INTRODUCTION

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DIRECT INFUSION of -adrenoreceptor agonists into the bronchial artery in sheep produces marked increases in bronchial blood flow (br), demonstrating that -receptors are present on the bronchial arteries (22). Similarly, when administered via inhalation, isoproterenol has been found to increase tracheal mucosal blood flow (2). The relaxation of vascular smooth muscle resulting from administration of -agonists has conventionally been attributed to occur via activation of adenylate cyclase resulting in an increase in adenosine 3,5-cyclic monophosphate (cAMP) in the vascular smooth muscle cell (13, 16). However, it has recently been shown that this effect is attenuated by removal of the vascular endothelium (24, 26) or by pretreatment with inhibitors of nitric oxide synthase (NOS) (10-12, 23, 26). In view of the potential interaction between the -agonist and nitric oxide (NO) pathways, we hypothesized that increases in br in response to -agonists are partially mediated through synthesis of endogenous NO. Therefore, to determine the potential contribution of NO in -agonist-mediated effects in the bronchial circulation in vivo, we first administered isoetharine HCl via inhalation to adult, anesthetized sheep to establish a dose-response relationship in br. We then tested the effect of NOS inhibition on -agonist-mediated bronchial arterial vasodilation with a subsequent challenge of isoetharine HCl. The NOS inhibitor, N-nitro-L-arginine methyl ester (L-NAME) was administered intravenously or by inhalation to establish whether it produced similar effects on -agonist-induced alterations in br with these two different modes of administration. A sheep model was chosen for this study because of similarities in the tracheobronchial circulation to those in the human (19) and because the sheep has a dense submucosal bronchial vascular plexus (5).

	METHODS

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Surgical Preparation

A left thoracotomy was performed through the fifth intercostal space, the bronchoesophageal trunk was identified, and the common bronchial branch was isolated. An ultrasonic flow probe (2 mm, 2RS, Transonic Systems, Ithaca, NY) was placed around the common bronchial branch of the bronchoesophageal artery for determination of br. Caution was taken to avoid compression or torsion of the vessel. The pericardium was then opened, and a second flow probe (16 mm, 16SS) was placed around the pulmonary trunk for determination of cardiac output (T). After insertion, the flow probes were tested by connecting them successively to an ultrasonic blood flowmeter (Transonic Systems T201) to ensure that a strong pulsatile reading was being obtained. The chest was then closed, and the lung was reexpanded. A Silastic catheter for arterial blood sampling and arterial pressure measurement was placed in the left carotid artery. A balloon-tipped pulmonary artery catheter (Pentalumen Thermodilution 8-Fr, Abbott, N. Chicago, IL) was placed via the left jugular vein and advanced into the pulmonary artery. An 18-gauge needle was inserted in the endotracheal tube and connected to a calibrated transducer for assessment of airway pressure. Systemic arterial pressure (Psa) and pulmonary arterial pressure (Ppa) were measured with calibrated transducers (referenced to left atrium) and continuously recorded (model 2107-8890-00, Gould, Cleveland, OH). A continuous chart recording of vascular flows as well as hemodynamic pressure parameters was obtained. Arterial blood samples were drawn under anaerobic conditions from the carotid catheter using a heparinized syringe and were analyzed for pH, Pa_O2, and Pa_CO2 (Radiometer ABL-520, Copenhagen, Denmark).

Delivery of Inhaled Agents

Isoetharine HCl. A small-volume nebulizer (Salter Labs, Arvin, CA) was connected between the endotracheal tube and the ventilator tubing and secured with a T adapter. Isoetharine HCl inhalation solution (USP, 1%, Winthrop Pharmaceuticals, New York, NY) was placed in the nebulizer in graduated doses (2.5, 5.0, 10.0, and 20.0 mg) and diluted with sterile NaCl (0.9%) to achieve a total volume of 2.5 ml with each dose, thus yielding a final concentration range of 3.6 × 10³ to 2.9 × 10² M. The nebulizer was connected to an O₂ E cylinder and driven with O₂ at a flow rate of 8 l/min for a total of 8 min/dose, the time required to empty the nebulizer.

