Address of Corresponding Author

J Vasc Res 2006;43:70-85 (DOI: 10.1159/000089547)

 Outline

• Key Words
• Abstract
• Introduction
• Microsensor Construction and Amperometry
• NO Microsensor Performance
• Calibration and Detection Limit
• Specificity for NO
• Quality Testing of NO Microsensors
• Applications
• NO Measurements in Cell Suspensions
• NO Measurements in Cell Culture
• The Isolated Arterial Segment
• In vivo Measurements of NO Concentration
• Evidence that Microsensors Measure Physiological NO Concentration
• Diffusion of NO from the Site of Production to the Site of Action
• Evidence that Myograph Studies Estimate Intramural NO Concentration
• Conclusions
• Acknowledgements
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Nitric oxide microsensor
• L-arginine pathway
• Human vein endothelial cells

  Abstract

Nitric oxide (NO) plays an important role in the regulation of blood flow. Pharmacological tools and a series of other techniques have been developed for studying the NO/L-arginine pathway, but it has proved difficult to make a quantitative link between effect and tissue NO concentration. NO microsensors have been applied with success for the measurement of NO in suspensions of mitochondria and cells, such as platelets and leukocytes, and in cell cultures, which together with other interventions or measurements are particularly useful for the examination of cell signalling related to the NO/L-arginine pathway. In isolated vascular segments, studies using the NO microsensor have defined the relationship between NO concentration and relaxation and revealed residual NO release in the presence of NO synthase inhibitors. Moreover, simultaneous measurements of NO concentration and vasorelaxation in isometric preparations have shown that agonist-induced relaxation is L-arginine dependent and NO release is reduced in hypertension. By placing NO microsensors in catheters, it is possible to measure NO in the living animal and man. This approach has been applied for the measurements of NO concentration in relation to increases in flow, erection, in conditions of hypoxia, and in endotoxemia. However, further methodological development of NO microsensors is necessary to avoid the influence of changes in temperature, pH and oxygen on the measurements.

Copyright © 2006 S. Karger AG, Basel

 Introduction

The evidence for involvement of nitric oxide (NO) in certain physiological events is in many cases based on the use of pharmacological tools which inhibit the formation of NO by interaction either with NO synthase (NOS), scavenging of NO, inhibition of guanylyl cyclase, and recently, also by inhibition of smooth muscle phosphodiesterase, as reviewed by Andrews et al. [1]. These kinds of experiments yield valuable indications whether the NO pathway plays a role in vasodilation or neurogenic responses, and a clear advantage of most of the pharmacological tools for investigation of the NO pathway is that they can easily be applied under all kinds of physiological conditions. However, these pharmacological tools have other actions in addition to inhibition of NOS [2,3,4,5,6,7,8,9]. Another approach is the use of genetically modified animal strains including NOS gene deletion or the determination of NOS expression and activity. However, knockout of the NOS gene does not always exclude formation of NO by other isoforms or splice variants [10], and gene deletion may be accompanied by compensatory changes in other components of the pathway. Whilst all these methods have their place, only the measurement of the NO concentration allows direct conclusions relating the molecule to physiological events.

There are several techniques which have been developed to determine NO release such as bioassays [11], alteration in haemoglobin spectra and fluorescent dyes such as diaminofluorescein [12], Griess reaction spectrophotometry [13], ozone chemiluminescence [14], electron paramagnetic resonance spectroscopy [15], and measurement of NOS enzyme activity [13] or NOS activity estimated by determining ¹⁵N-arginine to ¹⁵N-citrulline labelling [16]. The amperometric NO sensor was developed to measure NO concentration at the low levels that are active physiologically in the cardiovascular system, and hence, to correlate the NO output with the corresponding physiological response. The present review focuses on the application of NO-sensitive microelectrodes for the investigation of NO release in the cardiovascular system, including the underlying principles and construction, in so far as these determine their applicability, preconditions to obtain optimal data, the range of applications for which they are useful, and advances in the understanding of physiology, drug action and disease that have emerged from the use of NO microsensors.

 Microsensor Construction and Amperometry

The first successful NO sensor was a miniature solid-state probe, based on the Clark electrode for the detection of oxygen, where the analyte is oxidized at a platinum electrode protected by a selectively permeable membrane [17], and this principle is applied in the gas-permeable NO probes (typically 2 mm in diameter) [18]. Malinski and Taha [19] developed a much smaller microsensor based on a carbon fibre with a double coating comprising polymerized metalloporphyrin and an outer layer of Nafion. Most of the published work with NO sensors uses one of these two types or more recent modifications of them (table 1).

Table 1. Technical parameters of the NO sensor and requirements for the best performance

The advantage of the coated carbon fibre sensor is that it can be small in size and brought close to the cells of interest where NO concentration is expected to be greatest. The original sensor was designed to record NO release from single cells and was made from a 7-µm-thick carbon fibre that was etched down to 0.5 µm in diameter at the tip [19]. For many applications, a slightly larger probe is preferred, which can either be made by combining several (3-10) carbon fibres giving an overall diameter of up to 30 µm [20] or by coating a carbon fibre that is 30 or 200 µm in diameter [21].

