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Cardiovascular Research 1998 38(3):655-667; doi:10.1016/S0008-6363(98)00051-0
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

Effects of vasodilators and perfusion pressure on coronary flow and simultaneous release of nitric oxide from guinea pig isolated hearts1

Satoshi Fujitaa, David L Roerigb,e, Zeljko J Bosnjaka,c,e and David F Stowea,c,d,e,*

aAnesthesiology Research Laboratory, Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
bDepartment of Pharmacology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
cDepartment of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA
dCardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226, USA
eVA Medical Center, Milwaukee, WI 53226, USA

* Corresponding author. Department of Anesthesiology, Medical College of Wisconsin, MEB 462C, Milwaukee Regional Medical Center, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA. Tel.: +1 (414) 456-5733; Fax: +1 (414) 456-8541; E-mail: dfstowe@post.its.mcw.edu

Received 12 October 1997; accepted 27 January 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aims were to validate the use of a direct reading NO electrode, to compare the effects of diverse acting drugs on altering coronary flow (CF) and NO release, and to examine the effects of altered perfusion pressure on flow-induced changes in NO concentration [NO] in the hemoglobin free effluent of guinea pig isolated hearts. Methods: Hearts were isolated and perfused initially at a constant perfusion pressure (55 mmHg) with a modified Krebs–Ringer's solution equilibrated with 97% O2 and 3% CO2 at 37°C. Heart rate, left ventricular pressure, CF, and effluent pH, pCO2, pO2, and NO generated current were monitored continuously on-line. Effluent was sampled for L-citrulline. Percent O2 extraction and O2 consumption were calculated. [NO] was quantitated with a sensitive amperometric sensor (sensitivity ≥1 nmol/l{approx}3 pA) and a selective gas permeable membrane. Results: The electrode was not sensitive to changes in solution pO2, flow, or pressure. The electrode was sensitive to pCO2 (–0.50 nmol/l/mmHg) and temperature (+24.5 nmol/l/°C), so coronary effluent pCO2 was measured to compensate for a small decrease in pCO2 that occurred with an increase in coronary flow, and effluent temperature was rigidly controlled. Serotonin, bradykinin, and nitroprusside increased NO release along with CF, whereas nifedipine, butanedione monoxime, zaprinast, and bimakalim comparably increased CF but did not increase [NO] or NO release. Increases in CF (ml/g/min) and NO release (pmol/g/min), respectively, were 5.0±1 and 100±17 for 1 µmol/l serotonin, 7.5±1 and 148±18 for 100 nmol/l bradykinin, and 7.8±1 and 173±28 for 100 µmol/l nitroprusside. The increases in effluent NO by bradykinin were proportional to the increases in L-citrulline. Tetraethylammonium decreased CF, but did not change NO release, indomethacin changed neither CF nor NO release, and NG-nitro-L-arginine methyl ester (L-NAME) reduced CF by 2.6±1 ml/g/min and NO release by 25±8 pmol/g/min. An increase of CF of 8.0±0.3 ml/g/min, produced by increasing perfusion pressure from 25 to 90 mmHg, increased [NO] by 30±4 nmol/l; L-NAME but did not reduce the pressure-induced increase in CF, but reduced the increase in [NO] to 10±5 nmol/l. Conclusions: This study demonstrates in intact hearts real-time release of NO by several vasodilator drugs and by pressure-induced increases in flow (shear stress) and attenuation of these effects by L-NAME.

KEYWORDS Bradykinin; Coronary endothelium; Nitroprusside; Serotonin; Vascular smooth muscle


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelium-derived relaxing factor [1]is most likely nitric oxide (NO) [2–4]. NO is normally generated in the circulation via stimulation of vascular endothelial NO synthase (eNOS) by endothelial mechanochemical receptors [4, 5]. NO can also be released from nitrosyl compounds such as nitroprusside (NP) at guanylyl cyclase [6, 7]. NO gas released abluminally stimulates vascular smooth muscle guanylyl cyclase to elicit vasodilation by increasing cGMP [4]. Many vasodilator drugs are thought to act predominately through either endothelium-dependent or -independent pathways.

It has been demonstrated directly and indirectly that NO is released from coronary endothelium and that it modulates coronary vascular tone, at least in vitro [8–11]. However, there has been no direct information on whether a given vasodilator stimulates NO production, how pressure-induced changes in coronary flow (CF) affect NO production, if NO contributes fo flow reulation, and how drug and mechanically induced NO production is affected by eNOS antagonism in the intact coronary vascular bed. Several methods have been reported that indirectly measure NO [10–16]. More recently, highly specific amperometric sensors for NO have been developed for real-time measurement of NO from cells or intact tissues [17–21].

To elucidate the role of NO on local control of the intact coronary circulation we quantitated, in real time, coronary effluent release of NO from intact hearts during increases in CF induced by endothelium-dependent and -independent drugs, measured NO release resulting from pressure-induced changes in CF (shear stress), and assessed effects of an eNOS antagonist and other antagonists on altering CF and NO responses. Advantages to the use of the crystolloid perfused, isolated heart were that effluent NO release could be monitored continuously on-line with CF and effluent pO2 in the absence of hemoglobin which inactivates luminally released NO, that effluent [NO] could be measured during either drug or flow-induced endothelial stimulation, and that effects of inhibitors on [NO] and NO release could be assessed.

