Reduction and S-nitrosation of the neuropeptide oxytocin: Implications for its biological function

Nitric Oxide 17 (2007) 82–90 www.elsevier.com/locate/yniox

Reduction and S-nitrosation of the neuropeptide oxytocin: Implications for its biological function Jean-Franc¸ois Roy a, Michelle N. Chre´tien a, Barbara Woodside b, Ann M. English a

a,*

Department of Chemistry and Biochemistry, Centre for Biological Applications of Mass Spectrometry, Concordia University, 7141 Sherbrooke Street W., Montreal, Que., Canada H4B 1R6 b Department of Psychology, Centre for Studies in Behavioural Neurobiology, Concordia University, 7141 Sherbrooke Street W., Montreal, Que., Canada H4B 1R6 Received 28 March 2007; revised 11 June 2007 Available online 4 July 2007

Abstract Oxytocin (OT; Cys-Tyr-Ile-Gln-Asn-Cys-Pro-leu-Gly), a posterior pituitary peptide hormone, is characterized by a Cys1Cys6 disulfide bond in its stable, isolated state. This paper describes a simple, one-step method for the production of OT in its reduced, dithiol form (OT dithiol), free of reducing agent. The effects of temperature, pH, and metal–ion chelators on the autoxidation of OT dithiol were examined to establish if this form is likely to persist under biological conditions. It was found that OT dithiol has a half-life of 1.8 h with respect to reformation of OT disulfide at 37 C and pH 6.9 in the presence of the copper chelators, DTPA and neocuproine. S-Nitrosation of OT dithiol by acidified nitrite at pH 3.0 was examined by absorption spectroscopy and HPLC-UV-MS, which revealed that both singly and doubly S-nitrosated OT are formed. These results suggest novel chemical aspects to OT signaling, the biological implications of which are discussed here.  2007 Elsevier Inc. All rights reserved. Keywords: Oxytocin; Thiol/disulfide; S-Nitrosation

Introduction Oxytocin (OT)1 has multiple functions in mammals. Traditionally, emphasis has been placed on the role that OT released from the neurohypophysis into the circulation plays in uterine contraction and milk letdown. Indeed, the

*

Corresponding author. Fax: +1 514 848 2868. E-mail address: [email protected] (A.M. English). 1 Abbreviations used: ACN, acetonitrile; DAD, diode-array detector; DMF, dimethylformamide; DTPA, diethylenetriaminepentaacetic acid; DTT, DL-dithiothreitol; eNOS, endothelial nitric oxide synthase; GnHCl, guanidine hydrochloride; GPCR, G-protein coupled receptor; GSH, glutathione; GSNO, S-nitrosoglutathione; GSSG, oxidized glutathione; NAYA, N-acetyl-L-tyrosinamide; nNOS, neuronal nitric oxide synthase; NOS, nitric oxide synthase; OT, oxytocin; OT–NO and OT–(NO)2, monoand di-S-nitrosated OT; OTR, oxytocin receptor; PVN, paraventricular nuclei; PBS, phosphate-buffered saline; RSNO, S-nitrosothiol; SON, supraoptic nuclei; TCEP, Tris(2-carboxyethyl)phosphine; Trx, thioredoxin; VWD, variable wavelength detector. 1089-8603/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2007.06.005

first usage of human pituitary extract to induce labor was reported in 1911 [1] and synthetic analogs of OT, Pitocin and Syntocinon, are still widely prescribed to induce and augment labor. Recently, considerable interest has been paid to the actions of OT within the brain. It has been shown to act centrally to modulate memory and learning [2], sexual arousal [3], social recognition [4], increased trust levels [5], social affiliation as well as in the suppression of stress and anxiety [6]. The known physiological effects of OT are believed to be mediated through its interaction with the oxytocin receptor (OTR), a member of the G-protein coupled receptor (GPCR) superfamily [7]. OTR is widely distributed in the brain, supporting the assignment of OT as a major central neurotransmitter. As expected, OTR is also expressed in the uterus, displaying maximum levels at parturition followed by a sharp decrease after birth. OTR is continually expressed in the mammary glands after parturition, demonstrating how OT switches its target organs throughout