The original coated fibre NO sensor had a dual coating consisting of a polymerized nickel porphyrin (which is electroconductive) and an outer layer of Nafion. The original rationale for using nickel porphyrin polymer was that it would be a specific electrocatalyst for NO [19]. However, it is uncertain whether electrocatalysis takes place, and the improved sensitivity may be due to modification of the surface properties of the carbon fibre [22, 23]. The porphyrin layer is applied by electropolymerization from a suitable monomer, and thus, its thickness and rate of deposition can be accurately controlled. The outer layer of Nafion, which has multiple acid residues and cation exchange properties, improves sensitivity and is also impermeable to anions; thus, it excludes nitrite and certain other potential contaminants that are oxidized at the same electrode potential as NO.

A series of integrated carbon fibre-based NO sensors are commercially available. These have a dual coating consisting of Nafion covered by a proprietary membrane (composition not revealed by the manufacturer). The proprietary membrane excludes cations and is claimed to increase sensitivity to NO [21]. These sensors are available in a range of sizes, from 200 to 0.1 µm tip diameter. The quoted sensitivity and detection limits are similar for the different sizes of sensors. The reference electrode is a silver layer applied to the sensor housing to make a very compact combination probe [21, 24]. An NO sensor fabricated from layers of carbon, platinum and silver deposited on a silicon chip substrate was reported to have improved sensitivity for NO, with a detection limit of 0.3 nM [25].

Operation of the NO microsensor requires setting the working electrode to a suitable poise potential relative to a reference electrode. The exact point of the optimal poise potential depends on the sensor construction and composition of the coating (table 1). In direct amperometry, the poise potential is set to its optimum and the generated current is monitored continuously. Differential amperometry involves rapid switching between two set potentials and measuring of the current difference, thus excluding baseline drift and increasing sensitivity. Voltammetry involves sweeping the electrode through a range of potentials to determine a voltammogram which would typically include the optimal poise potential. The voltammogram provides information about the substance that is being oxidized at the working electrode and can be used to confirm the identity of NO as previously described [22,26,27,28]. An alternative operating principle has been developed for measuring a concentration gradient of NO by switching the position of the sensor tip through a distance of 10 µm at a frequency of 0.3 Hz and measuring the difference in signal at these two points [29].

Several papers have described modifications of the original coated carbon sensor in an attempt to increase sensitivity and stability. Optimal sensor performance requires a stable, insoluble adherent film with suitable thickness, hydrophobicity and permeability properties. However, in general, the transport properties of polymers cannot be determined a priori, and this development work has been mostly done by trial and error. There has been extensive testing of novel electroactive polymeric coatings, selectivity barriers, and electrode substrates. Coatings made of o-phenylenediamine or m-phenylenediamine plus m-phenylenediol had improved selectivity compared with nickel porphyrin [23, 30]. Other successful coatings include nickel-sulphonated phthalocyanine [31] and Nafion and cellulose acetate [32]. An extensive compilation of published data from different laboratories compares many different types of coating and the resulting sensitivity and properties of the sensor [33].

There are several publications that have used a microsensor constructed from a platinum/iridium electrode coated with potassium chloride, nitrocellulose laquer and silicone [34, 35]. One study reported a linear calibration of this sensor with NO, and found that NO output increased with increasing perfusion rate when the sensor was inserted into the wall of a cannulated dog femoral artery perfused with Krebs buffer [36]. However, it has also been reported that this sensor did not give a voltammetric peak in response to NO and could not be calibrated with NO solution in water, but only with the NO donor S-nitroso-acetylpenicillamine. Moreover, the sensor was found to respond to nitrite [37]. Thus, there is doubt about the usefulness of this particular sensor.

An electrochemical NO sensor with a hydrogel coating has been developed for the measurement of NO in the gas phase, although its sensitivity and specificity were not reported [38]. A reticulated vitreous carbon and Nafion membrane-covered electrode, constructed for measurement in the gas phase, was reported to detect NO at 6 ppb [39].

For further information about specific types of NO sensors and how they are constructed, readers are referred to previous reviews [18, 22, 27, 30, 33,40,41,42]. The authors encourage those who have the expertise to develop their own NO sensors to improve on the performance and produce less expensive and more specific NO sensors than those commercially available.

 NO Microsensor Performance

 Calibration and Detection Limit

The microsensors are measuring very small currents and, in physiological applications, are usually working at the lower limit of detection. In practice, major limitations arising are noise, drift and temperature sensitivity (fig. 1, table 1). Noise and drift can be reduced by proper shielding and grounding of the equipment. However, individual NO sensors vary from one to another in their sensitivity and noise, even when made according to a standard fabrication protocol. Such variations are probably caused by small differences in the sensor area, surface electrochemical properties and coating thickness (table 1). Moreover, sensor responsiveness changes significantly from one day to the next when the same sensor is used again [5]. Consequently, the calibration of the NO has to be carried out on a daily basis and in conditions that closely match the experimental recording conditions. A solution of NO gas is the authentic reference standard; however, its manufacture requires considerable care, involving purging with argon and passing the NO gas through a strong base to remove higher oxides of nitrogen [5, 26]. Calibration with NO solution has the advantage that the NO will be subject to the same rate of destruction during calibration and measurement, and thus, the calibration should in theory compensate where changes in experimental conditions would change NO breakdown. The NO donor S-nitrosoacetyl penicillamine, in the presence of copper as a catalyst, provides a steady release of NO. This is a simple and convenient calibration procedure and is claimed to release NO quantitatively [18]; however, a rigorous quantitative comparison has not been made. NO can be generated stoichiometrically by the reduction of nitrite with iodide [18, 41], but this reaction requires a strongly acidic environment which is harmful to certain sensor coatings.