After extensive testing for artifacts, and after demonstrating the effectiveness of the electrode to measure NO when placed in the coronary effluent, our specific objectives were: (1) to measure and compare the effects of eight known vasodilators to release NO and increase CF; (2) to measure release of NO when CF was altered by step changes in coronary perfusion pressure; (3) to confirm drug-induced NO release by measuring release of L-citrulline, its co-product; and (4) to compare effects of NG-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, indomethacin (INDO), a prostaglandin synthesis inhibitor, and tetraethylammonium (TEA), a non-specific K+ channel blocker, on CF and NO production. The drugs selected were: serotonin (5-HT) and bradykinin (BK), eNOS-dependent dilators; nitroprusside (NP), a NO releaser at guanylyl cyclase; nifedipine (NIF), a Ca2+ channel blocker; butanedione monoxime (BDM), an intracellular Ca2+ inhibitor; zaprinast (ZAP), a phosphodiesterase (PDE) type IV inhibitor of cGMP breakdown; prostaglandin (PG) E1, a cAMP activator; and bimakalim (BIM), a KATP+ channel opener. Their putative pathways are depicted in Fig. 1.


Figure 1
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  		Fig. 1 Simplified drawing of postulated or known mechanism of action of 11 compounds that produce or inhibit (in parentheses) relaxation of vascular smooth muscle via endothelium-dependent and -independent pathways. Several of the agonists (e.g. PGI2) and antagonists (e.g. INDO, TEA) may have effects in both cell types. INDO, indomethacin; PGI2, prostaglandin I2; BDM, butanedione monoxime; 5-HT, 5-hydroxytryptamine (serotonin); BK, bradykinin; NP, nitroprusside; L-NAME, nitro L-arginine methylester; NIF, nifedipine; ZAP, zaprinast; TEA, tetraethylammonium; BIM, bimakalim. PL, phospholipids; PLA2, phospholipase A2; AA, arachidonic acid; COG, cyclooxygenase, PGS, prostaglandin synthase; L-arg, L-arginine; NOS, nitric oxide synthase; NO, nitric oxide; G, G (guanine nucleotide-binding) protein; cAMP, cyclic adenosine monophosphate; AC, adenylyl cyclase; cGMP, cyclic guanine monophosphate, SGC, soluble guanylyl cyclase; PDE IV, phosphodiesterase type IV.

 

   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Langendorff heart preparation
The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1995). After approval was obtained from the institutional Animal Studies Committee at the Medical College of Wisconsin, 10 mg of ketamine and 1000 U of heparin were injected intraperitoneally into 45 albino English short-haired guinea pigs (250–300 g). The description of the surgical preparation for this model has been reported in detail previously [22]. The animals were decapitated when unresponsive to noxious stimulation. After thoracotomy, the inferior and superior venae cavae were ligated and cut and the aorta was cannulated distal to the aortic valve. Each heart was perfused in retrograde fashion through the aorta with cold, oxygenated, modified Krebs–Ringer's solution equilibrated with 97% O2 and 3% CO2 and was then rapidly excised. The perfusate, a modified Krebs–Ringer's salt solution, was disk filtered (5 µm pore size) in-line and had the following control composition in mmol/l: Na+ 137, K+ 5, Mg2+ 1.2, Ca2+ 2.5, Cl– 134, HCO3– 15.5, H2PO4– 1.2, glucose 11.5, pyruvate 2, mannitol 16, EDTA (ethylenediaminetetraacetic acid) 0.05, and insulin 5 U/l. Perfusate, bath and O2 and NO electrode temperatures were maintained tightly at 37.2±0.1°C using a thermostatically controlled water circulator to jacketed glass tubing, bath, and aluminum heat exchangers.

Left ventricular pressure (LVP) was measured isovolumetrically with a transducer connected to a thin, saline-filled latex balloon (Hugo Sachs Electronic KG, March–Hugstetten, Germany) inserted into the left ventricle through the mitral valve from a cut in the left atrium. Balloon volume was adjusted to maintain a diastolic LVP of zero mmHg during the initial control period. Two pairs of bipolar electrodes (Teflon-coated silver, diameter 125 µm, Cooner Wire Chatsworth, CA) were placed in each heart to monitor intracardiac electrograms from which spontaneous atrial heart rate (HR) was determined from the right atrial beat-to-beat interval.

Coronary (aortic) inflow (CF) was measured at constant temperature and at a nominal aortic perfusion pressure of 55 mmHg by a self-calibrating in-line ultrasonic flow meter (Transonic T106X small animal blood flow meter; Transonic System, Ithaca NY) placed directly into the aortic inflow line. Basal flow for all studies was 6.3±2 ml/g/min. Maximal CF was elicited with adenosine (0.2 ml of 200 µmol/l stock solution) injected directly into the aortic root cannula during the initial control period and after the last control reading. Beginning and ending flow responses to adenosine (arrested hearts) were 16.3±2 and 12.5±3 ml/g/min, respectively, for all groups combined. In six hearts perfusion pressure was changed abruptly from 55 to 25, from 25 to 55, from 55 to 90, and from 90 to 55 mmHg for 5 min at each level to simultaneously measure steady-state changes in flow and [NO].

