Journal of Applied Physiology
Vol. 82, No. 2, pp. 686-692, February 1997
PULMONARY CIRCULATION AND LUNG FLUID BALANCE

Nitric oxide and -adrenergic agonist-induced bronchial arterial vasodilation

Nirmal B. Charan^1,3, Shane R. Johnson^1,3, S. Lakshminarayan^2,3, William H. Thompson^1,3, and Paula Carvalho^1,3

¹ Pulmonary Research Laboratory, Veterans Affairs Medical Center, Boise, Idaho 83702; ² Veterans Affairs Medical Center, Seattle, Washington 98108; and ³ Division of Pulmonary/Critical Care Medicine, Department of Medicine, University of Washington, Seattle, Washington 98195

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Charan, Nirmal B., Shane R. Johnson, S. Lakshminarayan, William H. Thompson, and Paula Carvalho. Nitric oxide and -adrenergic agonist-induced bronchial arterial vasodilation. J. Appl. Physiol. 82(2): 686-692, 1997.In anesthetized sheep, we measured bronchial blood flow (br) by an ultrasonic flow probe to investigate the interaction between inhaled nitric oxide (NO; 100 parts/million) given for 5 min and 5 ml of aerosolized isoetharine (1.49 × 10² M concentration). NO and isoetharine increased br from 26.5 ± 6.5 to 39.1 (SE) ± 10.6 and 39.7 ± 10.7 ml/min, respectively (n = 5). Administration of NO immediately after isoetharine further increased br to 57.3 ± 15.1 ml/min. NO synthase inhibitor N-nitro-L-arginine methyl ester hydrochloride (L-NAME; 30 mg/kg, in 20 ml saline given iv) decreased br to 14.6 ± 2.6 ml/min. NO given three times alternately with isoetharine progressively increased br from 14.6 ± 2.6 to 74.3 ± 17.0 ml/min, suggesting that NO and isoetharine potentiate vasodilator effects of each other. In three other sheep, after L-NAME, three sequential doses of isoetharine increased br from 10.2 ± 3.4 to 11.5 ± 5.7, 11.7 ± 4.7, and 13.3 ± 5.7 ml/min, respectively, indicating that effects of isoetharine are predominantly mediated through synthesis of NO. When this was followed by three sequential administrations of NO, br increased by 146, 172, and 185%, respectively. Thus in the bronchial circulation there seems to be a close interaction between adenosine 3,5-cyclic monophosphate- and guanosine 3,5-cyclic monophosphate-mediated vasodilatation.

bronchial circulation; bronchial blood flow; isoetharine; adenosine 3,5-cyclic monophosphate; guanosine 3,5-cyclic monophosphate

INTRODUCTION

NITRIC OXIDE (NO) and -adrenergic-receptor agonists (-ARAs) are two potent vasodilating agents. It is generally believed that -ARAs produce vasodilatation via activation of adenylyl cyclase and consequent stimulation of adenosine 3,5-cyclic monophosphate (cAMP) formation, whereas NO acts through guanosine 3,5-cyclic monophosphate (cGMP). However, recently it has been suggested that NO may also play a role in the -ARAs-induced vasodilatation. For example, it has been shown that removal of endothelium from the canine coronary arteries abolishes the relaxation caused by acetylcholine and decreases -ARAs-induced relaxation (21). This effect has now also been shown in other vascular beds (9, 17, 19). Furthermore, there is some evidence that NO, which acts through cGMP, can modify cAMP responses through regulation of phosphodiesterase activity (14). On the other hand, NO synthase has been shown to be induced by cAMP (10), and cAMP has also been reported to prolong the half-life of cGMP (25). The interaction between NO and -ARAs on the bronchial circulation has not been studied. Because -ARAs are commonly given by inhalation for the treatment of obstructive airway disease and, recently, NO has been used by inhalation in treatment of respiratory failure (18, 20), the present study was designed to investigate the interaction between inhaled NO and aerosolized -ARAs on the bronchial blood flow.

We postulated that inhalation of NO may result in diffusion of this gas across the airway epithelium into the bronchial vasculature and may cause a decrease in bronchial vascular resistance. Thus we first investigated the dose-dependent effects of NO on bronchial blood flow and bronchial vascular resistance. We then studied the interaction between inhaled NO and aerosolized isoetharine, a -ARA, by giving them repeatedly either in an alternate or sequential fashion before and after the use of a NO synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME). We chose a sheep model for this study because sheep have a favorable anatomy for flow probe placement and, additionally, have a dense submucosal bronchial vascular plexus (4).