L-NAME. L-NAME HCl (Sigma Chemical, St. Louis, MO), 10 mg/kg, was dissolved in 2.5 ml of sterile 0.9% NaCl. We were unable to use higher amounts because, at this dose, we had achieved a saturated solution. Therefore, at higher doses, the physical properties of the mixture impaired adequate delivery. L-NAME was administered via Salter nebulizer driven by O₂ at a flow rate of 8 l/min for 10 min.

Phenylephrine. Phenylephrine HCl (1% injection; Neo-Synephrine, Winthrop Pharmaceuticals, New York, NY), 10 mg in 2.5 ml of normal saline, was administered via continuous nebulization (Salter) driven by O₂ at a flow rate of 8 l/min for 10 min.

NO. A premixed cylinder of NO (Airco, BOC Group, Murray Hill, NJ) containing NO, 600 parts per million (ppm) in N₂, was connected to the nitrous oxide port of the anesthesia machine via a stainless steel regulator. The NO-N₂ mixture was blended with O₂ and air to achieve an approximate concentration of 100 ppm. Although the exact concentration of NO was not measured, each dose administered in this study was uniform.

Experimental Protocol

Experiment 1. Dose-response relationship of aerosolized isoetharine HCl on br (n = 6). Baseline physiological parameters, including br, T, Psa, end-inspiratory airway pressure (Paw), Ppa, and arterial blood gases, were obtained. Bronchial vascular resistance (BVR) was calculated using the equation (6): BVR = Psa  Ppa/br (Torr · min · ml¹), in which the mean values of Psa, Ppa, and br were used for each data point.

Experiment 2. Effect of NO on isoetharine HCl-induced bronchial arterial vasodilation (n = 4). In four of the above animals, NO challenge was given before and after the isoetharine dose-response protocol. Because -agonists are thought to act predominantly via production of cAMP (13, 16) and NO acts via guanosine 3,5-cyclic monophosphate (cGMP) (20), the purpose of this protocol was to investigate whether NO produces additional vasodilatory effects after maximal vasodilation has already been achieved with -agonists. NO was administered at 100 ppm via inhalation for 5 min before beginning the treatment protocol with nebulized isoetharine HCl. Physiological parameters and arterial blood gases were obtained before and after treatment with NO. After NO was discontinued, a stabilization period of 20 min was allowed before administration of the first dose of isoetharine HCl. Twenty minutes after the last dose of isoetharine was given, NO (100 ppm) was again administered for 5 min. Arterial blood gases and hemodynamic parameters were then obtained before and after administration of NO.

Experiment 3. Effect of NOS blockade on isoetharine HCl-induced alterations in br (n = 10). In six animals, a single dose of isoetharine HCl was administered via nebulizer (20.0 mg, nebulizer volume 2.5 ml), and parameters were obtained during the 20-min stabilization period as before. L-NAME (30 mg/kg in 20 ml of NaCl) was then administered intravenously over 1 min via a peripheral vein (4), and parameters were observed for a 20-min stabilization period in three animals and for 80 min in three other animals. After the stabilization period, a subsequent challenge with nebulized isoetharine HCl (20.0 mg, nebulizer volume 2.5 ml) was then given as before, and parameters were obtained until stable.

Experiment 4. Response to isoetharine HCl after pretreatment with phenylephrine (n = 7). To further determine whether the inhibitory effect of NOS on isoetharine-induced bronchial arterial vasodilation depends on the increased vascular tone, additional animals were treated with phenylephrine as a bronchial arterial vasoconstrictor. Three animals were given phenylephrine by inhalation (10 mg in 2.5 ml of NaCl over 10 min) to determine the extent and duration of the effect on br and BVR. Hemodynamic parameters and blood flows were obtained at frequent intervals until they returned to baseline, an additional 30-80 min. Four separate animals were then treated with nebulized phenylephrine for 10 min as before, immediately followed by nebulized isoetharine (20 mg over 8 min), and parameters were observed until stable.

