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Direct measurement of nitric oxide release from the rat hippocampus
Analytica Chimica Acta 415 (2000) 127–133
Direct measurement of nitric oxide release from the rat hippocampus Yuezhong Xian, Wen Zhang, Jian Xue, Xiangyang Ying, Litong Jin∗ Department of Chemistry, East China Normal University, Shanghai 200062, China Received 16 November 1999; received in revised form 29 February 2000; accepted 16 March 2000
Abstract A novel, sensitive, selective and reproducible nitric oxide (NO) microsensor is developed for direct measurement of NO in vitro and in vivo. Using the microsensor, the first direct measurements NO release from the rat hippocampus are made. NO levels are increased by stimulated with l-arginine and acetylcholine. l–Nω -nitro-arginine, a NO synthase inhibitor, decreases the NO increase induced by l-arginine and acetylcholine. The effect of sodium nitroprusside, a NO donor, is also performed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide; Hippocampus; Microsensor
1. Introduction In recently years, nitric oxide radical (NO) has been increasingly recognized as an important intraand inter-cellular messenger molecule, which plays a physiological role in vascular relaxation, platelet activation, neurotransmission, and immune response [1–6]. This bioradical, a potent vasodilator or endothelium-derived relaxing factor, is synthesized from l-arginine (l-Arg) and molecular oxygen in various cell types by three distinct isoforms of the enzyme NO synthase (NOS). Two of these, constitutive and calcium-dependent isoenzymes (cNOS), which have been observed in neuronal and endothelial cells produce a small amount of NO when calcium/calmodulin binding permits electron transfer from NADPH via flavin groups to an active site [7–9], whereas the other inducible, calcium-independent isoform (iNOS) [10] in macrophages, chondrocytes, and hepatocytes initiated by various immunological stimuli such as ∗ Corresponding author. Fax: +86-216245-1876. E-mail address: [email protected] (L. Jin)
endotoxin, microbes, and cytokines synthesizes high levels of NO. NO plays multifaceted and important roles in the brain. It is a neurotransmitter and a free radical, and it has been implicated in the regulation of cerebral blood flow and inflammation [11–15]. In the hippocampus, independent induction of long-term potentiation (LTP) or depression (LTD) has been reported [16,17]. The elevated concentration of this radical by NOS has been implicated in the pathogenesis of several disease states such as septic shock [18], arthritis [19], atherosclerosis [20], diabetes mellitus [21], graft rejection [22], and ischemia/reperfusion injury [23]. Thus, the precise measurements of this bioradical and its metabolites in the circulation are of increasing clinical importance. NO is a small, highly reactive, readily diffusible gas that can combine with most biologically active molecules. Thus, the measurement of NO is very difficulty. Over the past years, efforts have been made to directly and indirectly measure NO release in biological systems. In general, the measurement methods include: (a) NO is ‘trapped’ by nitroso compounds, or reduced hemoglobin, forming a stable
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adduct that is detected by electron paramagnetic resonance (EPR) [24,25]; (b) NO oxidizes reduced hemoglobin to methemoglobin, which is detected by spectrophotometry [26,27]; (c) NO interacts with ozone producing light, ‘chemiluminescence’ [28,29]; (d) NO is measured with NO-fluorescence probes, fiber-optic NO sensors, and chemical assay of NO cofactors using microseparation [30–35] and (e) NO is monitored by electrochemical methods through chemically modified microsensors [36–44]. Among those techniques, electrochemical methods are most advantageous because they are simple, relatively fast, sensitive, and can be applied on-line measurement. In addition, the explosive growth in the fabrication of ultramicroelectrodes [45] strongly stimulates the use of electrochemical sensors for in vivo NO detection. NO microsensor can be fabricated to extremely small dimensions (m or nm in diameter) and thus are ideal for insertion directly into the biological systems with minimal damages. In addition, chemically modified microsensors can electrocatalytically oxidize or reduce NO, therefore, diminishing the interference induced by some other substances in biological systems. Owing to those advantages, the chemically modified microsensor is widely used real-time and in vivo measurement NO. Although NO is a subject of great interest in brain research, to our knowledge, direct in vivo measurements of NO in hippocampus using electrochemical microsensor have not been performed. Here, we demonstrate the ability to study NO in vivo using an novel chemically modified NO microsensor, which is prepared by electrodeposition copper–platinum microparticles on carbon fiber (d=15 m) surface. It is used to measure the NO release from rat hippocampus modified by l-Arg, acetylcholine (Ach), l–Nω -nitro-arginine (l-NNA) and sodium nitroprusside (SNP).