2.2 Nitric oxide, oxygen and pH measurements
Coronary sinus effluent was collected by placing a small, gas impermeable cannula into the right ventricle through the pulmonary artery after ligating the superior and inferior venae cavae. Effluent nitric oxide (NO), pH and O2 tension were measured continuously on-line. Coronary outflow (coronary sinus) O2 tension and pH were measured continuously on-line with a miniature thermostabile Clark O2 electrode (Instech Laboratories, Model 203B, Plymouth Meeting, PA) and temperature compensated pH electrode (micro computer pro-vision pH meter, model 05669-20, pH electrode PHE 2121, Cole Palmer Instruments, Vernon Hills, IL). Coronary inflow and effluent pH and O2 and CO2 tensions were measured during each maneuver off-line at 37°C with an intermittently self calibrating analyzer system (Radiometer ABL-2, Medtron Chicago, Des Plaines, IL). Coronary effluent CO2 tension was calculated from the on-line pH signal with HCO3– assumed constant at 15 mmol/l; calculated effluent CO2 tension at a given pH was verified off-line with the gas analyzer.

NO concentration was measured as the change in redox current (pA) generated by a gas permeable, water impermeable NO electrode (ISO-NOP 2 mm, World Precision Instruments, Sarasota, FL). The electrode probe measures NO concentration in aqueous solutions polarographically [20]. The electric current is generated as NO diffuses through the membrane and becomes oxidized at the platinum electrode. The generated current is proportional to diffusion of NO through the membrane and diffusion is based on its partial pressure which, in turn, is proportional to [NO] at the probe tip. Current is measured with a sensitive amperometer during zero voltage suppression to expand the response range. Selectivity of the membrane for NO over other gases is determined by the potential applied to the electrode. The presence of NO2 gas (2NO+O2=2 NO2) could generate a current but in aqueous solution NO2 is highly unstable and at physiological pH degrades to NO2– and NO3– which do not penetrate the membrane. A possible effect CO2 on altering pH of the internal electrolyte solution was minimized by used a two-part buffered electrolyte solution (catalog no. 7521) furnished by the supplier.

NO calibration curves were generated by graded chemical production of NO at 37°C in a stirred bath where KI and H2SO4 are in excess: 2NaNO2+2KI+2H2SO4=2NO+I2+2H2O+K2SO4+2Na2SO4. Calibration was carried out 1 day after changing the gas permeable membrane and internal filling solution which were changed weekly. Five point calibration curves (n=14) of current (pA) as a function of [NO] (50–1000 nmol/l) gave a correlation coefficient (r2) of 0.99 (P<0.001), a slope of 2.92±0.16 (95% confidence intervals) and a y-intercept of 9.4±41 pA (P>0.1). For every pA increase in current, NO concentration therefore increased by 0.34 nmol/l. There was no significant change in NO electrode sensitivity (≥1 nmol/l) over a 1-week period. Transient time from the right ventricle (coronary sinus) cannula to the NO electrode determined by methylene blue infusion, was 0.5 s at a flow of 5 ml/min.

Percentage O2 extraction was calculated as the difference between inflow and outflow tensions multiplied by 100, and divided by inflow O2 tension. Percent O2 extraction was measured in all studies and used to assess direct vasodilatory responses separate from those due to an autoregulatory response, e.g. a decrease in CF secondary to decreased contractility. In the absence of O2 debt, an imbalance of O2 consumption to O2 delivery reflects a change in coronary vascular tone. Use of this measurement assumes that local metabolites are produced in proportion to myocardial O2 consumption and that local metabolites are major factors controlling autoregulation of coronary flow. O2 delivery (DO2) was calculated as inflow O2 tension in mmHg, multiplied by O2 solubility (24 µl/ml Krebs–Ringer's solution at 760 mmHg O2 and 37°C), multiplied by coronary flow (CF, ml/min), and then divided by the wet weight of each heart (1.90±0.06 g (s.e.m.)). O2 tension of the inflow perfusate was kept constant by maintaining pressure 5 mmHg above atmospheric pressure in the reservoir containers. Myocardial O2 consumption (MVO2) was calculated as O2 solubility multiplied by the difference between inflow and outflow O2 tensions times coronary flow per gram of wet heart tissue. Relative cardiac efficiency was calculated as LVP times HR divided by MVO2. DO2 divided by MVO2 defines the O2 supply to demand ratio. Electrograms, spontaneous heart rate, AV conduction time, outflow O2 (mmHg), (pH, in mV), coronary flow, systolic and diastolic isovolumetric left ventricular pressure (DLVP, SLVP), and NO electrode current (pA) were displayed continuously on a fast-writing (3 kHz), high resolution, eight channel chart recorder (Astro-Med, West Warwick, RI). NO (pA) and pH (mV) electrode signals were zero suppressed and amplified for continuous display.

2.3 Measurement of L-citrulline
L-Citrulline was measured in coronary effluent samples of eight hearts in the absence and presence of 0, 0.1, 1, and 10 nmol/l bradykinin by high performance liquid chromatography (HPLC). The HPLC system consisted of a Laboratory Data Control (LDC) Constametric III G pump, a Gilson Automatic Sampler Model 231, and an electrochemical detector (Bioanalytical Systems, BAS LC-4B). The column (Beckman Ultrasphere ODS 5 µ, 4.6 mmx25 cm) was perfused at a mobile phase flow rate of 1.5 ml/min. The detector potential was set at +0.7 V. The mobile phase consisted of 800 ml 0.1 mol/l sodium acetate, pH adjusted to 5.7, plus 260 ml acetonitrile. L-Citrulline was detected electrochemically as the o-phthaldialdehyde (OPA) derivative. The OPA reagent consisted of 25 ml 0.1 mol/l borate buffer (pH 9.5), 50 µl of 2-methyl 2-propanethiol, 2.5 ml methanol, and 135 mg of o-phthaldialdehyde. All chemicals were HPLC grade. Chromatographic data was collected on a Hewlett Packard 3393A integrator and stored on a Hewlett Packard 9122 disc drive. Coronary venous effluent was collected (2 ml) during drug-free control periods and during the last 30 s of BK infusion and frozen at –15°C. Samples were later prepared and analyzed as follows: to each 0.5 ml sample was added 25 µl of methyl-L-arginine (2 µg/ml) as an internal standard. Three ml ethanol was added to each sample, mixed, and centrifuged. The supernatant was transferred to a clean tube and evaporated to dryness under a stream of air at 40°C. The dried residue was redissolved in 2.0 ml of mobile phase and 400 µl was mixed with 40 µl of the OPA reagent for exactly 2.00 min prior to injection of 100 µl into the HPLC. L-Citrulline concentration was calculated from the standard curve of the respective peak height ratio versus concentration. Standard curve data were derived using perfusate that did not pass through the isolated heart. L-Citrulline standard curves was linear over the concentration range studied. The limit of detectability was 1 ng/ml perfusate. The absolute retention time for L-citrulline was 10.3 min.