J.-F. Roy et al. / Nitric Oxide 17 (2007) 82–90

pregnancy and post-parturition [8]. OTR has also been detected in a plethora of peripheral tissues including the ovary, testis, kidney, pancreas, heart, and vascular endothelium [7], suggesting that OT is likely involved in physiological functions that have yet to be established. OT and nNOS co-localization Since being identified as a regulator of vascular tone, NO has been shown to control a nearly limitless number of biological functions. NO biosynthesis from L-arginine is catalyzed by nitric oxide synthase (NOS) [9]. NOS was first discovered in the endothelial lining of blood vessels (eNOS) and a second isoform, neuronal NOS (nNOS), has been identified in neurons that generate NO. Significantly, the highest nNOS expression is found in the magnocellular neurosecretory neurons of the supraoptic nuclei (SON) and paraventricular nuclei (PVN), the major sites of OT synthesis [10], suggesting that NO production is topographically co-distributed with OT expression [11]. A functional relationship has been established between OT and NO based upon the observation that nNOS is upregulated in OT cells of the paraventricular and supraoptic nuclei of the rat hypothalamus when these cells are stimulated. Moreover, inhibiting NOS under these conditions increases the release of OT from the pituitary [12]. Conversely, OT appears to regulate the expression of NOS in the brain, thereby making NO a potential feedback inhibitor in the OT signaling system [12,13]. Co-localization of NO and OT production and their upregulation at parturition suggest possible chemical crosstalk between these signaling molecules and novel regulatory mechanisms of neuroendocrine function. No biochemical reactions between OT and NO have been reported to date, but there is mounting evidence that thiol S-nitrosation is a key regulatory mechanism in the mediation of the physiological effects of NO [14–16]. OT, a nineresidue peptide with cysteines at positions 1 and 6, could undergo single or double S-nitrosation in its reduced thiol form (Scheme 1).

83

Although the possible biological implications of OT S-nitrosation are intriguing, OT is normally isolated as the disulfide. OT immunodetection in tissues is performed with an antibody raised against the disulfide form as antigen [17], which may not be sensitive to reduced forms of OT. It was originally proposed that the biological activity of OT was due to thiol/disulfide exchange with free thiols on OTR [18], but the current view [7] is that OT–OTR binding does not involve a covalent interaction. OT and other neuropeptides are believed to have a 2-fold interaction with their receptors. The cyclic portion of OT (defined by the Cys1Cys6 disulfide linkage) binds to the transmembrane domain of the GPCR and the three-residue tail interacts with an extracellular loop [7,19–21]. Both modes of binding are thought to be important for biological signaling and this model is supported by molecular dynamics simulations of OT–OTR docking [22]. Based on the present model of OT–OTR interaction, the dithiol form of OT is not expected to be biologically active. However, this has not been tested to our knowledge, and OT dithiol has been little studied. Three literature reports [23–25] dating from the early 1990s describe a series of experiments that characterize the cis/trans conformational equilibrium across the Cys6Pro7 peptide bond [23] of OT dithiol, and the kinetics of OT thiol/disulfide exchange with glutathione and cysteine [24,25]. The exchange kinetics revealed strain in the OT disulfide bond since OT formed a mixed disulfide with GSH or cysteine close to two orders of magnitude faster than expected [24]. However, OT disulfide is rapidly reformed by intramolecular thiol/disulfide exchange such that at plasma concentrations of non-protein thiols, the OT mixed disulfide concentration is expected to be small [24]. Nonetheless, Cys1 and Cys6 of OT dithiol are not constrained to be closer together than in an unstructured peptide [24]. Given the reported strain in the OT disulfide bond [24], we hypothesized that the dithiol form would be stable in the absence of free thiols. We describe here an efficient method to prepare and isolate OT dithiol in the absence of reducing agents. OT dithiol autoxidation leading to disulfide reformation was examined in air in the absence of reductants, and the reactivity of OT dithiol to S-nitrosation was examined. The results are discussed in terms of possible chemical crosstalk between NO and OT signaling. Materials and methods Materials

Scheme 1. Proposed reactions of OT dithiol with acidified nitrite. At high nitrite concentrations, pathway (A) will be favored giving rise to OT– (NO)2 as the major product. At low nitrite concentrations, pathway (B) will dominate leading to OT disulfide and HNO (nitroxyl) formation. The sequence of OT is given and the proposed reactions are based on [40]. Photolysis of OT–(NO)x was prevented in the present study by carrying out all procedures in the dark.