2. Experimental 2.1. Chemicals NO standards were prepared by serial dilution of a saturated NO solution. Preparation of the saturated NO solutions involved meticulous exclusion of O2 , as NO was rapidly destroyed by O2 . To produce the saturated
NO solution (at 20◦ C, 2.05 mM NO) [46], deionized water solution (2 ml) was bubbled with nitrogen for 30 min to remove oxygen. Then the solution was bubbled with pure NO gas (Aldrich) for 30 min and kept under NO atmosphere until use. The concentration of saturated NO is about 2.00 mM. Standards were made fresh for each experiment and kept in a glass flask with a rubber septum. Dilutions of the saturated solution were made using deoxygenated water samples. Phosphate-buffered saline (PBS) containing 137 mmol/l NaCl, 2.7 mmol/l KCl, 8.0 mmol/l Na2 HPO4 and 1.5 mmol/l KH2 PO4 was prepared and adjusted to pH7.4. CuCl2 (Shanghai reagent factory, Shanghai), K2 PtCl6 (Chengdu reagent factory, Chengdu), l-Arg (Sigma, USA), l-NNA (Sigma, USA), Ach (Sigma, USA), SNP (Katayama Chemical, Japan) and other reagents were analytical reagents. 2.2. Electrochemical instrument Electrochemical experiments were performed on a CHI-832 electrochemical analyzer (CH Instruments Inc., Cordova, USA) at room temperature (25±2◦ C) in a three compartment cell with a saturated calomel reference electrode (SCE), an Au wire auxiliary electrode and a copper–platinum microparticles modified carbon fiber microsensor (15 m in diameter). 2.3. Preparation carbon fiber microelectrode and electrochemical procedures Carbon fiber microelectrodes were built according to previous description [47] with modifications. They consisted of one carbon fiber, 15 m diameter and 450 m length, protruding from a glass pipette. Electrical contact with the carbon fiber was assured either by a polyester resin mixed with carbon powder [48] and connection to the polarograph was assured by a copper wore, or a silver wire was glued to the carbon fiber by a silver conductive paint thus assuring the connection to the polarograph. In this case, the extremity of the pipette was filled with an epoxy resin in order to assure tightness. The carbon fiber were treated in PBS (pH 7.4), by passing down the electrodes triangular pulses (80 Hz) in two successive sequence of 20 s duration at +2.85 and +1.6 V, respectively. Electrode-
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position of copper–platinum microparticles and Nafion (5% solution, Aldrich) were successively achieved by immersing the carbon fiber microelectrode in a few microliters (4–5 l) of each solution placed in small loop of platinum wire (2 mm diameter) connected to the auxiliary and reference plugs of the Biopilse. 2.4. In vitro calibration Prior to NO measurements, the modified carbon fiber electrodes were placed in PBS, and the potential was cycled between +0.70 and −0.80 V at a rate of 50 mV/s until a steady response was obtained. For NO determination, a diluted NO solution was subsequently added with a gas-tight syringe. The current due to the electrocatalytic oxidation of NO by the microsensor was recorded after each addition. Differential pulse voltammetry (DPV) was performed with a potential sweep rate of 5 mV/s and 5 pulses/s (pulse height 50 mV, pulse width 60 ms). Differential pulse amperometry (DPA) was performed according to the procedure: the initial potential of the microsensor was set at 0.00 V for 1 s to clean the work electrode, then the potential was jumped from 0.00 to +0.70 V. At +0.70 V the applied potential was kept for 100 ms. After this potential delay, the potential subsequently was jumped from +0.70 to +0.80 V. At +0.80 V, the potential was maintained for 100 ms. The current measured was the difference between the values at +0.70 and +0.80 V. 2.5. Selectivity of the microsensor Before calibration with NO, microsensors were tested for selectivity against nitrite, ascorbate, dopamine (DA), norepinephrine (NE), epinephrine (E), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3acetic acid (5-HIAA), and 3,4-dihydroxyphenylacetic acid (DOPAC). The selectivity was calculated by comparing the change in current or charge measured from NO with the current measured after addition of interferences. 2.6. In vivo experiments Experiments were performed in male Sprague– Dawley rats (250–350 g). In a series, rats were
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anesthetized with an initial dose of chloral hydrate (500 mg/kg i.p.) supplemented by additional injections of 90 mg/kg of chloral hydrate every 90 min. Body temperature was monitored by a rectal probe and maintained at 37.0±1.0◦ C. Usually, 20–30 min after the end of the surgical preparation electrochemical signals was monitored in the hippocampus. l-Arg, Ach, l-NNA and SNP were diluted in PBS and were applied directly in the hippocampus. Differential pulse voltammetry or amperometry was used to monitor an analytical signal. In differential pulse voltammetry, a potential modulated with 40 mV rectangular pulses was linearly scanned from 0.30 to 1.00 V. The resulting voltammograms (alternating current–voltage plot) contained a peak due to NO oxidation. Maximum current of the peak was observed at a potential of 0.73 V versus the Ag/AgCl electrode, which was a characteristic potential for NO oxidation on copper–platinum microparticles/Nafion electrodes. Differential pulse amperometry was used primarily to verify that current measured was due to NO oxidation. In amperometric measurements, a potential modulated with 40 mV pulses was kept at a constant level of 0.70 V, and a plot of alternating current versus time was recorded. The amperometric method (with a response time better than 50 ms) provided rapid quantitative response to minute changes of NO concentration. Differential pulse voltammetry, which also provided quantitative information but required approximately 40 s for the voltammogram to be recorded, was used mainly for qualitative analysis. Differential pulse voltammograms were always recorded in the three-electrode system in order to obtain accurate and reproducible values of peak potentials. Either a two- or three-electrode system was used for the measurement of NO release in the hippocampus. The three-electrode system consisted of a NO sensor working electrode, an Au wire (0.25 mm) counter electrode and a Ag/AgCl reference electrode. The Ag/AgCl was omitted from the two-electrode system. The working electrode was stereotaxically implanted perpendicularly into the hippocampus at coordinates 5.3 mm posterior and 4.8 mm lateral to the bregma and the tip 7 mm below the dura [49]. The counter and/or reference electrodes were placed on the brain surface 1.0 mm posterior and 3.5 mm lateral to the bregma, respectively. NO was continuously
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measured for 4 h using amperometry. NO concentration was determined from the measured current by means of a calibration curve. All data were expressed as mean±S.E.