2.4 Protocol
Experiments to validate the NO electrode were conducted in the experimental setup, in the absence of hearts, using a flow restrictor between the inflow and outflow tubing to imitate the coronary circulation. Factors examined for their potential artifactual effects on the NO electrode were flow, pressure, O2 and CO2 tensions (pH), and temperature. Preliminary trials were then undertaken in 14 isolated hearts to tightly control heart and effluent temperature and to measure effluent pH with changes in flow (data not reported). After preliminary trials were completed, eight differently acting vasodilator drugs were examined for effects on CF, on other cardiac variables, and on release of NO, in 14 additional hearts. Adenosine was given to determine maximal flow, and at least 30 min was allowed to stabilize variables before obtaining initial control measurements.

Dose response curves were generated for randomly administered concentrations of 5-HT (0.1, 0.5, 1 µmol/l), BK (1, 10, 100 nmol/l), or NP (10, 50, 100 µmol/l). Maximally vasodilating concentrations of NIF (20 nmol/l), BDM (1 mmol/l), ZAP (10 µmol/l), PGE1 (50 nmol/l) or BIM (1 µmol/l) were also infused in random fashion. Only 5-HT, BK, or NP was given with one or two drugs of the other group in each heart to minimize any drug interactions or long term effects. CF and NO current returned approximately to the control level after washout of each drug. The putative site of action of several of these drugs on vasodilator pathways is shown in the simplified schema (Fig. 1).

The role of several inhibitors of endogenous vasodilators on basal CF and release of NO was examined in 11 additional hearts. The individual and combined effects of single concentrations of INDO (20 µmol/l), TEA, (10 mmol/l), and L-NAME (100 µmol/l) were assessed for their effects on lowering basal CF and altering NO. Basal (absolute) NO concentration was not measured, but was inferred after NOS inhibition by L-NAME (see Section 3). Effect of these inhibitors to blunt vasodilation induced by BK (1, 10, 100 µmol/l) was also examined. In 6 additional hearts perfusion pressure was altered to examine changes in flow-induced changes in [NO] in the presence and absence of L-NAME (100 µmol/l).

Measurements were obtained during the peak steady state change in CF during exposure to each concentration of a drug infused for 5 min. All drugs and concentrations were given in random fashion. Drug vehicles had no effects on any variable measured. There was a 10 min drug-free washout period between each drug administration. NO concentration (nmol/l) or release (pmol/g/min) were expressed as the change from the preceding control. After the last control period, adenosine was again injected at the same concentration into the aortic cannula to observe any change in maximal CF reserve.

2.5 Statistics
All data are expressed as means±s.e.m. or slope±95% confidence intervals. Individual drug responses were compared to the preceding control by paired Student's t-tests. Dose–response curves to bradykinin (BK) were compared by Tukey's comparison of means tests following ANOVA for repeated measures (Super Anova 1.11 software for Macintosh from Abacus Concepts, Berkeley, CA). Calibration curves for NO and NO electrode responses to flow, pressure, oxygen, temperature and CO2 (in the absence of hearts), and the CF to NO release relationship in the presence of several concentrations of vasodilatory drugs, were fitted by linear regression analysis to determine correlation coefficients, significance of slopes and confidence intervals. Equations are of the form Y=mX+B, where Y is a function of X, and m=slope, and B=Y slope intercept. Differences among means were considered statistically significant when P≤0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Validation of nitric oxide electrode
Within the physiological range of studies conducted in the isolated hearts, but in the absence of hearts, the NO electrode was insensitive to changes in flow between 0 and 30 ml/min (Y=0.007·flow +3.8; r2=0.15; P>0.1), O2 tension between 50 and 250 mmHg (Y=0.0001·pO2 (mmHg)+1.26; r2=0.01; P>0.1), and pressure above barometric between 10 and 100 mmHg at 10 ml/min constant flow (Y=0.005·mmHg+2.1; r2=0.01; P>0.1). The NO electrode was markedly sensitive to artificially induced changes in bath temperature in the absence of hearts (+24.5 nmol/l/°C) (Fig. 2). To control for this effect during isolated heart experiments, the temperature of the coronary effluent passing through the NO electrode chamber was held to a change of <0.05°C by the heat exchanger during a maximal range in flow of 4–18 ml/min. Thus there was no appreciable change in coronary effluent temperature to alter NO electrode sensitivity. The NO electrode was slightly sensitive to changes in CO2 induced by changing percent CO2 gas flow in the gas mixing chamber in the absence of hearts. The small effect of a change in effluent H+ concentration (–0.22 nmol/l/mV), or pCO2 (–0.50 nmol/l/mmHg), was factored in at each data point by continuously measuring coronary effluent pH (Fig. 3A,B). The maximal decrease in coronary effluent CO2 tension from control CF (6.5±0.2 ml/g/min) to a maximal steady-state increase in CF (13.3±2 ml/g/min) was 4 mmHg, i.e., an apparent increase in [NO] of about 2 nmol/l.