Oxytocin (CYIQNCPLG–NH2, disulfide) was purchased from CanPeptide (Montreal, Canada) as the trifluoroacetate salt (C43H66N12O12S2ÆCF3CO2). The peptide content was given as 90% and >98% of the peptide was confirmed to be OT by HPLC analysis. Tris(2-carboxyethyl)phosphine (TCEP) was purchased from Pierce (Nepean, Canada) either as the hydrochloride salt or as a 50% aqueous gel suspension containing agarose-immobilized TCEP (8 mM equivalent TCEP concentration). HandeeTM Spin columns (0.9 mL) equipped with a 10 lm polyethylene filter were also purchased from Pierce. DL-Dithiothreitol (DTT), phosphate-

J.-F. Roy et al. / Nitric Oxide 17 (2007) 82–90

buffered saline (PBS) tablets, neocuproine hydrochloride, diethylenetriaminepentaacetic acid (DTPA), oxidized glutathione (GSSG), sodium nitrite, N-acetyl-L-tyrosinamide (NAYA), and the mass calibrants, GluFib peptide, MRFA peptide (methionine–arginine–phenylalanine–alanine), and horse skeletal myoglobin were obtained from Sigma (Oakville, Canada). Formic acid was purchased from Anachemia (Montreal, Canada), ammonium formate from Fisher Scientific (Ottawa, Canada), N,N-dimethylformamide (DMF) from Aldrich (Oakville, Ontario), guanidine hydrochloride (GnHCl) from BioShop (Burlington, Canada), ethanol (>99%) from Commercial Alcohols (Montreal, Canada) and acetonitrile (ACN) from VWR (Mississauga, Canada). Water (HPLC-grade) was dispensed from a Millipore Simplicity system with a resistivity of 18.2 MX.

0.4 0.35 0.3

Absorbance

84

0.25 0.2 0.15 0.1 0.05

Chromatography and mass spectrometry

0 240

Chromatographic separations were carried out on a 4.6 mm · 150 mm (4 lm particles) Phenomenex SynergiFusion RP-80 analytical column coupled to an Agilent 1100 or 1090 HPLC. Mobile phases A and B consisted of 10% and 90% ACN, respectively, in 0.1% formic acid. Gradient elution was performed with 030% mobile phase B over 10 min at a flow rate of 1 mL/min. The peptide signal was monitored at 273 nm using a variable wavelength (VWD) or diode-array detector (DAD). OT disulfide reduction was monitored by MS in positive-ion mode on a Waters Micromass Q-ToF2 mass spectrometer running MassLynx software. The sample was infused at flow rate of 1 lL/min directly into the nano Z-spray source, and analyzed using the instrument parameters given in the legend of Fig. 2. Time courses of disulfide reformation (Fig. 3) were monitored by HPLC-UV as described above. HPLC-MS analysis of OT dithiol S-nitrosation was performed on a ThermoFinnigan SSQ 7000 mass spectrometer coupled to an Agilent 1090 HPLC. Samples were injected manually into a 100 lL sample loop with a Hamilton Gastight 100 lL syringe, and 30 lL/min of the split HPLC eluate was directed to the ESI source of the SSQ 7000 operating under the conditions given in the legend of Fig. 5. Spectra were acquired in the m/z 1901900 range at a rate of 1 per 3 s.

Molar absorptivities of OT disulfide, OT dithiol, and OT–(NO)2 The spectrum of OT disulfide in 6 M GnHCl is shown in Fig. S1 (Supplementary data). Since the commercial OT sample contained only 90% peptide, the molar absorptivity at 280 nm in 6 M GnHCl (eGnHCl) of OT disulfide was calculated by summing the literature eGnHCl values of its two residues with 280 nm absorption, tyrosine (1.280 mM1 cm1) and cystine (0.120 mM1 cm1) [26]. Additionally, eGnHCl of OT disulfide was estimated from the eGnHCl values obtained from Beer’s law plots (Fig. S2, Supplementary data) for NAYA (1.315 mM1 cm1; see UV spectrum in Fig. S1), a model compound for tyrosine residues in peptides, and GSSG (0.114 mM1 cm1). The eGnHCl of OT disulfide used here is the average (1.415 mM1 cm1) of the two estimates (1.400 and 1.429 mM1 cm1). The molar absorptivity at the near-UV maximum (273 nm) in water (e273) of OT disulfide was calculated to be 1.5 mM1 cm1 from e273 = eGnHCl · (AH2 O =AGnHCl ) where AH2 O =AGnHCl ¼ 1:06: To obtain the latter ratio, 1 mg/mL of the trifluoroacetate salt of OT was dissolved in water, diluted 5-fold with 6 M GnHCl or water and the absorption spectra in a 1 cm cell at 25 C were recorded on a Beckman DU-800 spectrophotometer. To determine e273 for OT dithiol, the 1 mg/mL OT disulfide stock was diluted to 200 lM in water and in 10 mM TCEP (which does not absorb above 240 nm). After 15 min incubation at room temperature, the spectra were recorded from 240 to 350 nm (Fig. 1) and, based on the estimated e273 (1.5 mM1 cm1) of the disulfide, e273 of OT dithiol was calculated to be 1.3 mM1 cm1. This is in agreement with the e273 reported for NAYA (1.28 mM1 cm1) [26] as expected since only Tyr2 contributes to the 273 nm absorption of OT dithiol (Fig. 1). Values of e273 for the S-nitrosated forms of the peptide, OT–NO and OT–(NO)2, were not specifically