3. Results and discussion Fig. 1 presented a typical cyclic voltammogram of preparation copper/platinum microparticles modified carbon fiber microelectrode by scanning the electrode in solution containing 1.0×10−3 mol/l CuCl2 and 1.0×10−3 mol/l K2 PtCl6 between +0.70 and −0.80 V (versus SCE). Fig. 2 was the differential pulse voltammogram of Nafion/copper/platinum microparticles chemically modified microsensor in PBS with different concentration of NO. From Fig. 2, we found that the oxidation potential of NO was +0.73 V (versus Ag/AgCl). Fig. 3 was the differential pulse amperometry response of 1.0×10−7 mol/l NO in PBS. The measured current was linear with the NO concentration over the range of 8.0×10−8 –4.8×10−6 mol/l. The correlation coefficient was 0.9975, and the detection limit was 3.0×10−8 mol/l. For the NO measurement, the electrode must be sensitive to NO oxidation and it is equally important for the electrode
Fig. 1. Preparation copper–platinum microparticles modified microsensor by continuous scanning carbon fiber microelectrode in 1.0×10−3 mol/l CuCl2 and 1.0×10−3 K2 PtCl6 solution. (sweep rate: 50 mV/s).
Fig. 2. Differential pulse voltammogram of the copper–platinum microparticles modified carbon fiber microelectrode to the successive addition of NO. (a) 0; (b) 1.2; (c) 2.0; (d) 2.8; (e) 3.6 and (f) 4.4 mol/l.
to be insensitive to other electroactive molecules. Most endogenous substances are easily oxidized at a potential used for the measurement of NO. For example, the biological system contains a high extracellular concentration of ascorbic acid (AA), which
Fig. 3. Differential pulse amperometry of the copper–platinum microparticles to the successive addition of NO. Each addition of NO concentration is 1.0×10−7 mol/l.
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is oxidized at +0.32 V versus SCE. Other potential interferences in the brain, including the monoamine neurotransmitters, such as DA, NE, E, and their primary metabolites, can be oxidized at about +0.25 V. Nafion, a cation-exchange film, can prevent the diffusion of anions, such as ascorbate, uric acid, nitrite and metabolites of some neurotransmitters to the electrode thus suppressing the interference mentioned. We find that 3.2×10−5 mol/l nitrite, 3.5×10−4 mol/l ascorbate, 4.5×10−5 mol/l DA, 2.0×10−5 mol/l NE, 2.0×10−5 mol/l E, 2.5×10−5 mol/l 5-HT, 2.0×10−5 mol/l 5-HIAA, and 3.0×10−5 mol/l DOPAC showed no interference with the measurement of NO. Therefore, the microsensor can be used to monitor NO in real samples. When measurement of NO in vivo was performed, a stable background was monitored after implantation of the electrode in the hippocampus. The differential pulse voltammogram recorded at that time showed a small peak at 0.73 V, which may be attributed to basal NO concentration. However, a ratio of this peak current to the background current was lower than 3:1, which indicated that the basal NO concentration was below the sensor’s detection limit (≤3.0×10−8 mol/l). In order to observe the change of concentration of NO rapidly and precisely, DPA was performed. An increase of NO was observed immediately after 1 mmol/l l-Arg added, and a steady increase in NO concentration to a plateau of 452±12 nM (n=5) was observed after 3 min. After 10 min, NO concentration decreased below the detectable level (Fig. 4). In biological systems, there are
two types nitric oxide synthase (NOS). NO is formed directly from the guanidino nitrogen of the l-Arg by NOS through a process that consumes five electrons, and results in the formation of l-citrulline. NOS is an unusual oxidative enzyme, in that most other enzymes consume only one or two electrons for a similar function. The structures and functions of NOS have been clarified by molecular cloning of the cDNA from the brain (nNOS), endothelium (eNOS), macrophages and other type of inducible cells (iNOS). Three forms of the NOS enzyme are known: eNOS being initially isolated from the endothelium, nNOS being isolated from brain cerebellum, and iNOS being isolated from murine macrophages, human hepatocytes and chondrocytes. In hippocampus, the nitric oxide synthase is eNOS. When l-Arg was injected, the eNOS was activated by glutamate-induced increase in intracellular Ca2+ level, which in turn activated NOS via calmodulin. l-Arg was converted to NO in two successive steps of which a two-electron oxidation of l-Arg to Nω -hydroxy-l-arginine was the first step, then converted to NO and citrulline, utilizing one and half NADPH and O2 . Therefore, we observed the increase in NO concentration after l-Arg added. Pretreatment with 1 mmol/l l-NNA attenuated the l-Arg-induced increase in NO concentrations. From Fig. 5, we could observe the [NO]max was about 452±12 nM (n=5) after 1 mmol/l l-Arg added. When 1 mmol/l l-Arg and 1 mmol/l l-NNA were added simultaneously, the [NO]max was about 96±8 nM (n=5). l-NNA, as an analogue of l-Arg, was tolerated by enzyme. That is to say, l-NNA also has the binding affinity with NOS, which are competitive with l-Arg and disrupts
Fig. 4. Direct measurement of NO release from rat hippocampus. (a) Stimulated with 1 mmol/l l-Arg and (b) baseline.