Figure 2
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  		Fig. 2 Significant effect of temperature on apparent NO current. For a 1°C increase in electrode temperature apparent [NO] increased by 24.5 nmol/l. n=12 studies conducted in the Langendorff apparatus in the absence of hearts. The effect of increased coronary effluent flow to increase effluent temperature was circumvented by enclosing the NO electrode and flow through tubing in a water jacketed aluminum block so that temperature was maintained constant.

 

Figure 3
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  		Fig. 3 Significant effect of pH (A) and pCO2 (B) on apparent NO current. n=6 non-heart studies. In isolated heart studies as coronary flow increased, [H+] decreased maximally about 10 mV (60 mV/pH unit), corresponding to a 4 mmHg maximal decrease in pCO2. The apparent maximal increase of [NO] of 2 nmol/l with an increase in flow was factored out in calculating effective [NO].

 
3.2 Effects of vasodilators on coronary flow, NO release and cardiac function
Fig. 4 shows typical responses to 10 nmol/l bradykinin (BK). The time course of NO electrode current closely paralleled CF. The maximal increase in coronary effluent NO concentration was 23 nmol/l–2 nmol/l (due to the decreased pCO2 (increased pH) effect), or 21 nmol/l, for an increase in CF from 4.8 to 15.2 ml/min. Fig. 5A–C summarizes the increase in CF as a function of the increase in NO release elicited by three concentrations of each of two drugs believed to stimulate production of NO via NOS (5-HT, BK) and one drug that releases NO on contact with guanylyl cyclase (NP). The CF/NO slope was apparently steepest for BK (see Section 4). Fig. 6 displays the relationship between effluent concentrations of L-citrulline and NO during infusion of increasing concentrations of bradykinin. Both effects are concentration dependent. Fig. 7 shows that in contrast to responses to 5-HT, BK and NP, the vasodilators NIF, BDM, ZAP, PGE1, and BIM did not increase NO release with equivalent increases in flow. Fig. 8A–D displays other cardiac effects of the eight vasodilators. All drugs, except BDM and ZAP, variably increased HR. BDM and BIM greatly decreased LVP, whereas BK, NP, ZAP, and PGE1 slightly increased LVP. Each drug markedly decreased %O2E and MVO2 in approximate proportion to the changes in CF and LVP, respectively.


Figure 4
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  		Fig. 4 Representative tracings of pH (in mV), NO current, effluent O2 tension, coronary flow and left ventricular pressure before and during a 5 min infusion of 10 nmol/l bradykinin in one isolated heart experiment. The effluent NO response paralleled that of effluent O2 tension, pH and coronary flow.

 

Figure 5
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  		Fig. 5 Increase in NO release as a linear function of the increase in CF for three concentrations each of (A) serotonin (5-HT, 0.1, 0.5, 1 µmol/l), (B) bradykinin (BK, 1, 10, 100 µmol/l), and (C) nitroprusside (NP, 10, 50, 100 µmol/l).

 

Figure 6
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  		Fig. 6 Concentration-dependent increases in NO and L-citrulline concentration in coronary effluent with infusion of BK. Basal L-citrulline concentration was 10±3 nmol/l. Note the differences in scales.

 

Figure 7
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  		Fig. 7 Different effects of eight vasodilators on increasing coronary flow and NO concentration. 5-HT, BK, and NP increased [NO] along with coronary flow, whereas NIF (nifedipine), BDM (butanedione monoxime), ZAP (zaprinast), PGE1 (prostaglandin E1), and BIM (bimakalim) produced equivalent increases in coronary flow, but no significant increases in [NO]. NOS/GC, nitric oxide synthase/guanylyl cyclase.

 

Figure 8
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  		Fig. 8 Other cardiac effects of eight vasodilators; heart rate (A), LV pressure (B), % O2 extraction (C), and myocardial O2 consumption (MVO2) (D). See Fig. 1 for drug abbreviations.

 
Fig. 9A,B displays the effect of perfusion pressure-induced changes in steady-state coronary flow (shear stress) on altering [NO]. Decreasing (55 to 25 mmHg) and increasing (55 to 90 mmHg) perfusion pressure did not alter significantly the MVO2 (44±4 and 52±4, respectively; control 49±3 µl/g/min) because effluent pO2 decreased and increased proportionately. In the absence of L-NAME (control), [NO] changed proportionally with coronary flow during changes in perfusion pressure. In the presence of L-NAME, basal coronary flow and basal [NO] decreased significantly at the control perfusion pressure of 55 mmHg. Fig. 10 illustrates that [NO] varied as a function of the change in pressure-induced steady-state change in CF without L-NAME (control), but that [NO] did not vary as a function of a similar change in CF with L-NAME. When the change in [NO] was expressed as a linear function of change in CF over this pressure range, the slope was 4.0 (r2=0.87; P<0.01) without L-NAME and 1.1 (r2=0.31; P>0.1) with L-NAME. Overall, L-NAME reduced the flow-induced rise in [NO] over the pressure range from 30±4 to 10±5 nmol/l.