260

280

300

320

340

Wavelength/nm Fig. 1. Absorption spectrum of 200 lM OT disulfide (d) 200 lM OT dithiol (s) in water. The dithiol was generated in situ by incubation for 15 min in 10 mM TCEP (see Materials and methods), which has no absorption above 240 nm. Spectra were recorded in a 1-cm cuvette at 25 C.

evaluated but should not differ significantly from that of OT dithiol. Based on the e545 reported for GSNO [27], each SNO group is assumed to contribute 16 M1 cm1 to the absorbance of OT–(NO)x at 545 nm.

OT disulfide reduction To establish the conditions for efficient reduction, 250 lM OT disulfide was incubated with 10- or 40-fold molar excess of reducing agent (TCEP or DTT) in 10 mM ammonium formate buffer (pH 3.0) for 1560 min. Aliquots were diluted 10-fold with 0.1% formic acid and injected into the nano Z-spray source of a Waters Micromass Q-ToF2 mass spectrometer as described above. OT dithiol free of reductant was prepared using gel-immobilized TCEP. Typically, 200 lL of a 50% TCEP-gel suspension in water was pipetted into a spin column, centrifuged (1500g for 90 s at 4 C) to remove excess solvent, and incubated with a 200 lL aliquot of 2 mM OT disulfide in water for 12 h with continuous agitation. Mixing was required to prevent the gel from settling which led to incomplete reduction. OT dithiol was collected in an Eppendorf tube by centrifugation (1500g for 90 s at 4 C), diluted 10-fold into the mobile phase, analyzed by HPLC-UV (Fig. S3) to confirm complete disulfide reduction, and stored at 80 C.

Effects of pH, temperature, and chelators on OT dithiol stability in air The time course of disulfide reformation at 21, 37, and 50 C was monitored by HPLC-UV to establish the stability of OT dithiol to autoxidation. OT dithiol from a 1 mM stock solution in water was diluted to 250 lM into 20 mM PBS (pH 6.9), 20 mM PBS containing 100 lM DTPA plus 100 lM neocuproine (PBS/chelators), or water (final pH 2.9). The pH of PBS dropped to 6.9 on addition of the metal chelators, which are specific for CuII and CuI, respectively, and were added since trace copper promotes thiol autoxidation [28–30]. Aliquots were removed at different time points, diluted 10-fold into water (samples at pH 2.9) or into PBS/chelators, injected onto the HPLC column, and detected with the VWD at 273 nm. Note that the samples at pH 2.9 were not exposed to higher pH at any point. The fraction of OT dithiol remaining was calculated from the integrated peak area of the dithiol divided by the sum of the dithiol and disulfide integrated peak areas. To minimize OT dithiol autoxidation during handling and analysis, the chelators were added to all samples following incubation and the tubes were stored in a thermostatted HPLC sample tray at 4 C prior to injection.

J.-F. Roy et al. / Nitric Oxide 17 (2007) 82–90

OT dithiol S-nitrosation by acidified nitrite The pH of 10 mM ammonium formate buffer was adjusted to 3.0 with 1.0 N HCl, and 100 mM sodium nitrite and 2 mM OT dithiol (measured spectrophotometrically; e273 = 1.3 mM1 cm1) in water were diluted into the buffer. S-Nitrosation of 0.5 mM OT at a nitrite/OT ratio of 2:1 was monitored at 545 nm over 40 min at 25 C in a 1-cm cuvette in the lightprotected compartment of a Beckman DU-800 spectrophotometer and by HPLC-UV-MS. S-Nitrosation of 0.5 mM OT at a nitrite/OT ratio of 20:1 was also monitored by HPLC-UV-MS. Aliquots were removed after 5, 15, 30, and 45 min incubation, diluted 10-fold into the formate buffer, and injected onto the HPLC column (Fig. 5).