Statistical Analysis

	RESULTS

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Experiment 1

View larger version (18K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig. 1.   A: effect of normal saline (NaCl) and isoetharine HCl (Iso), administered by inhalation, on bronchial blood flow (br) in 6 animals. B: effect of NaCl and Iso, administered by inhalation, on bronchial vascular resistance (BVR) in 6 animals. Values are means ± SE; solid bars, duration of each treatment. * Significantly different compared with baseline values, P < 0.001.

There were no dose-related effects in T with increasing doses of isoetharine HCl (Table 1). With a control dose of nebulized 0.9% NaCl or increasing doses of isoetharine HCl, there were no changes in Psa, Ppa, Pa_O2, or Paw (Table 1). Pa_CO2 increased from 38 ± 1 Torr immediately before isoetharine to 42 ± 2 Torr at the end of the dose-response study (P < 0.05). With administration of isoetharine HCl, BVR progressively decreased, reached a plateau at ~30% of baseline levels (P < 0.001) after the 10-mg dose, and did not change with the subsequent 20-mg dose (Fig. 1B).

View this table: [in this window] [in a new window]   Table 1.   Effect of inhaled isoetharine on T, Psa, Ppa, Paw, arterial PO2, and arterial PCO2

Experiment 2

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Effect of nitric oxide (NO), administered by inhalation, on br before and after Iso in 4 animals. Values are means ± SE; solid bar, duration of treatment with NO;  and solid line, increase in br resulting from administration of NO before pretreatment with Iso;  and dashed line, increase in br resulting from administration of NO after treatment with Iso. * Significantly different compared with response in br before pretreatment with Iso, P < 0.01.

Experiment 3

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View larger version (26K): [in this window] [in a new window]   Fig. 3.   Effect of NaCl and Iso, administered by inhalation, on br before and after intravenous N-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg) in 6 animals. Values are means ± SE; solid bars, duration of treatment; vertical hatched bar, administration of L-NAME. * Significantly different compared with pre-L-NAME values, P < 0.001.

View larger version (19K): [in this window] [in a new window]   View larger version (18K): [in this window] [in a new window]   Fig. 4.   A: response in cardiac output (T; dashed line with ) and br (solid line with ) to intravenous L-NAME (30 mg/kg) in 6 animals. B: response in mean systemic arterial pressure (Psa; dashed line with ) and mean pulmonary arterial pressure (Ppa; solid line with ) to intravenous L-NAME (30 mg/kg) in 6 animals. Values are means ± SE; solid bars, duration of treatment with L-NAME. * Significantly different compared with pre-L-NAME values, P < 0.05.

View this table: [in this window] [in a new window]   Table 2.   Effect of isoetharine on T, Psa, Ppa, and Paw before and after L-NAME

In contrast to L-NAME administered via the intravenous route, L-NAME delivered via inhalation (n = 4) resulted in a much smaller and delayed decrease in br of 22 ± 5% from baseline values. The decrease in br with inhaled L-NAME was only evident at ~60-80 min after initial administration. In the two animals pretreated with nebulized NaCl, br increased by 6 ± 3% over baseline at 20 min. With the subsequent isoetharine challenge after treatment with nebulized L-NAME in these two animals, br increased by 30 ± 4% over baseline. In the two sheep that were pretreated with nebulized isoetharine HCl, br increased by 105 ± 25% over baseline values (P < 0.001). After treatment with nebulized L-NAME, however, the response in br was attenuated, only increasing by 25 ± 5% with the second isoetharine challenge (P < 0.001). There were no significant differences in arterial blood gases, T, Psa, Ppa, and Paw after treatment with nebulized L-NAME.