Figure 9
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  		Fig. 9 Effect of changing perfusion pressure from control (55 mmHg) on steady-state NO concentration (A) and coronary flow (B) in the absence (control) and presence of 100 µmol/l L-NAME. Peak transient changes in flow (not shown) were approximately twice that of steady-state flow. During control, induced decreases in perfusion pressure decreased flow and [NO] while induced increases in perfusion pressure increased flow and [NO]. In the presence of L-NAME, basal flow and [NO] decreased during control (55 mmHg) and induced decreases and increases in perfusion pressure produced similar changes in flow as in control, but changes in [NO] were significantly reduced.

 

Figure 10
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  		Fig. 10 Changes in coronary flow and [NO] induced by changes in perfusion pressure from the preceding pressure. Starting pressure was 55 mmHg (0 mmHg change). Note that [NO] increased proportionally with flow during control, but not in the presence of L-NAME. See Fig. 9A,B for additional details.

 
Fig. 11A,B shows individual and combined effects of inhibition of PG synthesis by INDO, non-specific K+ channel blockade by TEA, and inhibition of NO production by L-NAME on decreasing basal CF and NO release. As shown, INDO had no effect on CF or NO release, TEA decreased CF by 18% without an effect on NO release, and L-NAME decreased CF by 24% and NO release by 25 pmol/g/min. Together, these drugs reduced basal CF by up to 40% and NO release by up to 42 pmol/g/min, but NO release was not different (P>1.0) with all three drugs than with L-NAME alone.


Figure 11
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  		Fig. 11 Effect of inhibitors of prostaglandins (INDO, 20 µmol/l), non-specific K+ channels (TEA, 10 mmol/l), nitric oxide synthase (L-NAME, 100 µmol/l), and their combinations, on decreasing basal coronary flow (A) and NO release (B). TEA and L-NAME singularly and together decreased basal coronary flow about 20%, but only L-NAME decreased NO release. INDO alone had no significant effect on coronary flow or NO release. See Fig. 1 for drug abbreviations.

 
Fig. 12A,B shows the effects of PG, NOS and non-specific K+ channel inhibition on BK-induced CF and effluent [NO]. INDO alone did not change CF; INDO+TEA decreased basal CF by 1.8±0.3 ml/g/min but had no effect on basal [NO]. L-NAME alone or with INDO and TEA decreased basal CF by about 2.6±0.2 ml/g/min from the drug free control and decreased basal [NO] by about 9.5±1.5 nmol/l. On stimulation by BK alone (control), CF increased by up to 6.2±3 ml/g/min by 10 nmol/l BK, and [NO] increased by up to 27±2 nmol/l in a concentration-dependent manner as similarly shown in Figs. 5 and 6Go for NO release. INDO alone, or +TEA, had no significant effects on CF or [NO] responsiveness to BK, whereas L-NAME alone, or +INDO+TEA, abolished the increase in CF due to 0.1 nmol/l BK, and attenuated the increase in CF due to 1 nmol/l BK, whereas the increase in [NO] due to 0.1 and 1 nmol/l BK was abolished. Although L-NAME, alone or in combination with INDO or TEA, markedly attenuated the increase in [NO] induced by 10 nmol/l BK, CF was only slightly reduced from controls by L-NAME and by L-NAME+INDO in the presence of 10 nmol/l BK.


Figure 12
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  		Fig. 12 Effect of inhibitors of prostaglandins (INDO, 20 µmol/l), non-specific K+ channels (TEA, 10 mmol/l), nitric oxide synthase (L-NAME, 100 µmol/l), on altering coronary flow (A) and NO concentration (B) in the absence and presence of three concentrations of BK. In the absence of BK (0 nmol/l) INDO alone had no effect on coronary flow or [NO], TEA and L-NAME (with INDO) decreased coronary flow, and L-NAME, but not INDO and TEA, decreased [NO]. Increasing concentrations of BK increased coronary flow and [NO] in the presence of INDO alone or with TEA, but in the presence of L-NAME (with TEA and/or INDO) coronary flow did not increase with 0.1 and 1 nmol/l BK, but increased with 10 nmol/l BK, while [NO] increased slightly only at 10 nmol/l BK. See Fig. 1 for drug abbreviations.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Validation of nitric oxide electrode
Our study demonstrates the capability for real-time detection and quantification of nitric oxide in hemoglobin-free coronary effluent of guinea pig isolated hearts by recording the change in current generated by a sensitive NO electrode placed within the pulmonary outflow (coronary sinus) effluent. We observed that the electrode was itself insensitive to flow, pressure, and O2 within the physiologic ranges obtained in the isolated heart studies. However, because we found that the electrode is quite sensitive to a change in temperature and is moderately pCO2 sensitive, it was important to rigidly control coronary effluent temperature with a heat exchanger and to compensate for the small decrease in pCO2 that occurs with an increase in CF. During extensive testing we found that NO electrode sensitivity to pCO2 is determined by the buffering capacity of the internal filling solution. We were able to reduce CO2 sensitivity so that a maximal physiological decrease in pCO2 of 4 mmHg increased the apparent NO sensitivity less than 10%. However, we also found that a decrease in CO2 sensitivity is a trade off for a shorter usable period before the membrane and filling solution must be changed and the system recalibrated. Consequently, the electrode membrane was changed and the electrode recalibrated once per week. Calibration curves were very reproducible from week to week.

