Direct In Situ Measurement of Nitric Oxide in Mesenteric Resistance Arteries
Increased Decomposition by Superoxide in Hypertension
Abstract The endothelium plays a critical role in maintaining vascular tone by releasing vasoconstrictor and vasodilator substances. Endothelium-derived nitric oxide is a vasodilator that can be rapidly inactivated by superoxide (reaction rate constant, K=3.6×109 L/mol per second). The measurement of nitric oxide concentration in biological systems is a challenging analytic problem because nitric oxide is also rapidly inactivated by Fe(II), Fe(III), and O2, all of which are found in great abundance in biological systems. To date, no currently used instrumental technique has been suitable for direct in situ measurement of NO in isolated resistance arteries. We designed the present study to perform for the first time direct in situ measurements of NO in rat mesenteric resistance arteries and to delineate the effects of hypertension on the release of NO and/or its interaction with superoxide. We describe here an adaptation of the recently published design of a porphyrinic sensor for direct in vitro measurement of NO in a single cell. The most significant advantage of this modified porphyrinic microsensor is that its small size makes it ideal for NO measurement in resistance arteries with an internal diameter of 200 μm or less. Small segments of the third-order branch of the mesenteric artery were isolated from normotensive Wistar-Kyoto rats and stroke-prone spontaneously hypertensive rats and placed in an organ chamber filled with Hanks’ balanced salt solution buffer (2 mL, 37°C). The tip of the porphyrinic microsensor was inserted into the lumen of an isolated vascular ring, and NO release was monitored in situ after maximal stimulation of NO synthase with the receptor-independent agonist calcium ionophore A23187 (10 μmol/L). Maximal surface concentration of NO measured after A23187 administration was significantly smaller in 15-week-old hypertensive rats (0.28±0.03 μmol/L, n=10) than in age-matched normotensive rats (0.38±0.03 μmol/L, n=10, P<.03). However, in the presence of the superoxide scavenger superoxide dismutase (100 U/mL), the peak NO level from the hypertensive rats was 0.37±0.04 μmol/L (n=10), which was comparable to that observed for the normotensive rats in the absence and presence of superoxide dismutase. In summary, our results demonstrate that in rat mesenteric resistance arteries hypertension is associated with increased NO decomposition by superoxide, whereas NO release remains unaffected. This may be important in the pathogenesis of hypertension and its cardiovascular complications.
Peripheral vascular resistance is primarily regulated by arteries with an internal diameter of 200 μm or less, whereas the contribution of large arteries is relatively minor.1 The endothelium plays a critical role in maintaining vascular tone via generation of vasoconstrictor and vasodilator substances.2 An important endothelium-derived relaxing factor is NO (EDNO).3 Furthermore, endothelial cells are a source of superoxide that can mediate endothelium-dependent contractions by the breakdown of NO.4
The endothelium is a major target for cardiovascular risk factors. Hypertension is one of the most important risk factors and associated with an imbalance of endothelium-derived relaxing and contracting factors.5 The mechanism of endothelial dysfunction differs, however, in different models of hypertension and in different vascular beds. In the aorta of spontaneously hypertensive rats, the reduced response to acetylcholine is related to the production of a cyclooxygenase-dependent constricting factor,6 whereas in other forms of experimental hypertension, reduced formation of EDNO may predominate.7 Recently, it has been suggested that an increase in the generation of superoxide by inactivating EDNO could lead to an increase in peripheral vascular resistance and hypertension.8
The short half-life of NO in biological systems and its loss due to reaction with superoxide makes accurate quantitative measurements of NO difficult. Most current methods for NO detection are indirect, relying either on measurements of secondary species such as nitrite (an NO decay product) or on bioassays that rely on secondary effects.9 Recently, the design and application of a porphyrinic microsensor for the direct in situ electrochemical measurement of NO in a single cell have been published.10 However, this sensor, with a detection limit of 10−9 mol/L, was too fragile to be used in isolated resistance arteries. We describe here an adaptation of this porphyrinic sensor that because of its small size is well suited for direct in situ measurement of NO in resistance arteries with an internal diameter of 200 μm or less. It was our aim in the present study to perform for the first time direct in situ measurements of NO in rat mesenteric resistance arteries and to delineate the effects of hypertension on the release of NO and/or its interaction with superoxide.