Fig. 5. Effect of 1.0 mmol/l l-NNA on the NO release from rat hippocampus stimulated with 1.0 mmol/l l-Arg.
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Fig. 6. Direct measurement of NO release from rat hippocampus. (a) Stimulated with 1 mmol/l Ach and (b) baseline.
l-Arg binding. Therefore, we found that l-NNA could abolish the increase induced by l-Arg. The effect of endothelium-dependent vasorelaxant, Ach, on NO production was also performed. When 1 mmol/l Ach was added, the concentration of NO increased significantly compared to the baseline (Fig. 6). The [NO]max was about 252±10 nmol/l (n=5). When l-NNA was injected, this increase was abolished by the NOS inhibitor (Fig. 7). From Fig. 7, l-NNA 1.0 mmol/l decreased the oxidation currents of NO to 40%. The effect of the SNP, a NO donor on NO release was also examined. SNP, not only increased dramatically the NO concentration in untreated animals, but also restored the NO concentration efficiently after the NOS inhibitor l-NNA had decreased the concentration dramatically. When 1 mmol/l SNP was applied in untreated rat hippocampus, significant increase of
Fig. 8. (a) Effect of 1.0 mmol/l SNP on the NO release from rat hippocampus and (b) baseline.
Fig. 9. NO release from the rat hippocampus. (a) modified with 1 mmol/l l-Arg and 1 mmol/l l-NNA; (b) modified with 1 mmol/l l-Arg, 1 mmol/l l-NNA, and 1 mmol/l SNP.
the concentration of NO was observed (Fig. 8). In addition, when 1 mmol/l SNP, 1 mmol/l l-Arg and 1 mmol/l l-NNA were added at the same time, the concentration of NO increase is 50% compared with pretreatment with 1 mmol/l l-Arg and 1 mmol/l l-NNA (Fig. 9).
4. Conclusion
Fig. 7. Effect of 1.0 mmol/l l-NNA on the NO release from rat hippocampus stimulated with 1 mmol/l Ach.
The results show that carbon fiber electrode coated with copper–platinum microparticles and Nafion associated with DPA can be used as microsensors for NO in vitro and in vivo. The present study provides the first direct measurements of NO concentration in rat hippocampus. During the course of l-Arg added, the concentration of NO increases rapidly at the onset of l-Arg added followed by a decline of NO to control
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level immediately. When Ach is injected, we can observe the similar appearance. l-NNA, the inhibitor of NOS can abolish the increase of NO induced by l-Arg and Ach. In addition, SNP, as a NO donor, not only increase dramatically the concentration in untreated rat, but also restore the concentration efficiently after the l-NNA has decreased the NO induced by l-Arg dramatically. References [1] R.F. Furchgott, J.V. Zawadzki, Nature 286 (1980) 373. [2] I.J. Ignarro, Annu. Rev. Pharmacol. Toxicol. 30 (1990) 535. [3] R.M. Rapoport, M.B. Draznin, F. Murad, Nature 306 (1983) 174. [4] R.M. Palmer, A.G. Ferrige, S. Moncada, Nature 337 (1987) 524. [5] C. Nathan, FASEB J. 6 (1992) 3051. [6] O.G. Khatsenko, S.S. Gross, A.B. Rifkind, J.R. Vame, Proc. Natl. Acad. Sci. USA 90 (1993) 11147. [7] D. Bredt, S. Snyder, Proc. Natl. Acad. Sci. USA 87 (1990) 682. [8] U. Forstermann, H.H.H.W. Schmidt, J.S. Pollock, M. Heller, F. Murad, J. Cardiovasc. Pharmacol 17 (Suppl.) (1991) 557. [9] J.S. Pollock, U. Forstermann, J.A. Mitchell, T.D. Warner, H.H.H.W. Schmidt, M. Nakane, F. Murad, Proc. Natl. Acad. Sci. USA 88 (1991) 10480. [10] D. Stuehr, H. Cho, N. Kwon, M. Weise, C. Nathans, Proc. Natl. Acad. Sci. USA 88 (1991) 7773. [11] F.M. Faraci, Am. J. Physiol 261 (1991) H1038. [12] F.M. Faraci, D.D. Heistad, J. Cereb. Blood Flow Metabol. 12 (1992) 500. [13] E. Kozniewska, M. Oscka, T. Stys, J. Cereb. Blood Flow Metabol. 12 (1992) 311. [14] C. Iadecola, Proc. Natl. Acad. Sci. USA 89 (1992) 3913. [15] D.L. Granger, J.B. Hibbs Jr., J.R. Perfect, D.T. Durack, J. Clin. Invest 85 (1990) 264. [16] E.M. Schuman, D.V. Madison, Science 263 (1994) 532. [17] B.L. McNaughton, J. Sher, G. Rao, T.C. Foster, C.A. Barnes, Proc. Natl. Acad. Sci. USA 91 (1994) 4830. [18] R.G. Kilbourn, J. Griffith, J. Natl. Cancer Inst. 84 (1992) 827. [19] R.M. Stefanovic, J. Stadler, C.H. Evans, Arthritis Rheum. 36 (1993) 1036. [20] H. Yamamoto, J. Bossaller, J. Cartwright, P.D. Henry, J. Clin. Invest. 81 (1988) 1752. [21] J.A. Corbett, J.R. Lancaster, M.A. Sweetland, M.L. Macdaniel, J. Biol. Chem. 266 (1991) 21351. [22] J.R. Lancaster, J.M. Langrehr, H.A. Bergonia, N. Murase, R.L. Simons, R.A. Hoffman, J. Biol. Chem. 267 (1992) 10994.
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Direct measurement of nitric oxide release from the rat hippocampus Yuezhong Xian, Wen Zhang, Jian Xue, Xiangyang Ying, Litong Jin∗ Department of Chemistry, East China Normal University, Shanghai 200062, China Received 16 November 1999; received in revised form 29 February 2000; accepted 16 March 2000