4.2 Ex vivo measurement of nitric oxide
For the vast majority of studies on the role of NO on endothelial and vascular function investigators have used inhibitors of eNOS and measures of guanylyl cyclase activity or cGMP production as research tools. But there are now several methods to measure NO indirectly or directly in solutions. Bioassay [12], chemiluminescence [2, 9, 13, 14], electron paramagnetic response spectra [15], and spectrophotometric [8, 16]methods have been used to quantitate NO levels. Compared to these assays, the NO sensitive electrode has several potential advantages in that it measures changes in NO-generated current continuously, i.e. in real time, in organ effluent or superfused tissues. There are a few reports in which NO electrodes have been used to measure [NO] in superfusate solution from brain and cultured vascular endothelial tissues [17, 18, 21]. In our experimental setup we were able to simultaneously measure coronary effluent NO, O2 and pH at constant temperature. Changes in NO current were recorded within 0.5 s. The half-life of NO in aqueous solution lies between 3.8 and 5.6 s, but may be as short as 0.1 s when NO is perfused in an oxyhemoglobin solution through an isolated heart [8]. In our model, the tubing and electrode chamber were sealed against gas leakage. However, it remains possible that there is a proportionally small loss of NO after its release into hemoglobin-free Krebs–Ringer perfusate while in transit to the electrode, which would lead to an underestimated [NO].

A direct measure of NO in isolated heart coronary effluent is useful to confirm the many indirect studies in which NO release has been implied by using inhibitors of eNOS such as L-NAME. More importantly, the NO electrode allows direct confirmation of which vasodilator drugs induce vasorelaxation via the eNOS–guanylyl cyclase pathway. There are several indirect reports, based on eNOS inhibition or perfusion of NO scavengers, that coronary flow in the guinea pig and dog is regulated by NO [8, 23–25]. A potential limitation of our method is that vascular luminal release of NO may not reflect abluminal release of NO across interstitial fluid to the vascular smooth muscle cell. However, it is likely that NO luminal release is at least proportional to abluminal release in the coronary vascular bed perfused with a hemoglobin-free perfusate that imitates the interstitial fluid. The mean decreases in [NO] (9.5±2 nmol/l) and NO release (33±8 pmol/g/min, or 59±9 pmol/min) when L-NAME was infused suggests that these are basal (unstimulated) values for [NO] and NO release. Indeed, the decrease in [NO] by L-NAME was associated with a decrease in basal CF of about 20%. If this approximation is true, then stimulated endothelial production of NO, e.g. by 5-HT, increased absolute [NO] from about 10 nmol/l basally to about 30 nmol/l, a three-fold increase (data of Figs. 7 and 12Go). Kelm and Schrader [8]collected the coronary effluent of guinea pig hearts perfused with 4 µmol/l oxyhemoglobin and used a differential-spectrophotometric assay in which methemoglobin was produced in proportion to NO as NO combined with oxyhemoglobin. They estimated that basal release of NO was about 160±10 pmol/min, more than twice that estimated in our study. Ellwood and Curtis [9]assessed NO production indirectly from frozen aliquots of coronary effluent from isolated guinea pig hearts by a chemiluminescence method. They reported a 5- to 10-fold greater increase in NO release with 5-HT and NP than we did using our real-time electrode measurement. It is unclear if our NO measurements are underestimated, or those of the other investigators are overestimated. Our companion studies of L-citrulline measurements show a three-fold larger concentration that that of NO (Fig. 6).

4.3 Drug and pressure-induced changes in coronary flow and NO release
Our data shows that CF increases proportionately with NO release by 5-HT, BK and NP (Figs. 5 and 7Go), whereas NIF, BDM, ZAP, PGE1 and BIM similarly increased flow but not [NO] (Fig. 7). These results clearly confirm indirect experiments by others that 5-HT, BK, and NP produce vasodilation via the eNOS, and for NP the guanylyl cyclase, pathways and demonstrate, moreover, that vasodilation by NIF, BDM, ZAP, PGE1 and BIM is not related to eNOS activity or NO release. Kelm and Schrader [8]reported that 100 nmol/l of BK increased NO release about 300 pmol/min. This agrees with the maximal (Fig. 7) release ([NO]·CF) of NO (146±19 pmol/g/min, or 225 pmol/min at the heart weight average 1.6 g) we obtained with 100 nmol/l BK. A major difference in our studies is that we did not perfuse hearts with the NO scavenger oxyhemoglobin so that NO effluent levels measured in our preparation more likely reflect interstitial (abluminal) production of NO. Moreover, we have measured the more stable metabolite of NOS, i.e. L-citrulline in coronary effluent and report that L-citrulline and NO concentration increase together with infusion of bradykinin (Fig. 6). Since L-Citrulline concentrations were about three times greater than the corresponding NO concentrations. This could result from endothelial re-uptake of NO, loss of NO on transit to the NO electrode, or NO degradation at guanylyl cyclase.

Along with our examination of effects of different exogenous vasodilators on stimulating release of NO and CF, we examined effects of changes in perfusion pressure (wall stretch) on coronary flow (shear stress)-induced [NO] as well as effects of endogenous vasodilatory factors that contribute to maintenance of basal coronary flow by blocking several pathways that lead to vasodilation. It has been reported that NO modulates coronary autoregulatory responses during changes in coronary perfusion pressure [8, 23–25]. An increase in laminar flow over cultured endothelial cells induces NO release [26]but NO release to continuous shear stress is transient [21]. Autoregulation, although incomplete between 25 and 90 mmHg (Fig. 9B), was evidenced by a marked reduction in peak flow of about 50% induced by abrupt changes in perfusion pressure. Decreased and increased CF resulting from changes in perfusion pressure were associated with decreased and increased effluent [NO], respectively, but in the presence of L-NAME the NO responses were significantly attenuated by approximately 67% while the pressure-induced flow responses were not. Our results indicate that [NO] indeed varies with pressure-induced changes in coronary flow (shear stress) and that L-NAME reduces basal [NO] and CF at normal perfusion pressure. However, our observation that L-NAME greatly attenuates changes in [NO], but not changes in CF resulting from changes in perfusion pressure, suggests that factors in addition to NO contribute to autoregulation of CF. It is also possible that L-NAME does not completely block NO production and release.