Ten male WKY and 10 male SHRSP were obtained from colonies in the Department of Anatomy and Cell Biology at the University of Michigan. These were inbred colonies derived from a stock supplied by the National Institutes of Health, Bethesda, Md. Rats were treated according to the Guiding Principles for Laboratory Animal Care of the American Physiological Society and used for experiment at 15 weeks of age. Systolic pressure was measured in conscious rats by the tail-cuff method (W+W Electronics). Before the tail-cuff blood pressure determination, rats were heated with an infrared lamp for half an hour. Then the tail cuff and pressure sensor were secured on the tail, and blood flow was occluded by compression. Systolic pressure was recorded at the first detection of blood flow upon gradual release of the occlusion. At least three recordings of systolic pressure were made, and the mean values of these were taken as final readings.
Preparation of Mesenteric Resistance Arteries
On the day of the experiment rats were decapitated, and the mesentery was removed and placed in cold (4°C) modified Hanks’ balanced salt solution (pH 7.4) of the following concentration (mmol/L): NaCl 137, Tris-HCl 10, MgCl2 1, KCl 5, CaCl2 0.9, MgSO4 0.8, KH2PO4 0.44, Na2HPO4 0.33, and l-arginine 0.1. A small segment of the third-order branch of the mesenteric artery (3 mm long, 200-μm ID) was isolated and cleaned of adhering tissue under a dissection microscope (M3C, Wild AG).
NO Microsensor Fabrication
The NO microsensor was produced by threading an array of seven carbon fibers (Amoco Performance Products, Inc) through a pulled end of an L-shaped glass capillary, with a 6.0 mm length of the fibers left protruding. A copper wire was inserted into the opposite end of the glass capillary, which was sealed with conductive silver epoxy (AI Technology). Then the tip of the glass capillary was sealed with beeswax. A conductive polymeric film was deposited on the surface of the carbon fibers from a 0.25 mmol/L solution of nickel (II) tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin in 0.1 mol/L NaOH under N2 as previously described.10 After drying, the active tip of the sensor was immersed in 1 wt % Nafion solution in alcohol (Aldrich Chemie) and then allowed to dry again.
Experimental Setup and NO Measurement
A three-electrode system was used for measurement of NO release. The three-electrode system consisted of an NO sensor working electrode, a platinum wire counter electrode (diameter, 0.5 mm), and a saturated calomel reference electrode. Differential pulse amperometry was used as previously described to monitor the analytic signal.11 The amperometric method (with a response time of 0.1 millisecond in the 1 μmol/L NO range and 10 milliseconds near the detection limit of 10−9 mol/L NO) provides a rapid quantitative response to changes of NO concentration. Differential pulse amperometric experiments were performed with a potentiostat/galvanostat (model 273, EG&G PAR) interfaced to a computer (Gateway 2000 P4D-66) with custom data-acquisition and control software.
Immediately before NO measurements, isolated ring segments from WKY and SHRSP were placed in an organ chamber with fresh Hanks’ balanced salt solution (2 mL, 37°C), and the active tip (length, 6 mm; diameter, 30 μm) of the NO microsensor was inserted into the lumen of the resistance arteries (Fig 1⇓). Then, 20 μL of a 1 mmol/L solution of calcium ionophore A23187 was injected to reach a final concentration of 10 μmol/L in the organ chamber (maximal stimulation of the NO synthase). Experiments were then repeated in the presence of SOD (100 U/mL).
Calcium ionophore A23187, SOD, and chemical components of the modified Hanks’ balanced salt solution were obtained from Sigma Chemical Co.
The maximal NO concentration produced (micromoles per liter) was measured, and data are given as mean±SE. In each set of experiments, n equals the number of rats studied. Statistical evaluation was done by ANOVA followed by Scheffé’s F test. Means were considered significantly different when the probability values were less than .05.
Blood Pressure, Body Weight, and Vessel Dimensions
Blood pressure was markedly higher in 15-week-old SHRSP (199±5 mm Hg, n=10) than in age-matched normotensive WKY (110±6 mm Hg, n=10, P<.05). Body weight was significantly less in SHRSP (318±14 g, n=10) than in WKY (409±28 g, n=10, P<.05). Intraluminal vascular diameter was nearly identical in WKY (209±7 μm, n=10) and SHRSP (206±9 μm, n=10, P=NS).