METHODS

Surgical Preparation

Sheep were ventilated with a tidal volume of 10 ml/kg, and the tidal volume was not altered throughout the experiment. Respiratory rate was kept between 12 and 16 breaths/min to maintain a systemic arterial PCO₂ of ~40 Torr. Animals were given supplemental oxygen to keep the systemic arterial PO₂ >100 Torr. After initial ventilator adjustment to achieve the above-mentioned blood gases, the ventilator settings were not altered, but intermittent blood-gas measurements were obtained to make sure that satisfactory blood gases were maintained throughout the experiment.

Delivery of NO by Inhalation

Delivery of Aerosolized Isoetharine

2

Calculation of Bronchial Vascular Resistance

Protocol

Statistics

RESULTS

NO Dose-Response Curve

1

The control mean systemic arterial pressure was 73 ± 11 Torr, and the mean pulmonary arterial pressure was 12 ± 1 Torr, and these vascular pressures did not change significantly even when higher concentrations of inhaled NO were used. Similarly, the cardiac output during the control experimental period was 3.2 ± 0.3 l/min and it did not change significantly with NO. The control Pa_O2 was 201 ± 17 Torr and, after 15 min of receipt of NO, it was 193 ± 16 Torr. The control Pa_CO2 was 34 ± 4 Torr and, at 15 min of receipt of NO, it was 33 ± 3 Torr.

After pretreatment of animals with methylene blue (n = 2), the increases in bronchial blood flow with each concentration of NO were only about one-half of the control value. For example, when 100 ppm of NO was administered, the bronchial blood flow increased from 27.9 ± 4.1 to 32.6 ± 5.6 ml/min (only a 17% increase), suggesting that methylene blue partially blocked the vasodilator response to inhaled NO.

Alternate Administration of NO and Isoetharine (n = 5)

1

2

Table 1. Effects of alternating treatment with inhaled NO and aerosolized isoetharine on physiological parameters before and after L-NAME Experimental Condition BVR, ml · mmHg1 · min CO, l/min   mmHg   mmHg Control 2.98 3.61 ± 0.38  88 ± 5  9 ± 1  NO   5 min on 2.02 3.78 ± 0.46  88 ± 5  9 ± 1    5 min off 2.87 3.60 ± 0.38  88 ± 5  8 ± 1  Isoetharine (10 min) 2.04 4.15 ± 0.26  90 ± 3  9 ± 1  NO   5 min on 1.29 4.12 ± 0.24  83 ± 6  9 ± 1    5 min off 1.49 4.16 ± 0.24  83 ± 7  9 ± 1  L-NAME 7.19 3.19 ± 0.37  116 ± 8  11 ± 1  NO   5 min on 2.70 3.20 ± 0.38  106 ± 7  9 ± 1    5 min off 5.50 3.04 ± 0.35  101 ± 7  9 ± 1  Isoetharine (10 min) 5.56 2.98 ± 0.37  96 ± 4  10 ± 1  NO   5 min on 1.86 2.93 ± 0.41  87 ± 5  9 ± 1    5 min off 3.01 2.89 ± 0.42  87 ± 5  9 ± 0  Isoetharine (10 min) 2.76 3.09 ± 0.41  87 ± 5  9 ± 0  NO   5 min on 1.32 3.16 ± 0.41  85 ± 6  9 ± 0    5 min off 1.74 3.17 ± 0.40  84 ± 6  9 ± 0  Isoetharine (10 min) 1.57 3.30 ± 0.40  86 ± 7  10 ± 1  NO   5 min on 1.28 3.33 ± 0.42  86 ± 7  9 ± 1    5 min off 1.30 3.25 ± 0.41  86 ± 7  9 ± 1  L-NAME 1.25 3.34 ± 0.45  84 ± 6  9 ± 1 Values are means ± SE; n = 5 sheep. Isoetharine (2 ml) was diluted with 3 ml of saline (1.49 × 102 M concentration). NO, nitric oxide (100 parts/million); BVR, bronchial vascular resistance; CO, cardiac output; mean systemic arterial pressure; mean pulmonary arterial pressure; L-NAME, N -L-arginine methyl ester.