Abstract A novel, sensitive, selective and reproducible nitric oxide (NO) microsensor is developed for direct measurement of NO in vitro and in vivo. Using the microsensor, the first direct measurements NO release from the rat hippocampus are made. NO levels are increased by stimulated with l-arginine and acetylcholine. l–Nω -nitro-arginine, a NO synthase inhibitor, decreases the NO increase induced by l-arginine and acetylcholine. The effect of sodium nitroprusside, a NO donor, is also performed. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Nitric oxide; Hippocampus; Microsensor
1. Introduction In recently years, nitric oxide radical (NO) has been increasingly recognized as an important intraand inter-cellular messenger molecule, which plays a physiological role in vascular relaxation, platelet activation, neurotransmission, and immune response [1–6]. This bioradical, a potent vasodilator or endothelium-derived relaxing factor, is synthesized from l-arginine (l-Arg) and molecular oxygen in various cell types by three distinct isoforms of the enzyme NO synthase (NOS). Two of these, constitutive and calcium-dependent isoenzymes (cNOS), which have been observed in neuronal and endothelial cells produce a small amount of NO when calcium/calmodulin binding permits electron transfer from NADPH via flavin groups to an active site [7–9], whereas the other inducible, calcium-independent isoform (iNOS) [10] in macrophages, chondrocytes, and hepatocytes initiated by various immunological stimuli such as ∗ Corresponding author. Fax: +86-216245-1876. E-mail address: [email protected] (L. Jin)
endotoxin, microbes, and cytokines synthesizes high levels of NO. NO plays multifaceted and important roles in the brain. It is a neurotransmitter and a free radical, and it has been implicated in the regulation of cerebral blood flow and inflammation [11–15]. In the hippocampus, independent induction of long-term potentiation (LTP) or depression (LTD) has been reported [16,17]. The elevated concentration of this radical by NOS has been implicated in the pathogenesis of several disease states such as septic shock [18], arthritis [19], atherosclerosis [20], diabetes mellitus [21], graft rejection [22], and ischemia/reperfusion injury [23]. Thus, the precise measurements of this bioradical and its metabolites in the circulation are of increasing clinical importance. NO is a small, highly reactive, readily diffusible gas that can combine with most biologically active molecules. Thus, the measurement of NO is very difficulty. Over the past years, efforts have been made to directly and indirectly measure NO release in biological systems. In general, the measurement methods include: (a) NO is ‘trapped’ by nitroso compounds, or reduced hemoglobin, forming a stable
0003-2670/00/$ – see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 0 ) 0 0 8 6 3 - 1
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adduct that is detected by electron paramagnetic resonance (EPR) [24,25]; (b) NO oxidizes reduced hemoglobin to methemoglobin, which is detected by spectrophotometry [26,27]; (c) NO interacts with ozone producing light, ‘chemiluminescence’ [28,29]; (d) NO is measured with NO-fluorescence probes, fiber-optic NO sensors, and chemical assay of NO cofactors using microseparation [30–35] and (e) NO is monitored by electrochemical methods through chemically modified microsensors [36–44]. Among those techniques, electrochemical methods are most advantageous because they are simple, relatively fast, sensitive, and can be applied on-line measurement. In addition, the explosive growth in the fabrication of ultramicroelectrodes [45] strongly stimulates the use of electrochemical sensors for in vivo NO detection. NO microsensor can be fabricated to extremely small dimensions (m or nm in diameter) and thus are ideal for insertion directly into the biological systems with minimal damages. In addition, chemically modified microsensors can electrocatalytically oxidize or reduce NO, therefore, diminishing the interference induced by some other substances in biological systems. Owing to those advantages, the chemically modified microsensor is widely used real-time and in vivo measurement NO. Although NO is a subject of great interest in brain research, to our knowledge, direct in vivo measurements of NO in hippocampus using electrochemical microsensor have not been performed. Here, we demonstrate the ability to study NO in vivo using an novel chemically modified NO microsensor, which is prepared by electrodeposition copper–platinum microparticles on carbon fiber (d=15 m) surface. It is used to measure the NO release from rat hippocampus modified by l-Arg, acetylcholine (Ach), l–Nω -nitro-arginine (l-NNA) and sodium nitroprusside (SNP).