Experiment 4

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	DISCUSSION

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The systemic circulation in the lung is known to contain -adrenoreceptors (1, 3). Parsons et al. (22) have shown that direct injection of isoproterenol into the bronchial artery in sheep induces an increase in br. Similarly, it has also been shown that administration of -agonists in the tracheobronchial circulation results in decreased tracheal vascular resistance (17) and increased tracheal mucosal blood flow (2, 7). In this study, we have also found that isoetharine, a -adrenergic agonist, increases br, thus confirming the presence of -receptors in the bronchial vasculature of sheep (Fig. 1A).

To simulate the clinical condition, we administered a -adrenergic agonist by inhalation to intact animals to determine the local vascular effects in the bronchial circulation. We found that br markedly increased with the administration of inhaled isoetharine HCl, an effect that appeared to be independent of changes in hemodynamic parameters such as T, Psa, or Ppa. Additionally, when isoetharine was given as a single 20-mg dose, there were no changes in arterial blood gases. We also found that the increases in total br were dose related. These findings are in agreement with those of Barker and colleagues (2), who demonstrated that local exposure of the tracheal mucosa to inhaled isoproterenol resulted in a dose-dependent increase in tracheal mucosal blood flow. However, Wanner et al. (27) measured only the tracheal mucosal blood flow by the inert soluble gas technique using dimethyl ether, whereas we measured the total bronchial arterial blood flow directly with an ultrasonic flow probe, a technique that measures the total blood flow to the airway mucosa as well as that supplying the entire airway wall. br progressively increased with higher doses of isoetharine HCl and ultimately reached a plateau. With administration of the last dose of isoetharine HCl (20 mg), there were no further significant changes in br or BVR (Fig. 1, A and B), thereby suggesting that maximal -receptor stimulation had been achieved. However, when exogenous NO was then delivered via inhalation, an additional increase in br was seen, suggesting that exogenous NO produced further vasodilation (Fig. 2). This is not surprising, because NO causes vasodilation through stimulation of soluble guanylate cyclase and production of cGMP (20). It is also possible that prolongation of the half-life of cGMP by cAMP may potentially have contributed to this additional vasodilation (18, 25). However, the increase in br with NO after the last dose of isoetharine HCl was proportionally smaller than the increase in br we observed before pretreatment with isoetharine. A possible explanation for these findings could be that the near-maximal capacity for bronchial vascular engorgement had been achieved after -agonist treatment, thus further NO-induced increases in br were less pronounced.

Although the relaxation of vascular smooth muscle by -receptor agonists had previously been thought to occur via activation of adenylate cyclase and production of cAMP (13, 16), there have now been recent reports to suggest that NO may also have an important function in promoting vascular smooth muscle relaxation induced by -agonists (10-12, 21, 23, 26). For example, Rubanyi and Vanhoutte (24) reported that removal of the endothelium in canine coronary arteries attenuated isoproterenol-induced vascular smooth muscle relaxation. Other recent studies have shown that -receptor-mediated vascular smooth muscle relaxation may involve release of endogenous NO from the endothelium in isolated vascular preparations (11, 12, 21, 26) and that NOS is induced by cAMP (15). In isolated vascular ring preparations, isoproterenol has been shown to act through endothelial -receptors to raise levels of cAMP, an effect that is attenuated by inhibition of NOS (11, 12). These investigations on the role of the endothelium in -agonist-mediated vascular smooth muscle relaxation have been conducted in isolated vascular or cultured cell preparations (11, 12, 21, 26) or by injection in a regional vascular bed (10, 23, 26). In contrast, we investigated the effects of inhaled isoetharine HCl on bronchial arterial blood flow in the intact animal and found a rapid increase in br. Furthermore, inhibition of NOS with intravenous administration of L-NAME not only decreased br by ~80% but also resulted in further attenuation of isoetharine HCl-induced increases in br (Fig. 3). These findings suggest that isoetharine HCl stimulates release of endogenous NO in the bronchial circulation, thus confirming the findings obtained in prior in vitro vascular preparation and cell culture studies (11, 12, 21, 26). The responses in br after rechallenge with isoetharine HCl were comparable when isoetharine was given either 20 or 80 min after intravenous L-NAME, indicating that the effect of NOS blockade was of long duration. This is in accordance with findings by White et al. (28), who, in a preliminary study, showed that intravenous infusion of L-NAME in the dog produced an immediate fall in bronchial vascular conductance by ~50%, an effect that was sustained for 3 h.