NO and Isoetharine Given Sequentially (n = 3)

2

Table 2. Effects of sequential treatment with aerosolized isoetharine and inhaled NO and on physiological parameters before and after L-NAME Experimental Condition BVR, ml · mmHg1 · min CO, l/min   mmHg   mmHg Control 4.66 3.50 ± 0.27  81 ± 4  12 ± 2  NO   5 min on 2.87 3.67 ± 0.15  81 ± 4  10 ± 1    5 min off 4.69 3.61 ± 0.16  81 ± 4  11 ± 1  Isoetharine (10 min) 2.63 5.20 ± 0.50  96 ± 6  11 ± 1    After 30 min 2.65 5.05 ± 0.48  95 ± 10  12 ± 0  L-NAME 8.82 4.30 ± 0.48  102 ± 7  12 ± 0  Isoetharine (10 min) 8.78 4.11 ± 0.29  112 ± 14  11 ± 1    After 30 min 8.79 4.05 ± 0.44  106 ± 14  11 ± 1  Isoetharine (10 min) 8.11 4.10 ± 0.48  106 ± 13  11 ± 1    After 30 min 6.57 4.01 ± 0.61  101 ± 10  11 ± 1  Isoetharine (10 min) 6.84 4.13 ± 0.56  103 ± 11  12 ± 1    After 30 min 5.78 3.92 ± 0.47  96 ± 9  11 ± 0  NO   5 min on 2.20 3.83 ± 0.46  90 ± 9  10 ± 0    5 min off 3.22 3.74 ± 0.43  88 ± 8  10 ± 0  NO   5 min on 1.92 3.67 ± 0.39  87 ± 9  10 ± 0    5 min off 2.70 3.71 ± 0.40  87 ± 9  10 ± 1  NO   5 min on 1.83 3.72 ± 0.42  87 ± 9  10 ± 1    5 min off 2.35 3.84 ± 0.48  87 ± 9  10 ± 1  L-NAME 2.63 3.92 ± 0.51  89 ± 10  10 ± 1 Values are means ± SE; n = 3 sheep.

DISCUSSION

NO is a lipophilic gas with potent vascular smooth muscle-relaxing properties in both systemic as well as in pulmonary vascular beds and plays an important role in maintenance of normal vascular tone (24). Although the physiological effects of NO have been studied on the pulmonary circulation (18, 20), there have been only a few indirect studies investigating the role of NO in modulation of bronchial vascular tone. Recently, Sasaki et al. (23) found that infusion of N^G-nitro-L-arginine (L-NNA) resulted in ~64% decrease in bronchial blood flow, and it also attenuated the acetylcholine-induced increases in bronchial blood flow. From this study they concluded that endogenous production of NO from the endothelial cells mediates the baseline bronchial vascular tone as well as the acetylcholine-induced vasodilatation of the bronchial vasculature (23). White et al. (26), in a preliminary study in a dog model, found that infusion of L-NAME resulted in ~50% fall in bronchial flow conductance that was sustained for at least 3 h. These data suggest that, similar to other vascular beds, continuous release of endogenous NO from the endothelial cells is important in the maintenance of bronchial vascular tone. On the other hand, Alving et al. (1, 2) studied changes in bronchial blood flow in anesthetized pigs during administration of cigarette smoke, before and after intravenous infusion of a competitive inhibitor of NO synthase, L-NNA. They found that exogenous NO accounted for most of the bronchial vasodilation observed after the inhalation of cigarette smoke. Direct effects of exogenous NO on bronchial blood flow have not been systematically studied.

Thus, in the present study in a sheep model, we studied the direct dose-dependent effects of inhaled NO on bronchial blood flow in a sheep model. The bronchial vascular effects of NO were observed as soon as NO was administered, suggesting that NO diffuses rapidly into the airway mucosa and into the bronchial microvascular bed. We also found that there was a dose-dependent increase in bronchial blood flow when NO was administered by inhalation and that, at 100 ppm, NO increased bronchial blood flow by ~37% and decreased the bronchial vascular resistance by ~27% (Fig. 1). However, it is likely that if we had continued to give higher doses of NO, we would have seen further increases in bronchial blood flow. The sheep has a submucosal as well as a peribronchial vascular plexus around the airways, and it is possible that inhaled NO has a vasodilatory effect only on the submucosal plexus of the bronchial microvasculature and, hence, we saw only a 37% increase in the bronchial blood flow. It is also unlikely that exogenous NO would affect the larger bronchial vessels that are present outside the bronchial wall. This may explain why intravenous infusion of NO synthase inhibitors has such a profound bronchial vascular constrictor effect (23, 26) because intravenous infusion must be affecting both submucosal and peribronchial vascular plexus as well as the larger branches or bronchial arteries.