Fig. 1. NO microsensor (ISONOP30 connected to an NOMKII amplifier from World Precision Instrument) calibrated with NO and response to sodium nitrite (NaNO2) (a), and sensitivity to changes in oxygenation of organ bath (b), temperature (c), and pH (d) [Stankevicius et al., unpubl. data].

 Specificity for NO

It is important that microelectrodes are selective for NO and able to distinguish NO from the end products nitrite and nitrate. Therefore, it is essential to evaluate whether the electrodes oxidize nitrite (fig. 1), which was shown to be the case for the iridium alloy electrode [37]. Depending on the construction of the microsensor and the concentration, different substances can be oxidized at the carbon fibre, and hence, interfere with the NO measurements (table 1). Analytical selectivity of the NO sensors is primarily determined by the selective permeability and thickness of their polymer coatings. However, this is a double-edged sword, as increasing the polymer coating tends to increase the selectivity for NO, but impairs response time and sensitivity of NO in biological fluid.

 Quality Testing of NO Microsensors

From our experience with the 30-µm diameter polymer-coated carbon microsensor [5, 43], the characteristics vary significantly from one sensor to another and can change on a daily basis. Therefore, each sensor must pass a series of quality tests before being considered suitable for use (table 1). Each sensor on every experimental day should be (1) tested for stability and low noise on the voltage output and (2) calibrated with NO solution in a stirred closed vial using the same physiological salt solution, temperature and oxygenation as in the experiment. It is worth trying to recoat sensors failing any of these tests. A sensor from each batch of sensors should be tested for lack of response to nitrite and all the drugs and vehicles that are to be employed in the experiments. In between experiments, the microsensor should be rinsed and then stored dry. With careful treatment, the polymer-coated microsensor may be useable for up to 3-5 days (8 h use per day), while electrode lifetime was reported to be 10 h for the porphyrinic microsensor [19]. We found it necessary to have a considerable stock of sensors in order to guarantee that there will be a useable sensor for the experiment.

In summary, improvements are still needed in sensor design to increase reliability and stability. Nevertheless, provided NO sensors pass rigorous quality testing, they fulfill the main requirements for an ideal method of detecting NO under in vitro conditions (table 1). However, questions remain about the validity of data obtained in vivo, given the lack of quality testing for stability, sensitivity and specificity under in vivo conditions.

 Applications

 NO Measurements in Cell Suspensions

NO formation by platelets and leukocytes, which exist physiologically as dispersed cells within an aqueous environment, can be studied as cell suspension with a microsensor dipped into the solution. Another widely used technique is the measurement of cyclic GMP concentration; however, this does not necessarily reflect the NO concentration, since drugs and disease can alter cyclic GMP regulation, for example by changing phosphodiesterase [e.g., ref. [44, 45].

Platelets (suspension of washed platelets) were found to generate NO in response to collagen (approximately 2 nM NO), ADP, thrombin, and a thromboxane mimetic [35, 46, 47]. NO formation occurred after platelet adhesion had reached a plateau and was able to prevent subsequent recruitment of platelets added at this time [47]. NO formation was prevented by N^G-monomethyl-L-arginine [43] in endothelial NOS -/- mice [48] and was reduced in platelets from smokers [43]. Losartan and other angiotensin receptor antagonists were found to stimulate NO release from washed rat platelets, which correlated with inhibition of aggregation [49].

Macrophages activated with interferon- to have a large output of NO were plated as single cells or small cell groups. The NO concentration decreased with distance, as would be expected for diffusional spread in three dimensions. Significant NO concentration was detected at a distance of 50 µm for single cells and 400 µm for cell groups [29]. Thus, inflammatory cells present within or adjacent to the artery wall would be able to deliver significant quantities of NO, and perhaps also toxic metabolites of NO, through the depth of the artery wall.

The addition of leukocytes to a platelet suspension (with a ratio of 100 platelets:1 leukocyte) was found to increase thrombin-stimulated platelet aggregation, and there was also a reduction in NO production. However, at the much greater ratio of 5 platelets:1 leukocyte, there was inhibition of platelet aggregation accompanied by increased NO production [50].

A further application of this technique is to study NO concentrations in suspensions of subcellular organelles. Thus, it was demonstrated that mitochondrial NOS, which has homology to neuronal NOS, is functional and able to generate significant quantities of NO in isolated cardiac myocytes [51] and altered in pathophysiological conditions [52, 53].

In summary, measurements in cell suspension have the advantage that cell number can be independently varied in the cuvette, and physiological responses such as platelet aggregation can be studied simultaneously with NO measurement. It is a possible disadvantage that the sensor surface may activate or modify the activation of platelets or leukocytes.

 NO Measurements in Cell Culture

Cultured cells are particularly appropriate for the examination of cell signalling, proliferation and migration, and offer the opportunity to study human cells or transfected cells. The measurement of NO₂ in the culture medium is a widely used technique; however, this does not necessarily faithfully reflect NO availability [e.g., ref. [54, 55]. Moreover, the impact of NO on cell signalling encompasses pathways that are chemically distinct from activation of guanylate cyclase, including nitrosylation of the sarcoplasmic reticulum calcium pump [56], and the ability of NO to interact with alternative targets will depend on its concentration. NO microsensors in cell culture studies enable the link between NO availability and cell signalling to be studied (fig. 2).

Fig. 2. Measurement of NO with an NO microsensor placed above a primary culture of HUVEC on a gelatine-covered slide and stimulated with histamine in the absence (a) and the presence (b) of an inhibitor of NOS, i.e. ADMA (300 µM) [Stankevicius et al., unpubl. data].