2. Experimental 2.1. Chemicals NO standards were prepared by serial dilution of a saturated NO solution. Preparation of the saturated NO solutions involved meticulous exclusion of O2 , as NO was rapidly destroyed by O2 . To produce the saturated
NO solution (at 20◦ C, 2.05 mM NO) [46], deionized water solution (2 ml) was bubbled with nitrogen for 30 min to remove oxygen. Then the solution was bubbled with pure NO gas (Aldrich) for 30 min and kept under NO atmosphere until use. The concentration of saturated NO is about 2.00 mM. Standards were made fresh for each experiment and kept in a glass flask with a rubber septum. Dilutions of the saturated solution were made using deoxygenated water samples. Phosphate-buffered saline (PBS) containing 137 mmol/l NaCl, 2.7 mmol/l KCl, 8.0 mmol/l Na2 HPO4 and 1.5 mmol/l KH2 PO4 was prepared and adjusted to pH7.4. CuCl2 (Shanghai reagent factory, Shanghai), K2 PtCl6 (Chengdu reagent factory, Chengdu), l-Arg (Sigma, USA), l-NNA (Sigma, USA), Ach (Sigma, USA), SNP (Katayama Chemical, Japan) and other reagents were analytical reagents. 2.2. Electrochemical instrument Electrochemical experiments were performed on a CHI-832 electrochemical analyzer (CH Instruments Inc., Cordova, USA) at room temperature (25±2◦ C) in a three compartment cell with a saturated calomel reference electrode (SCE), an Au wire auxiliary electrode and a copper–platinum microparticles modified carbon fiber microsensor (15 m in diameter). 2.3. Preparation carbon fiber microelectrode and electrochemical procedures Carbon fiber microelectrodes were built according to previous description [47] with modifications. They consisted of one carbon fiber, 15 m diameter and 450 m length, protruding from a glass pipette. Electrical contact with the carbon fiber was assured either by a polyester resin mixed with carbon powder [48] and connection to the polarograph was assured by a copper wore, or a silver wire was glued to the carbon fiber by a silver conductive paint thus assuring the connection to the polarograph. In this case, the extremity of the pipette was filled with an epoxy resin in order to assure tightness. The carbon fiber were treated in PBS (pH 7.4), by passing down the electrodes triangular pulses (80 Hz) in two successive sequence of 20 s duration at +2.85 and +1.6 V, respectively. Electrode-
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position of copper–platinum microparticles and Nafion (5% solution, Aldrich) were successively achieved by immersing the carbon fiber microelectrode in a few microliters (4–5 l) of each solution placed in small loop of platinum wire (2 mm diameter) connected to the auxiliary and reference plugs of the Biopilse. 2.4. In vitro calibration Prior to NO measurements, the modified carbon fiber electrodes were placed in PBS, and the potential was cycled between +0.70 and −0.80 V at a rate of 50 mV/s until a steady response was obtained. For NO determination, a diluted NO solution was subsequently added with a gas-tight syringe. The current due to the electrocatalytic oxidation of NO by the microsensor was recorded after each addition. Differential pulse voltammetry (DPV) was performed with a potential sweep rate of 5 mV/s and 5 pulses/s (pulse height 50 mV, pulse width 60 ms). Differential pulse amperometry (DPA) was performed according to the procedure: the initial potential of the microsensor was set at 0.00 V for 1 s to clean the work electrode, then the potential was jumped from 0.00 to +0.70 V. At +0.70 V the applied potential was kept for 100 ms. After this potential delay, the potential subsequently was jumped from +0.70 to +0.80 V. At +0.80 V, the potential was maintained for 100 ms. The current measured was the difference between the values at +0.70 and +0.80 V. 2.5. Selectivity of the microsensor Before calibration with NO, microsensors were tested for selectivity against nitrite, ascorbate, dopamine (DA), norepinephrine (NE), epinephrine (E), 5-hydroxytryptamine (5-HT), 5-hydroxyindole-3acetic acid (5-HIAA), and 3,4-dihydroxyphenylacetic acid (DOPAC). The selectivity was calculated by comparing the change in current or charge measured from NO with the current measured after addition of interferences. 2.6. In vivo experiments Experiments were performed in male Sprague– Dawley rats (250–350 g). In a series, rats were