The exogenous NO did not have any significant effects on systemic arterial pressures, cardiac output, pulmonary vascular pressures, and the arterial blood gases (Tables 1 and 2). This is not surprising because NO does not seem to have an effect on normal pulmonary vasculature except when it has been constricted with hypoxia or some other pathological condition (27). Furthermore, because NO is rapidly inactivated when it combines with hemoglobin (28), we did not see any changes in systemic arterial pressure (Tables 1 and 2). The bronchial vascular dilatory effects of NO were partially blocked by pretreatment of animals with methylene blue, confirming that the effects of NO on bronchial vasculature are mediated predominantly through cGMP (28). Discontinuation of exogenous NO resulted in a rapid decrease in bronchial blood flow. However, it can take up to 5 min before bronchial blood flow returns to normal. The exogenous NO has an immediate vasodilatory effect on bronchial vasculature and, thus, causes a decrease in bronchial vascular resistance. This finding may have some practical implications in the animal laboratory. For example, after placement of the flow probe around the bronchial artery, administration of exogenous NO for a brief period and finding that bronchial blood flow does increase with NO, seems to be a useful technique to validate that the flow probe is in fact around the bronchial artery. Thus this appears to be a rather simple and reliable method for confirming proper placement of the bronchial arterial flow probe. In our laboratory, we now use this technique routinely to confirm that the flow probe is, in fact, measuring the actual bronchial blood flow.

Both topical application as well as intravenous injection of -ARAs have been shown to increase bronchial blood flow by decreasing bronchial vascular resistance (6, 22). Because inhaled -ARAs are commonly used in patients with lung disease, we studied the interaction between inhaled NO and aerosolized -ARA isoetharine. We chose isoetharine for this study because it has a short duration of action. We also used a larger dose than generally given to patients to obtain maximal bronchial vascular dilatation. After having established from the dose-response curve (Fig. 1) that 100 ppm of NO produce a predictable vasodilatory response, we used a single 100 ppm dose of NO for this part of the study. As shown in Fig. 2, we found that 2 ml of 1% isoetharine solution had a bronchial vasodilatory effect that was comparable to that of 100 ppm of NO, and the effect seemed to last for at least 30 min. Interestingly, NO given after isoetharine had an additional vasodilatory effect. With intravenous L-NAME, given after bronchial vascular dilatation had been achieved with NO and -ARA (Figs. 2 and 3), there was a marked reduction in the bronchial blood flow accompanied by a marked increase in bronchial vascular resistance (Tables 1 and 2). This suggests that there is a continuous synthesis of NO in the bronchial microvasculature that is responsible for the maintenance of normal bronchial vascular tone and that synthesis of NO may play a role in -ARA-induced bronchial vasodilatation. When NO was given immediately after L-NAME, there was a 147% increase in bronchial blood flow and it was higher than control value (Fig. 2). This suggests that, in the absence of endogenous NO synthesis and after the vasculature has been pretreated with isoetharine, the bronchial vasculature becomes more sensitive to vasodilatory effects of exogenous NO.

NO given by inhalation after L-NAME had similar effect on bronchial blood flow to what was seen before L-NAME. However, when isoetharine was given after the animals had been treated with L-NAME (Fig. 3), there was no increase in bronchial blood flow, again confirming that most of the vasodilatory effects -ARAs are mediated through the synthesis of NO. Although both ₁- and ₂-receptors are present in the canine coronary circulation, the catecholamine-induced vasodilatation is primarily mediated through the ₁-receptors (24a). In contrast, in the femoral circulation the vasodilatation is mediated through the ₂-receptors (16). In the mesentery circulation of rats, only ₁-adrenoceptor activation causes relaxation via NO release (9). It has also been shown that in the canine coronary circulation both ₁- and ₂-receptors cause vasodilatation through synthesis of NO (17). Isoetharine is a sympathomimetic amine with a preferential affinity for ₂-adrenergic receptor sites and a lower order of affinity for ₁-adrenergic receptors. Thus it is likely that in the bronchial circulation vasodilatation is predominantly mediated through the ₂-receptors that stimulate synthesis of NO.

Interestingly, there was a progressive and significant increase in bronchial blood flow when the same dose of isoetharine was given repeatedly but only after treatment with NO (Fig. 2). This increase in blood flow could not have been due to endogenous synthesis of NO because NO synthase was blocked by L-NAME. These findings suggest that NO facilitates the vasodilatory effects of -ARA through some mechanism other than stimulation of NO synthase activity. On the other hand, NO administered after isoetharine also seemed to have an enhanced vasodilatory effect. For example, compared with a normal increase of ~40-50% in bronchial blood flow with 100 ppm of NO, the same dose of NO administered after isoetharine resulted in a 175% increase in bronchial blood flow (Fig. 2). This finding suggests that, after L-NAME, treatment with isoetharine makes vasculature more responsive to exogenous NO. The mechanism that is responsible for this interaction is not clear; however, a few explanations can be speculated. For example, recently it has been shown that -ARAs stimulate large-conductance calcium-activated potassium (K_Ca) channels, which may be important for -adrenergic relaxation of airway smooth muscles (15). It has also been shown that -ARAs regulate K_Ca channels in airway smooth muscle by cAMP-dependent and -independent mechanisms (13). The presence of K_Ca channels in the bronchial circulation has not been studied, but it is possible that alternate use of isoetharine and NO could have caused bronchial vascular vasodilatation by stimulation of these channels.

