INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

CENTRAL VENOUS INFUSION of adenosine (Ado) reduces pulmonary vascular resistance when administered over a short period (~15 min) in cardiac surgical patients without pulmonary hypertension (4). This vasodilation is selective for the pulmonary circulation without affecting systemic vascular resistance or mean systemic arterial pressure. Lack of an effect on the systemic circulation with central venous infusion of Ado reflects its rapid clearance from blood by adenosine deaminase found in vascular endothelial cells and red blood cells (13, 20). Because of this short half-life (4, 13, 18) and rapid clearance from the pulmonary circulation, Ado is a potential agent that can be administered locally in the pulmonary circulation without having undesired systemic effects.

A problem with the clinical utility of Ado is its potential to produce A₂-adenosine-receptor desensitization after sustained infusion. Although desensitization of the A₂-adenosine receptor has not been demonstrated in the pulmonary circulation, the downregulation of G_s protein, inhibition of adenylyl cyclase, and activation of phosphodiesterase have been observed in other cell lines as mechanisms of A₂-adenosine-receptor desensitization (2, 11, 12). We therefore sought to determine whether prolonged administration of Ado agonists results in sustained vasodilation or receptor desensitization in the pulmonary circulation.

On the basis of previous observations (3, 8, 16, 19, 25) in the precontracted pulmonary vascular bed, Ado-mediated vasodilation occurs after activation of the A₂- adenosine receptor, probably of the A_2b subtype (8), which is coupled through a G_s protein to adenylyl cyclase activation (19, 27, 28). Adenylyl cyclase activation increases intracellular cAMP and mediates vasodilation (25). In this study we investigate effect(s) of prolonged A₂-adenosine-receptor exposure to the nonmetabolizable nonselective Ado agonist 5-(N-ethylcarboxamido)adenosine (NECA) and the selective A_2a-adenosine-receptor agonist CGS-21680C on Ado-mediated vasodilation in the lung and on cAMP accumulation in pulmonary artery smooth muscle cell (PASMC) cultures from the Sprague-Dawley rat.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Materials. Ado, NECA, ANG II, and isoproterenol (Iso) were purchased from Sigma Chemical (St. Louis, MO). CGS-21680C was a kind gift from Ciba-Geigy (Summit, NJ), and indolidan (Ind) was a kind gift from Lilly Research Laboratories (Indianapolis, IN). All drugs were solubilized in normal saline except for NECA and CGS-21680C, which were dissolved in distilled H₂O, and Ind, which was dissolved in DMSO.

Isolated perfused lung. Male Sprague-Dawley rats (270-340 g) were anesthetized with Nembutal sodium (25 mg ip), and lungs were removed for extracorporeal perfusion as previously described (6, 17). A tracheostomy was performed that permitted ventilation with a Harvard rodent ventilator (model 683) at 55 breaths/min with a tidal volume of 2.5 ml and 2.0 cmH₂O positive end-expiratory pressure. The inspired gas mixture was 95% air-5% CO₂ (room air gas). During hypoxic challenges, lungs were ventilated with a 3% O₂-5% CO₂-balance N₂ (hypoxic gas) gas mixture. A median sternotomy was performed, heparin sodium (100 IU) was injected in the right ventricle, and cannulas were placed in the pulmonary artery and left ventricle. Heart, lungs, and mediastinal structures were removed en bloc and placed into a humidified chamber. Lungs were perfused by a Gilson Minipuls 2 peristaltic pump at a constant flow of 0.03 ml · g body wt¹ · min¹. Lungs were perfused with homologous blood (30 ml) previously collected in a heparinized syringe by cardiac puncture from three or four adult male Sprague-Dawley rats anesthetized with Nembutal sodium. The temperature of the recirculating blood was maintained at 37°C. Pulmonary arterial (P_pa) and venous pressures were continuously monitored with Cope pressure transducers (041-500-503) and were recorded on a Grass polygraph recorder (model 7E).