To determine whether the inhibitory effect of L-NAME on isoetharine-induced bronchial vascular dilation depends on the increased baseline vascular tone, we compared the effect of L-NAME with that of phenylephrine, a potent ₁-receptor agonist, which results in vasoconstriction without inhibition of NOS. We found that the vasodilatory response to isoetharine was not affected by concurrent treatment with phenylephrine despite a vascular tone comparable to that obtained with L-NAME. These results indicate that the effect of NOS blockade on isoetharine-induced bronchial arterial vasodilation appears to be independent of the baseline vascular tone.

We observed that L-NAME, when administered via the intravenous route, caused a rapid increase in BVR, whereas the effect of L-NAME given by inhalation was rather modest and delayed in onset. There may be several explanations for this difference. First, the dose of nebulized L-NAME was one-third of that given intravenously, and it is possible that delivery of the nebulized agent to the airways was not complete. Furthermore, it is expected that L-NAME administered intravenously would have rapid contact with the vascular endothelium and would thus have a rapid effect on inhibition of NOS. In contrast, L-NAME given by inhalation may require additional time to penetrate the bronchial mucosa to ultimately reach the submucosal plexus. It is also possible that L-NAME given by inhalation does not have a rapid effect on br, compared with other inhaled agents such as isoetharine HCl or other -agonists, because of its molecular structure and other chemical properties. However, although the effect of inhaled L-NAME on br was of a smaller magnitude, the vasodilatory effects of inhaled isoetharine with subsequent challenge were nonetheless attenuated.

In this model, br decreased with NOS inhibition because bronchovascular resistance increased and not because of alterations in the upstream or downstream pressures, since there were no appreciable changes from baseline values in Psa or Ppa after a period of stabilization. However, it is interesting to note that, immediately after intravenous infusion of L-NAME, br first rose acutely before its subsequent precipitous drop (Fig. 4A). This rise in br was associated with the transient drop in Psa, and it was only at ~4 min after completion of the infusion that br decreased and Psa increased (Fig. 4B). Although we do not have a conclusive explanation for this phenomenon of transient increase in br associated with a decrease in Psa after L-NAME, a few speculations can be made. It is conceivable that L-NAME caused a rapid release of NO from the endothelium and resulted in transient, generalized vasodilation before NOS was blocked. Although it was previously thought that NO-related factors were only released after their formation (14), recent studies have now provided evidence for the presence of preformed stores of NO-containing factors in the endothelium (8, 9). Thus, in view of the rapidity and transient nature of the resulting vasodilation after intravenous L-NAME, it can be speculated that this agent by itself or in conjunction with -agonists may have an initial effect on preformed stores of NO-containing factors. In this study, the vasodilatory effect on the bronchial vasculature was transient, since NO is rapidly inactivated by hemoglobin (20), and the profound vasoconstriction resulting from NOS inhibition then became evident.

In summary, we show that administration of isoetharine HCl, a -adrenoreceptor agonist, by inhalation in doses used clinically resulted in marked vasodilation in the bronchial circulation of the intact sheep. This increase in br occurred in a dose-dependent manner and was attenuated by inhibition of NOS. These data also suggest that, in the bronchial vasculature, -agonist-induced relaxation of vascular smooth muscle is partially mediated via synthesis of endogenous NO. It thus appears that both cAMP and cGMP pathways mediate -agonist-induced bronchial vascular relaxation.