After pretreatment with isoetharine, when NO administration was discontinued, the bronchial blood flow did not return to the pretreatment value (Figs. 2 and 3). There could be two explanations for these findings. It is possible that an alternate use of NO and isoetharine stimulated cAMP formation, which resulted in progressively increasing vasodilatation. It is also possible that in higher doses, isoetharine stimulated synthesis of cGMP. This concept is supported by the fact that there is some recent evidence that indicates that isoproterenol, a -ARA, can increase cGMP formation (25). The fact that there was no decrease in bronchial blood flow when L-NAME was given the second time (Figs. 2 and 3) suggests that the first injection of L-NAME had completely blocked the NO synthase activity and that isoetharine did not cause vasodilatation through synthesis of NO in this set of experiments. Indeed, White et. al. (26) have shown that the effects of L-NAME on bronchial circulation can last for at least 3 h.

Another possible explanation for our findings is that the increase in bronchial blood flow that was observed after L-NAME and alternate treatment with NO and isoetharine could have been due to the cumulative effect of isoetharine. To test this possibility, we conducted another set of experiments in which, after L-NAME, we gave three sequential doses of isoetharine and each time waited for 30 min to make sure that we had achieved a peak response with each dose. The results were somewhat surprising because, in this set of experiments, three sequential doses of isoetharine caused only a minimal change in bronchial blood flow (Fig. 3). On the other hand, three sequential treatments with NO caused a progressive increase in bronchial blood flow. Similar to the previous experiment, three inhalations of NO given sequentially after isoetharine caused 146, 172 and 185% increases in bronchial blood flow, respectively (Fig. 3). These data suggest that sequential treatments with NO might have resulted in stimulation of cAMP, which could have been responsible for progressive increases in bronchial blood flow. It is also possible that, as suggested by Kume et al. (13), isoetharine and NO could have stimulated K_Ca channels in the bronchial microvasculature.

The other interesting finding of this study is that when NO was given by inhalation, initially bronchial blood flow increased but, after discontinuation of NO, it returned close to control value in ~5 min (Figs. 2 and 3). However, when NO was given after isoetharine and then discontinued, the bronchial blood flow remained high. Recently, cAMP has been reported to prolong the half-life of cGMP (25). Therefore, a simple explanation for this finding could be that high intracellular cAMP levels produced by isoetharine competitively prevent cGMP degradation by inhibiting nonspecific phosphodiesterases, thereby enhancing the actions of NO, an agonist of cGMP. Thus these data also suggest that there seems to be cross talk between cAMP and cGMP.

Recently, it has been shown that NO can be detected in exhaled air in normal human subjects and that the concentration of NO is much higher in patients with asthma and bronchiectasis (11, 12). It is generally assumed that NO in the exhaled air is being secreted by the airway epithelium and the inflammatory cells within the respiratory tract. Our data suggest that bronchial circulation produces a significant amount of NO. Thus a significant portion of NO in the exhaled air could be coming from the bronchial microvasculature. This view is supported by the fact that intravenous L-NMMA decreases NO concentration in the exhaled air (8), which could be due to the fact that L-NMMA decreased the bronchial blood flow. Furthermore, increased levels of NO have been reported in patients with chronic lung infections such as bronchiectasis (12), diseases that cause marked hypertrophy of the bronchial circulation (3).

In conclusion, this study shows that inhaled NO has a dose-dependent vasodilatory effect on the bronchial circulation and that there is an interaction between the vasodilatory effects of NO and aerosolized isoetharine. Further studies are needed to delineate the mechanisms of interaction between these two vasodilating agents.

ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Program Project Grant HL-24163, the John Butler Lung Foundation, and the Medical Research Service of the Department of Veterans Affairs.

FOOTNOTES

Address for reprint requests: N. B. Charan, Chief, Section of Pulmonary/Critical Care Medicine, VA Medical Center, 500 West Fort St., Boise, Idaho 83702-4598 (E-mail ncharan{at}micron.net).

Received 29 July 1996; accepted in final form 15 October 1996.

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