NO release from cultured endothelial cells, grown as a confluent monolayer, has been studied by positioning the original porphyrinic microsensor 5 µm above the endothelial cell surface by means of a micromanipulator. Application of calcimycin, acetylcholine and bradykinin from a nanoinjector - also located 5 µm from the human umbilical vein endothelial cell (HUVEC) surface - produced a brief spike of NO that was back to baseline in 10-20 s [57, 58], and cerivastatin gave a wave-like release of NO that lasted 3 min [58]. Application of drugs to the bulk solution gives a more prolonged NO response; thus, in confluent cells where a porphyrinic microsensor was positioned 10-15 µm above the endothelial surface, histamine and thrombin gave an NO signal lasting approximately 1 min [46]. This response is still very brief in contrast to the continuous output of NO that endothelial cells can generate in intact artery preparations. The decay of the NO signal did not appear to be caused by local destruction of NO, and it was concluded that in cultured endothelial cells, histamine and thrombin initiate a brief pulse of NO. However, NOS in these cells remains capable of generating sustained NO release, as was observed on administration of thapsigargin [42]. It appears that the NO release characteristics of endothelial cells change when they are grown in culture, and this point will have to be considered when interpreting such data.

The NO concentration gradient has a more rapid fall-off away from isolated single cells compared with continuous cell layers. Adherent single endocardial cells had a microsensor placed 10 µm from the cell surface. Calcimycin gave a spike of NO with a peak of 520 nM and a duration of approximately 10 s. Testing at various distances showed that the NO signal declined rapidly (to half maximum at 10 µm) [59]. Similar data were obtained with rat ventricular myocytes which were dissociated and then allowed to settle on glass coverslips (NO microsensor positioned on the cell surface). NO was released in response to calcimycin to give a transient spike of approximately 900 nM at the surface, lasting about 2 s. The NO spike was diminished to half at approximately 10 µm away from the myocyte [60]. The ventricular myocytes also released NO in response to noradrenaline (enhanced by 3-isobutyl-1-methylxanthine) and by dibutyryl cyclic AMP [60]. These data suggest that positioning the microsensor at a distance of 10 µm from a confluent endothelial cell layer should measure NO close to the value at the cell surface.

A puzzling point that emerges when comparing papers that measure NO release from cultured cells using the porphyrinic microsensor positioned 5-15 µm above the endothelial surface is the variability in the concentration of NO reported. Thus, some studies report relatively low NO signals, e.g., in HUVEC, histamine at maximal concentration generated 60 nM and ionomycin generated 26 nM [61, 62], while other studies report that calcium ionophore 0.1 µM stimulated a transient pulse of NO reaching a peak of approximately 500 nM in HUVEC [57,63,64,65,66]. The disparity in the amplitude of the NO signal, which is also found in studies of pinned-out intact artery (see below), remains unexplained, and the resolution of this point is an important goal.

Cell culture studies have given considerable insight into the signalling pathways linked to NO. Repeated stimulation with histamine resulted in tachyphylaxis of the intracellular calcium signal; however, the NO response was maintained, and thus, it was concluded that NO formation was initiated by membrane, not cytosolic calcium [61, 62]. Evidence for the involvement of calcium calmodulin-dependent protein kinase II in NO formation has been obtained by showing slight inhibition of NO formation following the addition of a blocker of the enzyme [67]. Induction of inducible NOS (iNOS) gave a 5-fold increase in the capacity of cultured internal mammary artery smooth muscle cells to generate NO in response to L-arginine [42]. Endothelium stimulants such as acetylcholine and calcimycin can generate superoxide, thus reducing the net output of NO [64, 65,68,69,70]. More recently, a probe has been developed that incorporates separate microsensors for detection of superoxide and peroxynitrite in addition to NO (the 'tandem microsensor'). The basal NO concentration was 21 nM in HUVEC from white patients compared with 9 nM in cells from black patients. Calcium ionophore 0.1 µM stimulated a transient pulse of NO reaching a peak of approximately 500 nM in white patients but a slower and lower peak of approximately 250 nM in black patients. Based on the increased NOS activity levels found in black patients, it was proposed that the production of both NO and superoxide are increased in endothelial cells from black compared with white patients [66].

Cell culture studies have proved valuable for pharmacological studies with novel molecules or drug candidates. For example in HUVEC, it has been shown that the peptide angiotensin-(1-7) and its analogue AVE0991 each stimulated NO release, the latter giving a more prolonged action [71]. Parathyroid hormone [72] and 17-oestradiol also stimulated NO formation. Substances that stimulate cyclic AMP and cyclic GMP formation also stimulate NO release from endothelial cells, including forskolin [72] and YC-1 [73]. In cultured rat glomerular endothelial cells, the novel -adrenoceptor antagonists, carvedilol and nebivolol, caused formation of NO [74], while nifedipine was reported to release NO from cultured endothelial cells to levels about 30% of those generated by calcimycin [64]. A -adrenoceptor antagonist with a nitroxy adduct was shown to release NO in the presence of cultured endothelial cells, but not into the medium in the absence of cells [75].

Stimulation of NO production by surface shear force has been demonstrated in cultured endothelial cells. A porphyrin/Nafion-coated carbon fibre sensor was placed parallel to the surface of confluent bovine aortic endothelial cells in a parallel plate flow chamber at 22°C. Initiating solution flow caused shear stress which generated a spike of NO followed after 15 min by another spike. The shear-induced NO spikes were blocked by application of exogenous NO [76]. The NO sensor was calibrated with NO solution under flow conditions [76].