Kinetics of NO Release
Fig 2⇓ presents typical amperometric (current-concentration versus time) curves obtained for in situ ex vivo measurements of NO in mesenteric resistance arteries of 15-week-old WKY and age-matched SHRSP. Immediately after A23187 (10 μmol/L) administration, an initial rapid increase of NO concentration was observed. The rates of concentration increase were 188 and 128 nmol/L per second for WKY and SHRSP, respectively (Fig 2a⇓ and 2b⇓). The peak concentration of 394 nmol/L was reached 2.1 seconds after administration of calcium ionophore into WKY mesenteric artery. However, the peak concentration was significantly lower for SHRSP (281 nmol/L), with a slightly longer time (2.2 seconds) required for NO concentration to reach the maximal level. The average rates of decay of NO concentration (calculated as the percent decrease of peak concentration per second) were 15.0 and 11.2 nmol/L per second for WKY and SHRSP, respectively. In the presence of the superoxide scavenger SOD (100 U/mL), no significant changes of the kinetics of NO release were observed for WKY after stimulation with calcium ionophore (data not shown). However, the peak concentration (356 nmol/L) for NO release from SHRSP mesenteric resistance arteries (Fig 2c⇓) was 27% higher in the presence of SOD and was similar to that observed for WKY. Also, for SHRSP the rate of NO release (162 nmol/L per second) was much faster in the presence than in the absence of SOD (128 nmol/L per second). Furthermore, the average decay rate of NO concentration was slower for SHRSP (10.3 nmol/L per second) in the presence of SOD.
Fig 3⇓ summarizes data from 10 experiments and shows a statistically significant difference between NO release from mesenteric artery of WKY (0.38±0.03 μmol/L, n=10) and SHRSP (0.28±0.03 μmol/L, n=10).
The average release from SHRSP in the presence of SOD was 0.37±0.04 μmol/L, which was comparable to that observed for WKY in the absence and presence of SOD.
To compare the concentration of NO released from mesenteric resistance arteries with that released from large arteries, we performed additional measurements of NO from the aorta under identical experimental conditions. In the presence of A23187 (10 μmol/L), the NO concentrations released from the aorta were 0.90±0.12 and 0.69±0.08 μmol/L for WKY and SHRSP, respectively. The ratio of maximal NO concentrations ([NO]WKY/[NO]SHRSP) in the aorta was 1.3, which was identical to that observed in mesenteric artery. However, the maximal NO concentration in mesenteric artery was less than one half that measured in aorta.
The present study provides for the first time direct in situ measurements of NO in rat mesenteric resistance arteries and shows its enhanced inactivation by superoxide with hypertension. These studies also show that the concentration of NO released by the endothelium from mesenteric resistance arteries is significantly lower than that from the aorta. This indicates that the concentration of the NO synthase enzyme is lower in endothelial cells of mesenteric resistance artery compared with those of aorta.
The measurement of NO concentration in biological systems is a challenging analytic problem.12 The currently used instrumental techniques for NO measurements are spectroscopic and electroanalytic.9 Mass spectrometry and gas chromatography have been used occasionally for NO detection but are much less sensitive.9 The choice and efficient use of a technique depend on several factors, including anticipated NO concentration, possible interference, and sample size. From an analytic point of view, the detection of NO in the location with the highest concentration, the surface of the cell membrane, will be the most efficient and accurate. To date, however, no currently used instrumental technique was suited for direct in situ measurement of NO in isolated resistance arteries. The most significant advantage of the porphyrinic NO sensor described above is its small size. It can be placed exactly in the location where NO is produced and is therefore ideal for NO measurement in resistance arteries with an internal diameter of 200 μm or less.