After a 30-min equilibration period, all lungs were challenged with 0.1 g ANG II injected as a bolus in the pulmonary artery. Ten minutes after the ANG II challenge, lungs were ventilated with the hypoxic gas mixture for 10 min, and the first hypoxic pressor response (HPR1) was assessed. Room air gas was reinstituted, and lungs were allowed to equilibrate for 10 min (Table 1). This was followed by two 25-min sequential hypoxic challenges (HPR2, HPR3, n = 16 each). At the peak (~10 min) of HPR2, NECA (10 µM, final perfusate concentration) was administered to the reservoir, and the change in P_pa was monitored for an additional 15 min. At the peak of HPR3, NECA (10 µM) was administered (total of 20 µM NECA in perfusate) and P_pa was again monitored for 15 min (group A). With the use of the identical protocol, five lungs previously exposed to 20 µM NECA were washed with 100 ml of a 4 g/100 g albumin-physiological salt solution and reperfused with fresh homologous blood (30 ml) in a recirculating fashion as described above (group B). After a 10-min equilibration period, lungs were subjected to a fourth hypoxic challenge (HPR4). At the peak of HPR4, NECA (10 µM, final perfusate concentration) was administered to the reservoir, and the decrease in P_pa was compared with the decrease in P_pa observed after NECA administration during the peak of HPR2.

In group A, 11 lungs that demonstrated an attenuation of NECA-mediated vasodilation were subjected to HPR4, and either Ado (7.5 mM, n = 4), the nonselective agonist, Iso (1 µM, n = 3), or the cGMP-inhibitable cAMP phosphodiesterase inhibitor (7) Ind (1 µM, n = 4) was administered, and any change in P_pa was observed over a 15-min period (Table 1). In control lungs that were subjected to four sequential hypoxic challenges and not administered NECA (group C), studies were performed with Ado (7.5 mM, n = 3), Iso (1 µM, n = 3), and Ind (1 µM, n = 3) administration during the plateau of HPR4 for comparison with NECA-pretreated lungs from group A that were administered either Ado, Iso, or Ind. Whereas Iso and Ind were administered in the perfusate reservoir, Ado was infused in the pulmonary artery at a rate 0.125 ml/min.

In the final group of lungs, the selective A_2a-adenosine- receptor agonist CGS-21680C was added to the perfusate in a concentration of either 10 µM (n = 4, group F) or 1,000 µM (n = 4, group G) during HPR2 (45 min before 10 µM NECA administration). NECA was administered at the peak of the HPR3 as described above, and any change in P_pa was observed over 15 min. This change in P_pa seen with NECA was compared with that seen with NECA administration during HPR2 and HPR3 in group A.

PASMC. Main pulmonary arteries were isolated from anesthetized rats, and ~2-mm-large tissue fragments were placed immediately in 1:1 F-12 nutrient mixture and DMEM mixture supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (GIBCO-BRL Products, Grand Island, NY). After 3 days under standard incubation conditions the tissue pieces were removed, and cells that had migrated from the explants were grown to confluency. Culture medium was changed every 3 days. Cells from passages 4-8 were harvested using a 0.05% solution of trypsin (GIBCO) and were seeded in 24-well plates (Costar, Cambridge, MA) with 1 × 10⁵ cells in 2 ml of medium per well. The plates were used for experiments after 3-4 days when the cells reached confluency. Smooth muscle cell phenotype was confirmed by the presence of smooth muscle-specific -actin as detected by fluorescein-labeled specific antibodies (Sigma). Cell counts were determined by particle counter (Coulter Electronics, Luton, UK).

Measurement of cAMP content. Assessment of cAMP content was performed using standard RIA (Biomedical Technologies, Stoughton, MA). After PASMC were grown to confluency as described above, experiments were conducted using DMEM, with pH balanced to 7.4 and osmolality equal to 285-305 mosmol/kgH₂O. Agonists were added for the time periods indicated, cells were washed with DMEM, and cells were solubilized (reactions stopped) using 1 M NaOH. After assessment of cAMP concentrations, results were standardized to cell counts (10⁶ cells).

Statistics. Results are presented as means ± SE. Statistical analyses were performed using the paired and unpaired Student's t-test and one-way ANOVA. Tukey's test (29) was used for multiple comparisons when ANOVA indicated statistically significant differences between groups. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Isolated lung. Initial studies were designed to assess whether prolonged Ado receptor activation results in desensitization of the A₂-adenosine receptor. In group A, HPR2 and HPR3 were 22.3 ± 1 and 20.8 ± 1 cmH₂O, respectively. The administration of NECA (10 µM) during HPR2 resulted in a 12.7 ± 0.7 cmH₂O decrease in P_pa below the peak HPR2 at 2-4 min and an 8.7 ± 1 cmH₂O decrease in P_pa at 15 min. In contrast to the initial 57% vasodilatory response with NECA, repeat administration of NECA resulted in a 0.6 ± 0.3 cmH₂O decrease in P_pa at 2-4 min and a 2.4 ± 0.4 cmH₂O increase in P_pa above the initial peak P_pa at 15 min (Fig. 1).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Effects of sequential administration of 5-(N-ethylcarboxamido)adenosine (NECA, 10 µM) on the hypoxic pressor response (HPR). HPR is the increase in pulmonary arterial pressure (Ppa) above the baseline Ppa when lungs were ventilated with a hypoxic (fraction of inspired O2 = 0.003) gas mixture. NECA (10 µM) was administered to the perfusate reservoir at the peak of the second (HPR2) and third (HPR3) HPRs. HPR + NECA represents the change in HPR from the peak response after NECA administration. * Significantly different at P < 0.001. NS, not significant.