Cell culture studies are particularly useful in the evaluation of signalling pathways relating to NO. However, questions remain as to whether the NO-releasing properties of endothelial cells are altered when they are cultured. Moreover, the marked differences in time course and amplitude of the NO signal in different reports, even when studied with apparently similar methodology, need to be explained.

 The Isolated Arterial Segment

Electrochemical NO measurements can be performed in arteries that are cut open longitudinally and pinned on the bottom of an organ chamber filled with buffer, the electrode being positioned on the endothelial cell layer [20, 37, 85, 90, 92]. In case of smaller resistance arteries, other approaches have been applied such as threading one end of vascular segments on the NO-sensitive microelectrode [82] or cannulating the vessel segments with glass pipettes, where one pipette is connected to a pressure transducer and the NO-sensitive microelectrode is located within the other pipette [83, 84]. The advantage of working on vessel segments is that the endothelial cells are studied in their original location within the vascular wall, and interactions with the smooth muscle layer can be addressed. Thus, this approach has allowed the direct demonstration that there is an increased decomposition of NO due to superoxide in arteries from spontaneously hypertensive animals [20, 85]. In human atherosclerotic carotid artery, there was less NO formation compared with non-atherosclerotic internal mammary artery [86]. However, the pinned or threaded unstretched vascular segments are also contractile, and in case the electrode is placed too close to the vascular segments, contraction can result in bending of the microfibre and increases in current, which are not due to increased NO concentration. It has been reported that aspirin can release NO from the endothelium of pinned-out pig coronary artery (EC₅₀ = 50 nM aspirin), which might thus contribute to the cardiovascular protective actions of micoraspirin [87]. This method did not allow simultaneous measurement of force, and in parallel experiments, aspirin did not relax coronary artery rings; thus, there remains some doubt about the applicability of these data.

Introduction of an NO-sensitive microelectrode in the lumen of arterial segments mounted for isometric tension recording allows simultaneous measurements of NO concentration and vascular contractility, and hence, to establish direct relationships between them and address whether the release or response to NO is altered [5, 43, 55, 88] (fig. 3). Moreover, the combination of NO measurements with smooth muscle cell force in vascular segments provides an extra control for the reliability of the results, since the contractility of the preparation is followed at the same time as NO measurements are performed. The increase in NO is independent of change in force [5]. The properties of the microelectrode tip are the major limitations for measuring in smaller arteries. Although smaller commercial electrodes with tip sizes from 0.1 to 7 µm are currently available, these electrodes either do not reflect increases in NO in the intact vascular segment or the signal to noise ratio is low [Simonsen and Wadsworth, unpubl. observation]. Therefore, electrodes with 30-µm tips have been applied in almost all the studies of simultaneous measurements of force and vascular contractility [5, 43, 55, 88]. In addition to the microelectrode tip, two holding wires with sizes of 20-100 µm are in the arterial lumen, and hence, exclude simultaneous measurements of NO and vascular contractility in arteries with lumen diameters below 100 µm; so far, simultaneous measurements of NO concentration and contractility have only been reported for arteries with lumen diameters of 200 µm or more [89].

Fig. 3. Setup for simultaneous measurements of NO concentration and vasorelaxation in intact arteries (a). b Traces showing changes in force (upper trace) and NO concentration (lower trace) when noradrenaline (NA, 1 µM) and acetylcholine (ACh, 10 µM) are added.

Based on studies in pinned vascular segments, it was suggested that residual NO formation takes place, despite the presence of high concentrations of NOS inhibitors such as N^G-nitro-L-arginine methyl ester [90]. The simultaneous measurements of NO concentration and relaxation in rat superior mesenteric artery have been useful by providing direct evidence that residual NO causes relaxation [5]. Thus, simultaneous measurements after the addition of acetylcholine suggested that the rise of NO concentration is related to relaxation [5]. This was supported by the observation that inhibitors of NOS, N^G-nitro-L-arginine (L-NOARG) and asymmetric dimethylarginine (ADMA), caused significant inhibition of both acetylcholine-induced increases in NO concentration and relaxation [88].

Simultaneous measurements of NO concentration and relaxation have also revealed that not only iNOS depends on influx of L-arginine, but endothelium-derived NO concentration and vasorelaxation are also increased by L-arginine supplementation in segments of the rat superior mesenteric artery [43]. The extra increase in NO concentration evoked by acetylcholine in the presence of L-arginine appeared to involve L-arginine uptake through the Y⁺ transporter which is converted to NO primarily by NOS localized in caveolae.

The increases in NO concentration induced by acetylcholine in the presence of L-NOARG or ADMA are low and close to the detection limit [5, 88], but acetylcholine relaxation persisting in the presence of one of the NOS inhibitors, L-NOARG or ADMA, was reversed by oxyhaemoglobin, and this treatment also caused a time-related lowering in the NO concentration measured with NO-sensitive microsensors [5, 88]. These results may suggest that the acetylcholine relaxation persisting in the presence of NOS and cyclooxygenase inhibitors can be ascribed to residual NO release [5]. Although this is not the case in human subcutaneous small arteries [89], later studies performed in rat mesenteric small arteries in the presence of NOS and cyclooxygenase inhibition confirmed that residual NO appears to mediate part of acetylcholine hyperpolarization [91] and part of bradykinin hyperpolarization in porcine coronary arteries [92]. These studies underline that it is important to perform simultaneous measurements of NO concentration and relaxation [see also ref. [ 93] or use tissue from mice with a double knockout for cyclooxygenase and NOS [94], before concluding that relaxation persisting in the presence of NOS and cyclooxygenase inhibition can be ascribed to an endothelium-derived hyperpolarizing factor or myoendothelial gap junctions.

