female whereas 43% of patients in the reference group were female (P < 0.01). In the DT group the age at diagnosis of WG was significantly lower in comparison with the reference group (P < 0.05).
Table 2Patient characteristics of nine patients with Wegener’s granulomatosis who underwent DT for SGS or TS compared with the reference group of 182 patients who were diagnosed with WG in the same period without evidence of SGS or TSWG patients who underwent DT (n = 9)WG patients without evidence of SGS or TS (n = 182)P valueMale/ female number (%)1/8 (11%/89%)104/78 (57%/43%) <0.01Age at diagnosis of WG (years, mean ± SD)41.6 ± 11.253.3 ± 17.0<0.05ANCA specificityPR3-ANCA: n = 8 (89%)MPO-ANCA: n = 1 (11%)PR3-ANCA: n = 160 (88%)MPO-ANCA: n = 13 (7.1%)HNE-ANCA: n = 1 (0.5%)ANCA-negative: n = 8 (4.4%)Not significantWG Wegener's granulomatosis, DT dilatation tracheoscopy, SGS subglottic stenosis, TS tracheal stenosis, ANCA antineutrophil cytoplasmatic autoantibodies, PR3 proteinase 3, MPO myeloperoxidase, HNE human neutrophil elastase
Follow-up of the DT-group
In the nine patients identified with SGS or TS due to WG, a total of 22 DT’s were performed (Table 3). The mean follow-up after treatment was 25.4 ± 14.1 months (Table 3). Two patients (numbers 7 and 8) did not experience a recurrence of significant stenosis. Six patients (numbers 1, 2, 3, 4, 6, 9) required a second DT without recurrence of local disease activity after the second DT. One of these patients (patient number 6) had acute WG in the trachea, proven by biopsy (Fig. 4). She was treated with high dose prednisolone (intravenous methylprednisolone 1000 mg on three consecutive days) and intubation for a week. She could be detubated a week later (Fig. 5). Two patients needed a tracheostoma (patient numbers 2 and 5). One patient (number 5) developed a cricoid stenosis 5 years after the diagnosis of WG. Initially the stenosis was treated with two DT’s. During a pregnancy the patient required an emergency tracheotomy because of a threatened airway. After pregnancy she was treated with a CO2 laser and three DT’s. Unfortunately, however, she required a definitive tracheostomy. Currently the process of decannulation is taking place. One patient (number 2) became respiratory insufficient at diagnosis of WG and had a tracheotomy for 3 months. She subsequently developed a cicatricial SGS, but she has enough lumen to live without a tracheostomy (Table 3).
Table 3Follow-up after last DTPatient numberLocation of stenosisNumber of DT’sFollow-up after last treatment (months)Tracheotomy required1Subglottic219No2Subglottic221Yes, temporary3Trachea243No4Trachea214No5Cricoid812Yes6Subglottic216No7Subglottic154No8Subglottic126No9Subglottic224NoFig. 4Patient nr 6 with acute WG during biopsy of a subglottic stenosisFig. 5Patient nr 6 after pulse therapy and intubation. The picture was taken one week after Fig. 4
The effect of DT on peakflows was evaluated in five patients. In these patients the mean (±SD) peakflow increased from 164 ± 45.7 l/min before DT to 226 ± 69.6 l/min after dilatation. An example of the effect of DT on the peakflow is shown in Fig. 6. Two months after this patient had been diagnosed with WG she developed complaints of dyspnoea. Tracheoscopy revealed a SGS with signs of active vasculitis. After treatment with high-dose corticosteroids she had a regression of the WG activity. Fourteen and 28 months after first diagnosis of SGS a dilatation tracheoscopy was performed successfully.
Fig. 6Peak flow measurements after dilatation of patient number 9. The arrows indicate the DT procedures
Discussion
There are multiple causes of benign laryngeal or tracheal stenosis, the most common being traumatic. However, if there is no prior history of tracheal trauma, the aetiology of the stenosis may be obscure and difficult to determine, necessitating a systematic approach to make the diagnosis. Excluding trauma, the differential diagnosis of TS can be subdivided into four categories: congenital, neoplastic, infectious, and inflammatory.
Congenital TS is really quite rare and is often the result of posterior fusion of the tracheal rings, thereby forming complete rings. Other causes of congenital stenosis include vascular rings and other congenital cardiovascular anomalies such as an anomalous subclavian artery. These patients typically present at young age.
Primary benign tumours of the trachea such as chondromas, fibromas, squamous papillomas, hemangiomas, and granular cell tumours are also unusual causes of stenosis. In addition, extrinsic compression of the trachea can occur by thyroid neoplasms and goiters.
A number of infections of the bronchopulmonary tree can lead to TS. Fungal infections such as histoplasmosis and blastomycosis should always be considered when the aetiology of the stenosis is unclear. Serologic testing and histopathologic examination can be helpful in this regard. Other infectious causes of TS include rhinoscleroma, tuberculosis, syphilis, and diphtheria.
Non-infectious, inflammatory causes of TS include sclerosing mediastinitis, primary amyloidosis, and sarcoidosis. WG and relapsing polychondritis can also cause TS, but they are almost always seen in combination with other, more classic hallmarks of these diseases.
A laryngeal or tracheal stenosis is optimally diagnosed via tracheal visualization, which is generally performed by an otorhinolaryngologist. Indirect and fiberoptic laryngoscopy are non-invasive examination techniques that can be performed in the office, but usually do not show the entire trachea. A subglottic stenosis is not always visible, therefore direct rigid tracheoscopy in general anaesthesia is indicated in cases suspect of WG.