Results

85

1007.4 peak (Fig. 2b). A 10-fold molar excess of TCEP resulted in 50% OT disulfide reduction in 60 min (data not shown). Agarose-bound TCEP was used to prepare OT dithiol free of reducing agent. After 60 min incubation of 2 mM OT disulfide with the gel in water, the peptide was recovered from the spin column by centrifugation, and was shown by HPLC-UV to be fully in the dithiol form. Although the effective concentration of the gel-bound TCEP is 8 mM (4-fold molar excess over OT), a longer incubation time was required for complete OT reduction on the gel than in solution. Steric hindrance and limited diffusion of the immobilized reductant are expected to impact the efficiency of gel-bound TCEP as compared to free TCEP.

OT disulfide reduction OT dithiol stability Solutions of 250 lM OT were subjected to either 10- or 40-fold excess of reducing agent for 60 min in formate buffer (pH 3.0) to minimize disulfide reformation. The efficiency of two well-known disulfide reducing agents, DTT and TCEP, were compared by monitoring the time course of OT dithiol formation by mass spectrometry. Since OT disulfide (monoisotopic mass 1006.4 lm) and dithiol (monoisotopic mass 1008.4 lm) are mass-resolved, chromatographic separation was not employed prior to MS analysis in this case. The disappearance of the (M+H)+ ion of OT disulfide at m/z 1007.4 was used to confirm disulfide reduction. Both 10-fold (data not shown) and 40-fold molar excess of DTT (Fig. 2a) were ineffective in OT disulfide reduction over 60 min. In contrast, reduction was complete within 15 min in the presence of 40fold molar excess of TCEP as indicated by the loss of the m/z

The time course of OT disulfide reformation in air in the absence of reducing agents was monitored by HPLC-UV (Fig. S3, Supplementary data). Fig. 3b reveals that the disulfide fully reforms within 20 min at pH 6.9 and 37 C. Lowering the temperature slows the rate of autoxidation but more dramatic effects are observed on lowering the pH to 2.9 (Fig. 3a) or adding chelators (Fig. 3b, dashed lines). The times for 50% and >95% disulfide reformation are summarized in Table 1. OT dithiol S-nitrosation by acidified nitrite Although TCEP is reported to be highly selective for disulfides [31], its reactivity with RSNOs has not been

Fig. 2. Time course of OT disulfide reduction with 40-fold excess DTT and TCEP. ESI mass spectra of 250 lM OT solutions incubated with: (a) 10 mM DTT and (b) 10 mM TCEP. Samples were incubated in 10 mM ammonium formate buffer (pH 3.0) for the times indicated, diluted 10-fold with 50% ACN/ 0.1% formic acid, and infused directly at flow rate of 1 lL/min into the nano Z-spray source of a Micromass Q-ToF2 mass spectrometer. Operating parameters in positive-ion mode were: source temperature 80 C, capillary voltage 3.2 kV, cone voltage 35 V, collision-cell voltage 10 V, and TOF voltage 9.1 kV. The instrument was calibrated externally to a mass accuracy of 60 ppm with the Glu-Fib peptide and the samples were analyzed at an average resolution of 8000.

86

J.-F. Roy et al. / Nitric Oxide 17 (2007) 82–90 100

0.005

Absorbance (545 nm)

80

60

40

0.004

0.003

0.002

% Disulfide reformed

20

b

0 0

20

40

60

80

100

120

100

0

10

20

30

40

Time /min Fig. 4. Increase in SNO absorbance at 545 nm over time in the reaction between 0.5 mM OT dithiol and 1 mM acidified nitrite at pH 3.0. Data points were recorded continuously after mixing in a 1-cm cuvette at 25 C. The line is not a mathematical fit of the data.

80

60

40

20

a 0 0

4

8

12

16

Time /h Fig. 3. Effects of pH, temperature, and chelators on OT disulfide reformation. Two hundred and fifty micromolar OT dithiol at 21 C (d), 37 C (s), and 50 C (j) in: (a) 20 mM PBS at pH 6.9 with (dashed lines) or without (solid lines) 100 lM neocuproine plus 100 lM DTPA; (b) water at pH 2.9. Each data point represents the fraction of OT disulfide reformed as determined by HPLC-UV (see Materials and methods). The lines are not a mathematical fit of the data. Note that the x-axis in (a) is 020 h but 0120 h in (b).