One of the remarkable findings from simultaneous measurements of NO concentration and vascular contractility is the observation that the free NO concentration is not increased in arteries, where iNOS is expressed following exposure to lipopolysaccharide [43, 54, 55]. The concentration of free NO is low despite hyporeactivity to agonists [55]. However, the formation of the NO end product nitrite is markedly increased in preparations treated with lipopolysaccharide [54], probably as a consequence of increased formation of superoxide and/or hydrogen peroxide from iNOS and a reaction with NO generating peroxynitrite [54, 55]. In part, the increased reductive formation of superoxide from iNOS can be ascribed to the lack of the substrate L-arginine [55], which has also been shown to be the case in animals with septic shock [95].

The simultaneous measurements of NO concentration and relaxation have also revealed that in addition to decreased bioavailability of NO described in arteries from hypertensive animals [20], the release of NO from the endothelial cell layer appears to play a role for the reduced vasorelaxation in arteries from animals with renal hypertension [88]. Examining the NO concentration in groups of animals with a certain pathophysiology requires that the experimenter is blinded, since small currents are measured from the electrode and bias cannot be excluded despite best intentions. Moreover, the examination of the arteries should be randomized to avoid that electrode differences contribute to variability, and hence, preclude showing differences among arteries from control versus diseased animals.

In addition to NO concentration induced by agonists or drugs, it is possible to estimate the basal concentration by an NO scavenger such as oxyhaemoglobin and observe the decrease in NO concentration keeping the preparation at baseline tension [5, 55]. However, the pH of the oxyhaemoglobin solutions should be carefully controlled, and in addition, it is important to take into account that oxyhaemoglobin is oxidized, thus limiting the observation period to 5-8 min. Therefore, for these kinds of experiments, it would be useful with improved NO scavengers.

 In vivo Measurements of NO Concentration

A sensor has been devised for in vivo use that is made from a porphyrin-coated carbon fibre [19] mounted in a 0.5-mm diameter syringe needle protected by a slightly longer plastic catheter [96] (fig. 4). A standard three-electrode circuit is used, where a silver/silver chloride reference electrode and a platinum wire counter-electrode is attached nearby on the skin or organ in the living animal. The electrode assembly was placed within the parenchyma of the organ of interest, within the wall of a major artery, inside a blood vessel lumen, or close to the adventitia of the vasculature.

Fig. 4. Illustration of the working electrode for in vivo measurements (a), and typical recording of the effects of electrostimulating the cavernous nerve (SCN) for 1 min on the NO electrochemical signal (top), arterial pressure (AP), and intracavernous pressure (ICP) (b). Modified from Prof. Manuel Mas et al. [114, 115], with permission from Elsevier and Blackwell Publishing.

Electrochemistry has allowed direct assessment of NO concentration in physiological fluid [77,78,79]. NO is the main neurotransmitter involved in penile erection [80]. By use of NO sensitive electrodes, Mas et al. [79] measured changes in NO levels in the corpus cavernosum of anaesthetized rats during and after stimulation of the cavernosal nerves [81]. Following electrical stimulation of the cavernous nerve, they found an increase in intracavernosal pressure and then an increase in NO levels, but the NO levels remained elevated when the erectile responses had subsided [78, 81]. The authors demonstrated that a NOS inhibitor, N^G-nitro-L-arginine methyl ester, inhibited the change in electrochemical current, and this inhibition was reversed by L-arginine [78]. Therefore, these measurements suggest the release of NO or an NO-containing neurotransmitter, but further methodological development of NO-sensitive electrodes and/or other approaches are required to relate the release of NO with increases in intracavernosal pressure.

A series of studies have addressed whether flow is associated with increased NO formation in the mesenteric vascular bed [97, 98]. In the studies by Bohlen et al. [97, 99, 100], NO concentration was measured by use of an NO-sensitive gold microelectrode, which was developed by Buerk et al. [101], and situated close to the vessel wall of the intestine. The authors were able to show that L-arginine increases NO, and that NOS inhibitors such as L-NOARG decrease NO [97]. Moreover, in rats, absorption of glucose was associated with an increased flow associated with doubling of the measured NO concentration [97, 99]. In dogs, where NO concentration was measured by use of a 200-µm carbon microsensor, the flow induced by occlusion of adjacent mesenteric arteries was also associated with a doubling of the NO concentration [98]. The application of two different microsensors shows that it is possible to obtain physiologically relevant information showing that increased flow is associated with increased NO concentration.

Only a few studies have attempted to measure NO concentration in human tissue, but this was successful in one of the first in vivo studies where porphyrinic microsensors sensitive for NO were inserted in the hand vein of 5 healthy males [77]. The study demonstrated that bradykinin increased NO and inhibitors of NOS were able to inhibit both bradykinin- and acetylcholine-evoked increases in NO [77]. In this study, it is remarkable that the sensor tip was placed in the middle of flowing blood. Despite the surrounding red blood cells filled with the NO scavenger oxyhaemoglobin, the NO-sensitive sensor apparently reflects changes in endothelium-derived NO, although contributions from stimulated NOS containing platelets cannot be excluded. Later studies performed in dogs, where either an NO-sensitive porphyrinic microsensor [102, 103] or a more recently developed carbon fibre sensor with a diameter of 700 µm from Innovative Instruments was used [36], have also demonstrated that the NO-sensitive microsensors reflect the vascular NO. However, in these studies, inhibitors of NOS only partially inhibit the increase in NO concentration induced by agonists [36, 103], and further studies should address whether the increase in microelectrode current persisting in the presence of the NOS inhibitors can be ascribed to (1) an inadequate inhibition of NOS by L-arginine analogues as described in isolated arteries (see above), (2) to other sources of NO, besides L-arginine, or (3) whether the infusion of agonists results from increased flow directly increasing the microsensor current by mechanical interactions.