Recurrent SGS is a well recognized but uncommon feature of WG. Patients with WG who develop laryngotracheal disease have usually already been diagnosed as such because of the presence of disease in other organs at the same time or at a previous occasion [6]. Isolated involvement of the subglottic larynx is conspicuously rare—only a few cases have been reported in the literature [2, 7, 8]. In our series, however, three patients had SGS or TS as presenting symptom of their WG. We do not have an explanation for this rare presentation.
Another particular finding is that eight of our nine patients were female. This contrasts significantly with the male/female ratio in the reference group that consisted of WG patients that were diagnosed in the same period as the DT group but who did not have evidence of SGS or TS. It is difficult to speculate on the reason for this female preponderance. Anatomically the female airway is narrower than the male, making it more prone for post-intubation stenosis. However, we even had females presenting with a SGS or TS. More research in larger series has to be performed to explain this phenomenon.
In cases where the diagnosis of WG is suspected, ANCA testing should be part of the routine laboratory evaluation [9]. C-ANCA has been shown to be a specific marker for WG with rare false-positive results; p-ANCA testing is much less specific. Only the particular laboratory findings of c-ANCA reacting with PR3 and p-ANCA reacting with myeloperoxidase (MPO) are specific for the autoimmune vasculitides [9, 10]. Notably, seven of our nine patients that underwent DT were PR3-ANCA positive and one patient was MPO-ANCA positive.
All patients had involvement of additional organs. In three patients the disease presented as a stenosis, and laryngotracheoscopy revealed the size and site of the lesion. Organs involved showed no pattern in which there should be additional suspicion for the development of SGS or TS. There was no relation between the number of relapses and the development of TS or SGS. The TS or SGS occurred more often in periods where the disease appeared to be less active.
Remarkably, TS or SGS predominantly became manifest in periods in which WG appeared to be inactive. Interestingly, seven out of our nine patients who developed TS or SGS had not been diagnosed previously with tracheal involvement of WG, neither at first presentation nor at relapse(s). We speculate that during active disease a subclinical tracheal involvement occurs which may subsequently heal with scar formation. To examine this hypothesis in patients, elective tracheoscopy should be performed in all patients with WG. The time interval between presentation of clinical stenosis in these seven patients (mean 63 months, median 60, range 2–122) favours watchful waiting.
DT with the Groningen dilatation tracheoscope is a safe, minimally invasive procedure for the treatment of Cotton-Myer grade II subglottic or tracheal stenosis of various origin. It is an elegant, self-standing surgical intervention, where no additional interventions are needed. For an overview of the effect of DT the reader is referred elsewhere (G.B. Halmos, F.G. Dikkers, Dilatation tracheoscopy in treatment of subglottic and tracheal stenosis, submitted for publication).
Nowadays, mitomycin-C, an alkylating agent that inhibits cell division, protein synthesis, and fibroblast proliferation [11], is increasingly used as adjuvant treatment in the management of selected cases of laryngeal and tracheal stenosis, for example luminal obstruction in fresh circular sutured wounds. However, the laryngeal and tracheal stenoses of WG patients are almost always of older age and display advanced scarring at the time of diagnosis. Dilating such “mature” stenoses will inevitably lead to damage of tracheal epithelium. However, this damage is considerably different from granulomatous scarification in sutured lumina and, therefore, we do not expect an additional favourable response to mitomycin. Therefore, we have not applied mitomycin-C in our WG patient cohort.
In cases where TS or SGS develops, there is always the question whether or not to intervene, and the question of timing of the intervention. There are two main parameters in these patients: complaints, and physical signs. One might see this as a two-by-two table, with complaints on the x-axis, and peakflow values on the y-axis. Progressive complaints and progressive decline of peakflow values (+/+) indicate intervention by DT. Absence of progressive complaints and unchanged peakflow values (−/−) indicate watchful waiting. Absence of progressive complaints with decline of peakflow values (−/+) need to be addressed when the values reach a critical level, in which it can be expected that a common cold might lead to severe stenosis of the airways. Finally, progressive complaints with unchanged peakflow values (+/−) indicate that pulmonary function tests should be performed, and, if negative, indicate that the patient has an incorrect perception of his physical potentials. This can then be addressed. Unfortunately, in only five of the nine patients in this retrospective study, we have peak flow measurements. A reason is that two patients have or had a tracheostomy.
We have registered no complications or deaths during or because of DT. We have three reasons to regard dilatation tracheoscopy as a minimally invasive intervention. We have experienced no complications related to dilatation tracheoscopy. It requires short hospitalization (generally 3 days). The intervention is not straining for the patients.
A variety of surgical techniques has been used to treat SGS or TS of other aetiologies [12, 13]. The success of these surgical techniques in upper airway stenosis related to WG has been variable [3, 14]. In our series two out of nine patients were treated once for upper airway stenosis with dilatation without any re-stenosis because of WG.
Unfortunately, two patients required a tracheostomy. One was temporary, but one patient needed a permanent tracheotomy (11%): this was the pregnant woman needing an emergency procedure. This number equals that of Gluth et al. [9].
Conclusion
WG as such is a rare disease, and SGS and TS are rare symptoms in patients with WG. DT can offer a simple and repeatable solution to this very serious symptom. Patient complaints and monitoring of peakflow values offer simple tools for the decision whether or not to intervene. However, a causative solution to WG should be the ultimate goal.

dilatation tracheoscopy
tracheal stenosis
wegener’s granulomatosis
subglottic stenosis
constriction