Table 1 Effects of temperature, pH, and chelators on OT disulfide reformation Temperature (C) Time for disulfide reformation

21 37 50

0.001

H2O (pH 2.9) 20 mM PBS (pH 6.9)

20 mM PBS + chelatorsa (pH 6.9)

50%

50%

>95%

51.2 h 115 h 24.5 min 50 min 4.7 h 17.2 h 35.9 h 8 min 20 min 1.8 h 8.6 h 18.2 h

17.5 h 6.5 h

>95% 50%

>95%

a One hundred micromolar DTPA and 100 lM neocuproine. Addition of the chelators lowered the pH of PBS to 6.9.

reported so OT dithiol free of TCEP was used here. Since cysteine S-nitrosation yields a primary RSNO with absorption at 545 nm [27], OT–(NO)x formation was monitored at this wavelength. Fig. 4 shows the absorbance increase at 545 nm over time during the reaction of 0.5 mM OT dithiol with 1 mM nitrite at pH 3.0. The absorbance reached a pla-

teau after 20 min and did not change over the next 25 min. Based on an e545 of 16 M1 cm1 [27], the steady-state SNO yield was assumed to be 0.3 mM after 20 min. The S-nitrosation reaction was also monitored by HPLC-UV-MS (Fig. 5) and four species were identified by ESI mass spectrometry as summarized in Table 2. The singly (M+H)+ and doubly protonated (M+2H)2+ ions of OT disulfide (peak 2) and OT dithiol (peak 3) are clearly observed in the mass spectra (Fig. 5d and e). OT–NO (peak 4) is assigned based on the detection of its (M+H)+ and (M+2H)2+ ions at m/z 1038.5 and 520.0, respectively. The observation of intense fragment ions resulting from NO loss, (MNO+H)+ at m/z 1008.7 and (MNO+2H)2+ at m/z 505.0 (Fig. 5f and Table 2), support this assignment since we have previously demonstrated that the SNO bond readily undergoes homolysis in ESI sources [32,33]. OT–(NO)2 (peak 5) was also identified based on the observation of its (M+H)+ (m/z 1067.7), (M+2H)2+ (m/z 534.2), (M2NO+H)+ (m/z 1007.6), and (M2NO+2H)2+ (m/z 504.4) ions (Fig. 5g and Table 2). The OT disulfide concentration essentially doubled between 5 and 45 min in the 1 mM nitrite incubation (Fig. 5a and b). This dramatic acceleration of disulfide formation at pH 3.0 in the presence of nitrite compared to that seen in the absence of the anion (Table 1) must be induced by S-nitrosation. Since OT–(NO)2 is the dominant product and the OT disulfide yield is low in the 10 mM nitrite incubation (Fig. 5c), mono-S-nitrosation of OT dithiol must induce OT disulfide formation in the reaction with 1 mM nitrite.

Discussion Formation and stability of OT dithiol Thiol/disulfide exchange with reagents such as DTT, a dithiol, is commonly employed in protein chemistry to

J.-F. Roy et al. / Nitric Oxide 17 (2007) 82–90

87

Fig. 5. HPLC-UV-MS analysis of the reaction between 0.5 mM OT dithiol and acidified nitrite at pH 3.0. UV chromatograms (273 nm) after (a) 5 min incubation and (b) 45 incubation of 0.5 mM OT with 1 mM nitrite. (c) Thirty minutes incubation of 0.5 mM OT with 10 mM nitrite. The mass spectra of OT disulfide (peak 2), OT dithiol (peak 3), OT–NO (peak 4), and OT–(NO)2 (peak 5) are shown in (d–g). HPLC separations were performed as described in Materials and methods. The column eluate was split and directed into the ESI source of the SSQ 7000 at a flow rate of 30 lL/min. The source was operated in positive-ion mode with a nitrogen sheath gas pressure of 40 psi and a spray voltage of 4 kV. The source and heated capillary temperatures were 70 and 180 C, respectively. The instrument has unit-mass resolution, and was calibrated externally with the MRFA peptide and horse skeletal myoglobin to a mass accuracy of 300 ppm.