The alterations in NO concentration during limb ischaemia/reperfusion injury in skeletal muscle of rabbits have been studied by insertion of an NO-sensitive microelectrode which was either placed directly on the surface of a microvessel or implanted in the femoral artery wall [104, 105]. Ischaemia produced an increase in NO which peaked at 12 min (basal 52 nM, peak 125 nM NO) and then decayed to fall below basal. During reperfusion, there was a further fall in NO to undetectable levels by 100 min of reperfusion [104, 105]. The NO concentration at reperfusion was increased by administration of nitrosylated human serum albumin [106], by L-arginine, or by L-arginine plus a mixture of antioxidant vitamins [105].

Insertion of microelectrodes in the cortex or other regions of the brain has also allowed in vivo measurement of changes in NO concentration during ischaemia or hypoxia [107,108,109]. In a model of focal ischaemia in cats, occlusion of the middle cerebral artery reduced cerebral blood flow, and NO concentration increased simultaneously in the regions of the brain where depolarization takes place during ischaemia, and then, similar to the situation in limb ischaemia, decreases even below baseline levels over the next 60 min [107]. In contrast, in newborn piglets where NO was measured with a 200-µm carbon microsensor, hypoxia (6%) caused only a decrease in NO concentration followed by supranormal NO levels during reoxygenation with 100% oxygen [109]. It is likely that species differences and different experimental protocols are important, but it cannot be excluded that electrode properties to some degree explain these opposing results. Ischaemia, hypoxia and reperfusion will each alter oxygen levels, pH and temperature of the previously perfused organ. All these parameters are known to influence current recordings from NO-sensitive microsensors (fig. 1), and therefore, it is important to control these parameters simultaneously with the induction of ischaemia in vivo.

iNOS is thought to play a pivotal role in endotoxemia, and NO concentration has been measured in endotoxemia by insertion of an NO-sensitive porphyrin microelectrode in the lung [110], in the aorta wall [111], or in the pulmonary artery wall [112]. Injection of lipopolysaccharide to induce endotoxemia was followed by a decrease in systemic arterial pressure, and a transient increase in NO (baseline 50 nM, peak 150 nM NO) which lasted 20 min was measured both in the aortic wall [110] and lung tissue [111]. A second slower rise was recorded in the pulmonary artery at 45 min [112]. The increase in NO concentration demonstrated to be protective in the acute phase of endotoxemia, since inhibition of the NO increase by NOS inhibitors shortens survival of the rats with endotoxemia [111]. The low levels of free NO concentration measured in endotoxemia correspond to the levels detected in isolated preparations in vitro as mentioned above [43, 54, 55], and hence, underline that it is possible to obtain reliable measurements of NO concentration in living animals.

Applications of NO microsensors are being developed in several other areas of cardiovascular research. A sensor assembly placed in the ventricular wall of anaesthetized rabbits reported NO levels in the beating heart to be 600 nM in early systole, increasing to 2.7 µM in diastole [96]. Intracerebroventricular injection of clonidine to anaesthetized rats caused an increase in NO (sensor stereotaxically located in nucleus tractus solitarius) of 128 nM which lasted about 40 min and correlated with the fall in blood pressure [113].

In summary, the animal studies describing the application of the NO microsensor provide additional and valuable information about the dynamics and kinetics of NO release in biological tissue, both in physiological and pathophysiological conditions. However, in studies where the microlelectrode is inserted in tissue, the role of tissue damage for the electrode measurements should be explored. The possibility of measuring NO in blood is interesting, and therefore, the role of whole blood for the measurements should be explored and modelled, with the perspective of developing a catheter including an NO-sensing probe that can be guided into the lumen of, for instance, arteries developing atherosclerotic lesions. Methodological development of NO-sensitive electrodes avoiding the influence of changes in temperature and pH on the measurements is also required to relate the release of NO with changes in, for instance, flow.

 Evidence that Microsensors Measure Physiological NO Concentration

When NO was established as an important signalling molecule, it was recognized that it is chemically and biologically reactive, and therefore, would be expected to be evanescent. As a molecule that is generated by one cell, but acts at other cells some distance away, its concentration will be greatest at the site of production and less at the site of action. Moreover, if an NO sensor is located at a distance away from the site of action, it may measure a concentration of NO that is less than the effective concentration that is delivered to the target cells. These considerations have led to a debate about whether the concentration of NO that is recorded by the NO sensors is close to the physiologically active concentration of NO.