In lungs from group B, repeat administration of NECA (10 µM) during HPR4 decreased the peak P_pa by only 3.9 ± 1.3 cmH₂O at 2-4 min and by 1.7 ± 2.2 cmH₂O at 15 min. This NECA response was significantly diminished (P < 0.05) compared with the decrease in P_pa observed during HPR2, which was 9.9 ± 1.3 cmH₂O at 2-4 min and 8.2 ± 1.2 cmH₂O at 15 min. Repeat administration of NECA in lungs from groups A and B resulted in an attenuation of NECA-induced vasodilation and is consistent with desensitization of the A₂-adenosine receptor.

In group A where NECA-induced vasodilation could no longer be demonstrated, Ado, Iso, and Ind were administered (Fig. 2). Similar to NECA, Ado decreased P_pa 1.3 ± 1.4 cmH₂O compared with the 15.4 ± 1.5 cmH₂O decrease in P_pa observed in group C (no NECA pretreatment). Administration of Iso and Ind to lungs that were pretreated with NECA and refractory to NECA-induced vasodilation resulted in an attenuation of HPR4 comparable to that observed in lungs not previously treated with NECA (Table 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Representative tracings of sequential hypoxic pressor responses (HPR2-HPR4). HPR1 is not shown. HPR is the observed increase in Ppa above baseline when lungs are ventilated with a 3% O2-balance N2 gas mixture. In all studies, the nonmetabolizable, nonselective adenosine (Ado) agonist NECA (10 µM) was added to the perfusate reservoir at the peak of HPR2, resulting in a decrease in Ppa that waned with time. On repeat administration of NECA during HPR3, lungs were noted to be refractory to NECA-induced vasodilation. Ado (7.5 mM) infused in the pulmonary artery at 0.125 ml/min resulted in a minimal decrease in Ppa that was similar to NECA. In contrast, vasodilation with the 2-adrenergic agonist isoproterenol (Iso, 1 µM) and the cGMP-inhibitable cAMP phosphodiesterase inhibitor indolidan (Ind, 1 µM) was maintained compared with controls (see Table 2).

To assess whether the A_2a-adenosine receptor was involved in Ado receptor desensitization, lungs were pretreated with the selective A_2a-adenosine-receptor agonist CGS-21680C (10 and 1,000 µM) during HPR2 and allowed to recirculate ~45 min before NECA administration. CGS-21680C (10 µM) did not mediate a vasodilatory response when administered at the peak of HPR2. In contrast, CGS-21680C (1,000 µM) caused a 32 ± 3% decrease in HPR2. Unlike the relatively complete desensitization observed in group A with sequential NECA administration, NECA resulted in a 13.6 ± 2.2 cmH₂O decrease in P_pa in lungs pretreated with CGS-21680C (10 µM), which was not significantly different from the 12.7 ± 0.7 cmH₂O decrease observed with HPR2 in group A. However, in lungs pretreated with CGS-21680C (1,000 µM) the decrease in P_pa seen with NECA was 2.6 ± 0.5 cmH₂O, which was significantly less than seen with HPR2 in group A and similar to the attenuation of NECA-mediated vasodilation observed with HPR3 in group A.