Table 2 Ions identified by HPLC-MS in OT S-nitrosationa Peak #a

Species (monoisotopic mass)

Ion

2

OT disulfide (1006.4)

3

m/z Calculated

Observed

(M+H)+ (M+2H)2+

1007.4 504.2

1007.6 504.5

OT dithiol (1008.4)

(M+H)+ (M+2H)2+

1009.5 505.3

1009.5 505.6

4

OT–NO (1037.4)

(M+H)+ (M+2H)2+ (MNO+H)+ (MNO+2H)2+

1038.4 519.7 1008.5 504.7

1038.5 520.0 1008.7 505.0

5

OT–(NO)2 (1066.4)

(M+H)+ (M+2H)2+ (M2NO+H)+ (M2NO+2H)2+

1067.5 534.2 1007.4 504.2

1067.7 534.2 1007.6 504.4

a

Peak numbers and experimental details are given in Fig. 5.

reduce disulfides. This is initiated by intermolecular nucleophilic attack of the DTT thiolate on one of the oxidized sulfur atoms to release a substrate thiol, which is followed by intramolecular attack and release of the second substrate thiol. DTT with thiol pKa values >9 requires neutral

or slightly basic pH for maximum efficiency [34], which also maximizes the reverse reaction and drives the equilibrium away from the reduced substrate, especially in the case of dithiols. For example, the thiol/disulfide exchange equilibria of OT with GSH and cysteine were observed to lie largely toward the OT disulfide [24]. TCEP, an air-stable phosphine, is essentially an irreversible reductant of disulfides [35] with the additional advantage that it can be used over a wide pH range (1.5–8) [31,36]. Reduction is initiated by attack of the phosphorus atom on a disulfide sulfur with the formation of a thiophosphonium cation and thiolate anion. Hydrolysis of the cation releases the second thiol and the stable phosphine oxide [31]. TCEP is the disulfide reducing agent of choice for a number of biochemical applications [37], including OT disulfide reduction as established here (Fig. 2). Moreover, use of gel-immobilized TCEP and spin columns rapidly produces OT dithiol free of reducing agent as compared to the slow electrochemical reduction method used previously [24]. Solution pH, metal chelators, and temperature were all shown to affect the rate of OT disulfide autoxidation (Table 1). Trace copper catalyzes disulfide bond formation via thiolate coordination [28–30]. Thus, lowering the pH or

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adding copper chelators greatly increases the stability of OT dithiol (Fig. 3). Notably, free copper-catalyzed OT disulfide reformation should be negligible in vivo, since the free copper concentration within cells is proposed to be extremely low [38]. Hence, its half-life in air at pH 6.9 and 37 C (1.8 h, Table 1) reveals that OT dithiol can clearly exist under physiological conditions.

of 0.5 mM OT dithiol with 10 mM nitrite (Fig. 5c), and OT disulfide is a minor product. S-Nitrosodihydrolipoic acid and S-nitrosoDTT (1,3- and 1,4-vicinal dithiols, respectively) decompose to their disulfides within minutes [40], and no S-nitrosation yields have been reported. Hence, ring strain in OT disulfide [24] may contribute to the higher stability of its S-nitrosated forms (Figs. 4 and 5) relative to those of dihydrolipoic acid and DTT.

S-Nitrosation of OT dithiol by acidified nitrite Biological implications It is well documented that OT and nNOS co-localize in the hypothalamo-neurohypophyseal system [10,12,39] raising the possibility that OT is S-nitrosated in vivo. Based on the 545-nm absorbance, a steady-state SNO concentration of 0.3 mM, corresponding to 30% of the initial thiol (1 mM), was detected after 20 min in the reaction of 0.5 mM OT thiol with 1 mM nitrite (Fig. 4). Assuming that all OT forms exhibit similar 273-nm absorbance, HPLCUV-MS analysis of the same reaction revealed that OT disulfide is the major product after 45 min, but combining the OT–NO and OT–(NO)2 signals (Fig. 5b) also revealed that the SNO yield is 30% consistent with Fig. 4. Significant (25%) OT dithiol (peak 3, Fig. 5b) remained after 45 min to maintain the steady-state SNO concentration (Fig. 4) arising from formation of S-nitrosated OT and its decay to the disulfide. Based on studies with DTT and dihydrolipoic acid [40], the expected reactions between OT dithiol and acidified nitrite are given in Scheme 1. Photolysis of the OT– (NO)x products was prevented by carrying out S-nitrosation and product analysis in the dark. Pathway B is expected to dominate at low nitrite/dithiol ratios, where mono-S-nitrosation promotes intramolecular nucleophilic attack on the SNO sulfur. This is consistent with the observation of OT-disulfide as the major product in the reaction with 1 mM nitrite after 45 min (Fig. 5b), and indicates that mono-S-nitrosation accelerates disulfide formation even at pH 3.0 despite the low reactivity of thiols under acidic conditions [41]. The concomitant release of HNO from OT– NO, as reported for other dithiols [40,42], would provide a mechanism for the biosynthesis of this highly thiophilic reagent [43–45]. Thus, the formation of hydroxylamine and N2O (indirect reporters of HNO) in OT incubations with nitrite and other NO donors is currently being examined. In contrast to OT dithiol, S-nitrosation of monothiols in acidified nitrite is typically fast and goes to completion at equimolar concentrations of thiol and nitrite. For example, S-nitrosation of the monothiol, N-acetylpenacillamine, was complete within 15 min at concentrations similar to those used here [46]. In addition, the product RSNOs are stable under acidic conditions [47], making acidified nitrite the reagent of choice for RNSO synthesis in vitro. However, excess nitrite will likely be required for full S-nitrosation of dithiols (pathway A, Scheme 1) because of competing intramolecular disulfide formation (pathway B, Scheme 1). Indeed, OT–(NO)2 is the main product in the reaction