 Diffusion of NO from the Site of Production to the Site of Action

The reaction of NO with dioxygen is dependent on the concentration of NO, and therefore, proceeds slowly in aqueous solution at the low concentrations of NO that occur physiologically, but in tissue, NO is subject to more rapid degradation. In tissue, superoxide is generated by several processes in the artery wall, including NAD(P)H oxidase, xanthine oxidase, cyclooxygenase and, under some circumstances, by NOS; it has a high rate constant for its reaction with NO. Scavenging of NO by superoxide appears to be physiologically important, since superoxide scavengers can increase endothelium-dependent (i.e. NO-dependent) relaxation. However, there is no agreement as to the quantitative effect that endogenous superoxide formation will have on the half-life of NO within the milieu of the artery wall. NO binds strongly to guanylate cyclase, which is present in high concentration in vascular smooth muscle and endothelial cells, and this binding may slow the apparent diffusion of NO through the artery wall. Again, there are no reliable quantitative estimates of the extent of this effect on the measurement of NO. Given these considerations, it is generally accepted that the diffusion of NO from the cells that generate it within the artery wall to its point of detection at an NO sensor will involve some degradation, although the extent to which this occurs is unclear.

NO diffusion in the artery wall has been modelled mathematically. The models show that, even assuming a short half-life for NO, NO should be able to travel distances equivalent to the thickness of the artery medial layer. Thus, with a half-life of 4 s, the concentration of NO was approximately half of the initial value at a distance of 160 µm [114]. With multiple NO sources (such as the cells of the endothelial layer), the model predicted that a site at 500 µm distance would reach steady state after 20 s [115]. Even assuming a brief pulse of NO generation (1-10 s) and a short half-life (0.5-5 s), it was predicted that NO would have a significant presence at a distance of 100 µm [115, 116]. A model with the endothelium and smooth muscle as concentric cylinders predicts that significant NO should be present at 100 µm away from the endothelium, even when allowance is made for the sequestration of NO by haemoglobin within the artery lumen. The effect of the haemoglobin in blood would be strongly affected by the thickness of the erythrocyte-deficient layer adjacent to the endothelium [117]. A microsensor measurement of NO in the wall of the rabbit aorta found that the NO concentration in the smooth muscle at 100 µm distance from the endothelium was 65% of the endothelial concentration [116]. Since the vascular endothelium has a large excess capacity for NO production [5], these models predict that endothelial NO will be able to diffuse to and physiologically activate smooth muscle cells throughout the artery wall. Moreover, sampling NO concentration with a microsensor should give a reasonable estimate of the physiological NO concentration, provided the sensor is located within a distance of approximately 100 µm (for steady-state conditions). Indeed, the original 'sandwich' experiments of Furchgott and Zawadzki [11] showed that endothelial-derived NO can be detected at a distance and after diffusion through an aqueous medium.

 Evidence that Myograph Studies Estimate Intramural NO Concentration

An NO sensor placed within the lumen of a rat superior mesenteric artery mounted on a wire myograph detected NO for threshold relaxation at around 5 nM, whereas for maximal endothelial activation, NO was 20 nM [5]. In these experiments, a comparison was made of NO generated in two ways. Activation of the endothelium by acetylcholine causes synthesis of NO within the endothelium, which has then to diffuse through the physiological salt solution to be detected by the NO sensor (a distance of 10 µm). The NO donor molecules S-nitroso-acetylpenicillamine and SIN-1 generate NO within the aqueous medium, and thus, on the surface of the NO sensor. The concentrations of NO detected by the NO sensor, for equivalent relaxation, were the same for the NO donors as for acetylcholine [5]. It was argued that the concentration of NO detected at the NO sensor (5-20 nM) is a reasonable estimate of the concentration within the smooth muscle layer, since, if NO were subject to significant degradation within the artery wall, the NO concentration generated in the medium (by the NO donors) would have been recorded at a high level compared with the remnant of NO escaping from the endothelium.

 Conclusions

Many of the key questions in cardiovascular physiology, pathophysiology and pharmacology require knowledge of the physiological NO concentration and not just the direction of change. The NO microsensor was originally devised to measure the concentration of NO in living cells; however, it has proved a challenging task to arrive at values of NO that are physiologically relevant. After more than 10 years of experience with these sensors, there remain differences in the literature concerning the time courses and concentrations of NO signals detected using different sensors and comparing cultured with native cells. It is important to establish which methodologies provide the best estimates of the physiological situation to achieve progress in this area. Improvements are needed in sensor characteristics so that NO microsensors can be more widely available to a broad range of cardiovascular research. Future development and improvement in the NO microsensor has the prospect of bringing dramatic advances in the knowledge of NO in cell signalling, NO in vivo, and hopefully, eventually NO in the clinical setting.

 Acknowledgements

We thank Prof. M.J. Mulvany for the helpful comments on the manuscript. R.W. was supported by the British Heart Foundation (PG/99177 and FS/99011). U.S. was supported by the Danish Medical Research Council and the Danish Heart Foundation (No. 04-10-B116-A304-22197). The authors have received no direct or indirect financial support from World Precision Instruments. The World Precision Instrument microsensor was described in this review because there have been extensive publications with this technology and it is readily available.

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  Author Contacts

Dr. Ulf Simonsen
Department of Pharmacology, University of Aarhus
DK-8000 Aarhus C (Denmark)
Tel. +45 8942 1713, Fax +45 8612 8804
E-Mail us@farm.au.dk

  Article Information

Received: May 17, 2005
Accepted after revision: September 23, 2005
Published online: November 4, 2005
Number of Print Pages : 16
Number of Figures : 4, Number of Tables : 1, Number of References : 118

  Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 43, No. 1, Year 2006 (Cover Date: December 2005)

Journal Editor: Pohl, U. (Munich)
ISSN: 1018-1172 (print), 1423-0135 (Online)

For additional information: http://www.karger.com/JVR

  Drug Dosage / Copyright

Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.