It is noteworthy that pressure-induced changes in flow (shear stress) produced changes in [NO], whereas the several eNOS/guanylyl cyclase-independent vasodilators, e.g. BIM or NIF, also similarly increased CF but did not increase [NO] (Fig. 7). However, NO release ([NO]·CF) increased with several of these drugs with the increase in CF. It appears, therefore, that the NO vs. CF relationship in the intact heart is different if CF is increased by increasing perfusion pressure or by infusing vasodilatory drugs. Vasodilator drugs that are either eNOS/guanylyl cyclase-independent or -dependent increase CF by reducing smooth muscle tone so that vessel diameter is actively increased and this may have no effect on shear stress despite the flow increase. On the other hand, pressure-induced changes in flow directly alter vessel diameter passively by a change in the pressure gradient and so may produce a proportionate change in shear stress. This may explain why a large increase in CF by BIM is not accompanied by an increase in [NO], whereas a comparable increase in CF – induced by an increase in perfusion pressure – is accompanied by an increase in [NO]. The rapidly attenuated initial peak CF response to an abrupt change in pressure could be due to myogenically mediated vasoconstriction.

There may be other complex reasons for these apparent differences in cause and effect between CF and [NO] [11, 27]. Feedback mechanisms are probably quite different [28]. In our model it is not possible to separate pressure (wall stretch) from flow (shear stress) alterations as contributors to NO production. It is likely that drug-induced vasodilation overrides the metabolic autoregulatory mechanisms that maintain basal CF and lead to decreased O2 extraction as we have observed, whereas pressure-induced vasodilation is counteracted by myogenic autoregulatory mechanisms so that CF is relatively maintained while causing coronary distension, increased transmural pressure, and decreased O2 extraction [29, 30].

Coronary vascular tone is modulated by many endogenous substances and one vasodilatory mechanism is clearly eNOS dependent. Other vasodilatory mechanisms can be unmasked with inhibitors. We found that non-specific blockade of K+ channels (TEA) and blockade of NO production (L-NAME) reduced basal CF, but that only L-NAME reduced [NO] and NO release. Together, indomethacin (to block PG synthase inhibition), TEA and L-NAME reduced basal flow maximally by 40%, indicating a significant portion of basal vasodilation can be accounted for by blocking specific vasodilator pathways. If abluminal levels of NO reflect interstitial levels at the guanylyl cyclase receptor site for NO in smooth muscle, the data suggests that NO contributes to about 20% of basal CF.

Bradykinin has been reported to produce vasodilation by several mechanisms [31–34]. There may be a direct vasodilator effect on vascular smooth muscle at high BK concentrations. We additionally tested the vasodilatory effect of BK on CF and NO in the presence of indomethacin, and/or TEA and/or L-NAME. In our experiments, indomethacin alone did not alter the BK-induced increase in CF or release of NO. However, the combination of indomethacin and L-NAME and that of indomethacin, L-NAME and TEA, depressed the ≤1 nmol/l BK-induced increase in CF and NO. Although 100 µmol/l of L-NAME completely blocked the release of NO to 10 nmol/l BK, it did not completely block the increase in CF. Thus in the presence of all three inhibitors of vasodilation, the BK-induced increase in CF could not be completely abolished. These results suggest that the major mechanism of vasodilation by BK below 1 nmol/l is dependent on NO release but at higher concentrations (10 nmol/l), BK exerts an additional mechanism of vasodilation, perhaps a direct effect on vascular smooth muscle.

In summary, these results demonstrate real-time release of NO directly into the vascular lumen of hemoglobin-free coronary effluent by endothelium-dependent drugs, serotonin (5-HT) and bradykinin, as well as by nitroprusside. The release of NO is proportional to the increase in coronary flow by the eNOS dependent drugs and by nitroprusside. Inhibition of endogenous vasodilation due to eNOS activity by L-NAME is evidenced by reduced [NO] and NO release along with a decrease in CF. Drugs thought not to produce vasodilation via an eNOS-dependent mechanism, i.e., butanedione monoxime, zaprinast, bimakalim, PGE1 and nifedipine, indeed do not increase [NO]. Pressure-induced changes in CF lead to proportional changes in effluent NO concentrations and these changes in NO, but not CF, are attenuated by eNOS antagonism.

Time for primary review 23 days.


   Acknowledgements
 
This research was supported by grants from: Veterans Administration Merit Review (8204-P) and the American Heart Association (WI 95-GS-85, and WI 94-POST-26). The authors wish to thank James S. Heisner, BS, WW Chung, BS, and Enis Novalija, MD, for their valuable assistance in conducting this project and William M. Chilian, PhD, for helpful suggestions.


   Notes
 
1 Portions of this work have appeared in abstract form (Anesthesiology 1995;83:A621, FASEB J 1996;10:A156 and 1996;10:A571 and Anesth Analg 1996;82:S119). Back


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
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