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anesthetized with an initial dose of chloral hydrate (500 mg/kg i.p.) supplemented by additional injections of 90 mg/kg of chloral hydrate every 90 min. Body temperature was monitored by a rectal probe and maintained at 37.0±1.0◦ C. Usually, 20–30 min after the end of the surgical preparation electrochemical signals was monitored in the hippocampus. l-Arg, Ach, l-NNA and SNP were diluted in PBS and were applied directly in the hippocampus. Differential pulse voltammetry or amperometry was used to monitor an analytical signal. In differential pulse voltammetry, a potential modulated with 40 mV rectangular pulses was linearly scanned from 0.30 to 1.00 V. The resulting voltammograms (alternating current–voltage plot) contained a peak due to NO oxidation. Maximum current of the peak was observed at a potential of 0.73 V versus the Ag/AgCl electrode, which was a characteristic potential for NO oxidation on copper–platinum microparticles/Nafion electrodes. Differential pulse amperometry was used primarily to verify that current measured was due to NO oxidation. In amperometric measurements, a potential modulated with 40 mV pulses was kept at a constant level of 0.70 V, and a plot of alternating current versus time was recorded. The amperometric method (with a response time better than 50 ms) provided rapid quantitative response to minute changes of NO concentration. Differential pulse voltammetry, which also provided quantitative information but required approximately 40 s for the voltammogram to be recorded, was used mainly for qualitative analysis. Differential pulse voltammograms were always recorded in the three-electrode system in order to obtain accurate and reproducible values of peak potentials. Either a two- or three-electrode system was used for the measurement of NO release in the hippocampus. The three-electrode system consisted of a NO sensor working electrode, an Au wire (0.25 mm) counter electrode and a Ag/AgCl reference electrode. The Ag/AgCl was omitted from the two-electrode system. The working electrode was stereotaxically implanted perpendicularly into the hippocampus at coordinates 5.3 mm posterior and 4.8 mm lateral to the bregma and the tip 7 mm below the dura [49]. The counter and/or reference electrodes were placed on the brain surface 1.0 mm posterior and 3.5 mm lateral to the bregma, respectively. NO was continuously
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measured for 4 h using amperometry. NO concentration was determined from the measured current by means of a calibration curve. All data were expressed as mean±S.E.
3. Results and discussion Fig. 1 presented a typical cyclic voltammogram of preparation copper/platinum microparticles modified carbon fiber microelectrode by scanning the electrode in solution containing 1.0×10−3 mol/l CuCl2 and 1.0×10−3 mol/l K2 PtCl6 between +0.70 and −0.80 V (versus SCE). Fig. 2 was the differential pulse voltammogram of Nafion/copper/platinum microparticles chemically modified microsensor in PBS with different concentration of NO. From Fig. 2, we found that the oxidation potential of NO was +0.73 V (versus Ag/AgCl). Fig. 3 was the differential pulse amperometry response of 1.0×10−7 mol/l NO in PBS. The measured current was linear with the NO concentration over the range of 8.0×10−8 –4.8×10−6 mol/l. The correlation coefficient was 0.9975, and the detection limit was 3.0×10−8 mol/l. For the NO measurement, the electrode must be sensitive to NO oxidation and it is equally important for the electrode
Fig. 1. Preparation copper–platinum microparticles modified microsensor by continuous scanning carbon fiber microelectrode in 1.0×10−3 mol/l CuCl2 and 1.0×10−3 K2 PtCl6 solution. (sweep rate: 50 mV/s).
Fig. 2. Differential pulse voltammogram of the copper–platinum microparticles modified carbon fiber microelectrode to the successive addition of NO. (a) 0; (b) 1.2; (c) 2.0; (d) 2.8; (e) 3.6 and (f) 4.4 mol/l.
to be insensitive to other electroactive molecules. Most endogenous substances are easily oxidized at a potential used for the measurement of NO. For example, the biological system contains a high extracellular concentration of ascorbic acid (AA), which
Fig. 3. Differential pulse amperometry of the copper–platinum microparticles to the successive addition of NO. Each addition of NO concentration is 1.0×10−7 mol/l.