Small arteries with a diameter of 200 μm or less play an important role in the regulation of peripheral vascular resistance.1 Endothelial cells modulate underlying vascular smooth muscle tone by releasing endothelium-derived relaxing and contracting factors.2 Under physiological conditions endothelium-derived relaxing factors (ie, EDNO3 ) appear to dominate. An imbalanced production of relaxing and contracting factors may initiate as well as sustain the abnormal vasoconstriction of hypertension.6 The mechanism of this endothelial imbalance is controversial in different models of hypertension and in different vascular beds.6 7 8 In mesenteric resistance arteries of adult spontaneously hypertensive rats the impaired relaxations to acetylcholine are due to the production of cyclooxygenase-dependent endothelium-derived constricting factor, most likely prostaglandin H2, which opposes the relaxing properties of EDNO.13
In the present study we have shown that the NO concentration measured after maximal stimulation of NO synthase with the receptor-independent agonist calcium ionophore A23187 was significantly smaller in adult SHRSP than age-matched WKY. Also, the rate of NO release was 30% slower in SHRSP compared with that in WKY. However, in the presence of the superoxide scavenger SOD, a similar maximal NO concentration could be detected in WKY and SHRSP. This indicates that in mesenteric resistance arteries of adult spontaneously hypertensive rats, a higher production of superoxide and/or diminished activity of SOD accounts for an increased degradation of NO, whereas the actual production and release of NO are normal. Since all experiments were performed in vitro in the presence of modified Hanks’ balanced salt solution, an enhanced binding to plasma proteins, which are known to form adducts with NO, can be excluded.14 These findings are in line with results obtained in mesenteric resistance arteries from prehypertensive (4-week-old) spontaneously hypertensive rats. In these vessels, endothelium-dependent relaxations were impaired by the production of a contractile factor (or factors) that appears to be superoxide.15 Our results also support the concept that an imbalance in the availability of endothelium-derived relaxing and contracting factors may initiate or sustain the abnormal peripheral resistance with hypertension. The altered endothelial function, however, is mainly due to an augmented production of endothelium-derived constricting factors, ie, superoxide and/or prostaglandin H2, and is not the consequence of a decreased release of EDNO.
In summary, our results demonstrate that in rat mesenteric resistance arteries hypertension is associated with increased decomposition of NO by superoxide and not an altered release of NO. This increased decomposition of NO by superoxide may be important in the pathogenesis of hypertension and its cardiovascular complications.
Selected Abbreviations and Acronyms
|EDNO||=||endothelium-derived nitric oxide|
|SHRSP||=||stroke-prone spontaneously hypertensive rat(s)|
M.R.T. was supported by a stipend of the Roche Research Foundation, F. Hoffmann–La Roche Ltd, Basel, Switzerland. We are grateful to Saul Grunfeld, MSc, and Stephen Patton, MSc, for discussion and expert technical assistance.
- Received August 9, 1995.
- Revision received August 25, 1995.
- Accepted September 6, 1995.
Vanhoutte PM, Lüscher TF. Peripheral mechanisms in cardiovascular regulation: transmitters, receptors and the endothelium. In: Tarazi RC, Zanchetti A, eds. Handbook of Hypertension: Physiology and Pathophysiology of Hypertension, Volume 8: Regulatory Mechanisms. Amsterdam, Netherlands: Elsevier Science Publishers; 1986:96-123.
Lüscher TF, Vanhoutte PM, eds. The Endothelium: Modulator of Cardiovascular Function. Boca Raton, Fla: CRC Press; 1990:1-215.
Katusic ZS, Vanhoutte PM. Superoxide is an endothelium-derived contracting factor. Am J Physiol. 1989;357:H33-H37.
Lüscher TF, ed. Endothelial Vasoactive Substances and Cardiovascular Disease. Basel, Switzerland: Karger; 1988.
Dohi Y, Thiel MA, Bühler FR, Lüscher TF. Activation of endothelial L-arginine pathway in resistance arteries. Hypertension. 1990;15:170-179.
Blatter LA, Taha Z, Mesaros S, Shacklock PS, Wier WG, Malinski T. Simultaneous measurements of Ca2+ and nitric oxide in bradykinin-stimulated vascular endothelial cells. Circ Res. 1995;76:922-924.
Archer S. Measurement of nitric oxide in biological models. FASEB J. 1993;7:349-360.
Diederich DA, Yang Z, Bühler FR, Lüscher TF. Impaired endothelium-dependent relaxations in hypertensive resistance arteries involve the cyclooxygenase pathway. Am J Physiol. 1990;258:H445-H451.
Keany JF Jr, Simon DI, Stamler JS, Jaraki O, Scharfstein JS, Vita JA, Loscalzo J. NO forms an adduct with serum albumin that has endothelium-derived relaxing factor-like properties. J Clin Invest. 1993;91:1582-1589.
Jameson M, Dai F-X, Lüscher TF, Skopec J, Diederich A, Diederich D. Endothelium-derived contracting factors in resistance arteries of young spontaneously hypertensive rats before development of overt hypertension. Hypertension. 1993;21:280-288.