The considerable stability of OT dithiol to autoxidation under physiological conditions presents the possibility of novel chemical signaling by the oxytocin–vasopressin family of neuropeptides. Since a common feature of this family is the disulfide bond between Cys1 and Cys6, many neuropeptides may have their messenger activity temporarily modified by disulfide reduction and subsequent thiol modification. In support of a physiological role for OT dithiol, thioredoxin (Trx) and Trx-related protein TRP14 were recently shown to turn-over NADPH in the presence of OT disulfide in vitro [48,49]. Thus, Trx and related proteins may generate OT dithiol in vivo and, although GSH promotes OT disulfide formation in vitro [24], this could be inhibited within cells by any number of mechanisms. Both chemical modifications of OT disulfide investigated here, reduction and S-nitrosation, could induce a localized, temporary, and site-specific modulation of OT hormonal activity. For example, S-nitrosation-induced disulfide formation in OT could lead to rapid formation of the active hormone. OT–(NO)x could modify signaling pathways by chemically modifying the OTR. In fact, it was recently demonstrated [50] that RSNOs can modulate GPCR signaling in a reversible and highly receptor-specific manner via trans-S-nitrosation of free cysteines on the receptor. Several potential S-nitrosation sites exist in the OTR, a GPCR. For example, there are two cysteine residues in the extracellular domain of the human OTR and nine more are located within its seven transmembrane domains [22]. We have recently demonstrated [43] that HNO reacts readily with protein-based thiols to form disulfides and stable sulfinamides (RSONH2). Therefore, HNO released on denitrosation of OT–NO in the vicinity of the OTR could modify the receptor and modulate its signaling in a temporary or persistent fashion. Tools to probe the different OT species in vivo are of interest. Since OT is detected in biological fluids by immunoassay, a first step would be to compare the crossreactivity of OT antibodies with the disulfide, dithiol, and the S-nitrosated forms of the hormone. Antibodies raised specifically against OT dithiol and OT–(NO)2 could be used to probe for these forms in vivo. The affinity and specificity of recombinant OTR [51] for the different OT species could be evaluated, and the reactivity of OTR cysteine residues toward HNO and OT–(NO)x could be established. In conclusion, we have developed a simple, rapid, onestep procedure to isolate OT dithiol free from reductants.

J.-F. Roy et al. / Nitric Oxide 17 (2007) 82–90

The effects of temperature, pH, and metal chelators on OT dithiol autoxidation were evaluated, and revealed that OT dithiol is stable under physiologically relevant conditions. S-Nitrosation of OT dithiol in acidified nitrite to give the mono- and di-S-nitrosated hormone was demonstrated by HPLC-UV-MS. Mono-S-nitrosation may be a route to the bioactive OT disulfide in vivo and/or a mechanism for HNO biosynthesis. Like other RSNOs [47,52,53], the relatively stable di-S-nitrosated hormone could act as a NO donor to the OTR.

[13]

[14]

[15]

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Acknowledgments The authors acknowledge generous financial support from NSERC and CIHR in the form of operating grants (AME and BW) and a post-doctoral fellowship (MNC). We also thank Alain Tessier from the Centre for Biological Applications of Mass Spectrometry, Concordia University, for technical assistance. AME holds a Concordia University Research Chair.

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Appendix A. Supplementary data [21]

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.niox.2007. 06.005.

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