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is oxidized at +0.32 V versus SCE. Other potential interferences in the brain, including the monoamine neurotransmitters, such as DA, NE, E, and their primary metabolites, can be oxidized at about +0.25 V. Nafion, a cation-exchange film, can prevent the diffusion of anions, such as ascorbate, uric acid, nitrite and metabolites of some neurotransmitters to the electrode thus suppressing the interference mentioned. We find that 3.2×10−5 mol/l nitrite, 3.5×10−4 mol/l ascorbate, 4.5×10−5 mol/l DA, 2.0×10−5 mol/l NE, 2.0×10−5 mol/l E, 2.5×10−5 mol/l 5-HT, 2.0×10−5 mol/l 5-HIAA, and 3.0×10−5 mol/l DOPAC showed no interference with the measurement of NO. Therefore, the microsensor can be used to monitor NO in real samples. When measurement of NO in vivo was performed, a stable background was monitored after implantation of the electrode in the hippocampus. The differential pulse voltammogram recorded at that time showed a small peak at 0.73 V, which may be attributed to basal NO concentration. However, a ratio of this peak current to the background current was lower than 3:1, which indicated that the basal NO concentration was below the sensor’s detection limit (≤3.0×10−8 mol/l). In order to observe the change of concentration of NO rapidly and precisely, DPA was performed. An increase of NO was observed immediately after 1 mmol/l l-Arg added, and a steady increase in NO concentration to a plateau of 452±12 nM (n=5) was observed after 3 min. After 10 min, NO concentration decreased below the detectable level (Fig. 4). In biological systems, there are
two types nitric oxide synthase (NOS). NO is formed directly from the guanidino nitrogen of the l-Arg by NOS through a process that consumes five electrons, and results in the formation of l-citrulline. NOS is an unusual oxidative enzyme, in that most other enzymes consume only one or two electrons for a similar function. The structures and functions of NOS have been clarified by molecular cloning of the cDNA from the brain (nNOS), endothelium (eNOS), macrophages and other type of inducible cells (iNOS). Three forms of the NOS enzyme are known: eNOS being initially isolated from the endothelium, nNOS being isolated from brain cerebellum, and iNOS being isolated from murine macrophages, human hepatocytes and chondrocytes. In hippocampus, the nitric oxide synthase is eNOS. When l-Arg was injected, the eNOS was activated by glutamate-induced increase in intracellular Ca2+ level, which in turn activated NOS via calmodulin. l-Arg was converted to NO in two successive steps of which a two-electron oxidation of l-Arg to Nω -hydroxy-l-arginine was the first step, then converted to NO and citrulline, utilizing one and half NADPH and O2 . Therefore, we observed the increase in NO concentration after l-Arg added. Pretreatment with 1 mmol/l l-NNA attenuated the l-Arg-induced increase in NO concentrations. From Fig. 5, we could observe the [NO]max was about 452±12 nM (n=5) after 1 mmol/l l-Arg added. When 1 mmol/l l-Arg and 1 mmol/l l-NNA were added simultaneously, the [NO]max was about 96±8 nM (n=5). l-NNA, as an analogue of l-Arg, was tolerated by enzyme. That is to say, l-NNA also has the binding affinity with NOS, which are competitive with l-Arg and disrupts
Fig. 4. Direct measurement of NO release from rat hippocampus. (a) Stimulated with 1 mmol/l l-Arg and (b) baseline.
Fig. 5. Effect of 1.0 mmol/l l-NNA on the NO release from rat hippocampus stimulated with 1.0 mmol/l l-Arg.
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Fig. 6. Direct measurement of NO release from rat hippocampus. (a) Stimulated with 1 mmol/l Ach and (b) baseline.
l-Arg binding. Therefore, we found that l-NNA could abolish the increase induced by l-Arg. The effect of endothelium-dependent vasorelaxant, Ach, on NO production was also performed. When 1 mmol/l Ach was added, the concentration of NO increased significantly compared to the baseline (Fig. 6). The [NO]max was about 252±10 nmol/l (n=5). When l-NNA was injected, this increase was abolished by the NOS inhibitor (Fig. 7). From Fig. 7, l-NNA 1.0 mmol/l decreased the oxidation currents of NO to 40%. The effect of the SNP, a NO donor on NO release was also examined. SNP, not only increased dramatically the NO concentration in untreated animals, but also restored the NO concentration efficiently after the NOS inhibitor l-NNA had decreased the concentration dramatically. When 1 mmol/l SNP was applied in untreated rat hippocampus, significant increase of
Fig. 8. (a) Effect of 1.0 mmol/l SNP on the NO release from rat hippocampus and (b) baseline.
Fig. 9. NO release from the rat hippocampus. (a) modified with 1 mmol/l l-Arg and 1 mmol/l l-NNA; (b) modified with 1 mmol/l l-Arg, 1 mmol/l l-NNA, and 1 mmol/l SNP.
the concentration of NO was observed (Fig. 8). In addition, when 1 mmol/l SNP, 1 mmol/l l-Arg and 1 mmol/l l-NNA were added at the same time, the concentration of NO increase is 50% compared with pretreatment with 1 mmol/l l-Arg and 1 mmol/l l-NNA (Fig. 9).
4. Conclusion
Fig. 7. Effect of 1.0 mmol/l l-NNA on the NO release from rat hippocampus stimulated with 1 mmol/l Ach.
The results show that carbon fiber electrode coated with copper–platinum microparticles and Nafion associated with DPA can be used as microsensors for NO in vitro and in vivo. The present study provides the first direct measurements of NO concentration in rat hippocampus. During the course of l-Arg added, the concentration of NO increases rapidly at the onset of l-Arg added followed by a decline of NO to control
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