PASMC culture. To confirm whether NECA and CGS-21680C activation of the A₂-adenosine receptor increased cAMP, PASMC cultures were exposed to either NECA (10 µM), CGS-21680C (10 µM), or vehicle over a 45-min time course (Fig. 3). Activation of the A₂-adenosine receptor by NECA increased cAMP 876% above control at 5 min. After 45 min of NECA, PASMC cAMP accumulation decreased to 169% of control. In contrast, activation of the A_2a-adenosine receptor with CGS-21680C produced no detectable increase in cAMP, indicating that activation of the A_2b-adenosine receptor most likely accounted for NECA-induced increases in cAMP.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   NECA produces a time-dependent increase in pulmonary artery smooth muscle cell (PASMC) cAMP content by activating the A2b receptor. Either NECA (10 µM) or CGS-21680C (10 µM) was administered to confluent cultures of PASMCs for the indicated time points. Whereas NECA produced a time-dependent increase in cAMP that peaked at 5 min, CGS-21680C did not increase cAMP at any time point. NECA nonselectively activates A2a and A2b receptors, and CGS-21680C selectively activates A2a receptors, indicating that A2b receptors are functionally linked to increased cAMP production in PASMCs. * Significantly different at P < 0.05; n = 6 of experiments using PASMCs/group.

In the isolated lung, an ~45-min exposure to NECA desensitized the pulmonary circulation to subsequent NECA-induced vasodilatory challenges. We next examined whether PASMC were similarly desensitized to NECA-induced cAMP production. To test this idea, PASMC were exposed to either vehicle or NECA (10 µM) for 45 min (as in Fig. 2) and subsequently rechallenged with either vehicle, NECA (10 µM), Iso (1 µM), or Ind (1 µM) for 3 min. Whereas vehicle pretreatment followed by application of NECA increased cAMP 239% above control, NECA pretreatment followed by application of NECA did not significantly increase cAMP content (Fig. 4). These data are consistent with desensitization of the NECA-induced cAMP signaling pathway observed in the pulmonary circulation.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   NECA desensitizes PASMCs to NECA-induced increases in cAMP. NECA application (3 min) to cultured PASMCs increased cAMP content (control vs. vehicle-NECA). However, pretreatment of PASMCs with 10 µM NECA for 45 min prevented subsequent NECA (40 µM; 3 min)-induced increases in cAMP. * Significantly different at P < 0.05; n = 6/group.

Interestingly, in PASMC culture, 45-min exposure to NECA significantly attenuated responsiveness to -adrenergic stimulation with Iso. Whereas vehicle pretreatment followed by application of Iso elicited a 340% increase in cAMP above control, NECA pretreatment followed by Iso produced a 234% increase in cAMP (Fig. 5). In contrast, NECA pretreatment did not influence cAMP responsiveness to Ind (Fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   NECA desensitizes PASMCs to Iso-induced increases in cAMP but not Ind-induced increases in cAMP. The -adrenergic agonist Iso (1 µM; 3 min) increased PASMC cAMP content; pretreatment with 10 µM NECA for 45 min diminished the Iso (1 µM; 3 min)-induced increase in cAMP. Inhibition of phosphodiesterase activity using Ind (1 µM; 3 min) increased cAMP content and was not affected by 45 min pretreatment with 10 µM NECA. * Significantly different at P < 0.05; n = 6/group.

We examined whether NECA desensitization of cAMP stimulation involved G protein activation by pretreating PASMC with either vehicle or NECA (10 µM) for 45 min followed by stimulation of G_s proteins using cholera toxin (10¹⁰ to 10⁸ M). NECA pretreatment attenuated responsiveness to cholera toxin 312%, consistent with desensitization of G_s proteins for activation of adenylyl cyclase (Fig. 6).

View larger version (13K): [in this window] [in a new window]   Fig. 6.   NECA desensitizes cholera toxin (CT)-induced cAMP accumulation in PASMC. Direct Gs activator, cholera toxin, was applied to PASMCs at the indicated doses. Whereas cholera toxin (3 min) produced a dose-dependent increase in cAMP, 45 min pretreatment with NECA abolished the Gs-dependent elevation in cAMP. * Significantly different at P < 0.05; n = 6/group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The purpose of this study was to investigate whether desensitization of the A₂-adenosine receptor occurs in the rat pulmonary circulation on prolonged exposure to the synthetic nonmetabolizable Ado agonist NECA. We found that sequential administration of NECA (10 µM each dose) in the isolated rat lung precontracted with hypoxic gas caused an acute vasodilatory response with a maximum decrease in P_pa 3-4 min after administration. However, ~45 min after the initial administration, repeat administration of NECA resulted in only a minimal vasodilatory response. This effect of NECA in the lung closely paralleled cAMP accumulation in PASMC culture. In PASMCs, the highest accumulation of cAMP occurred ~3-5 min after NECA administration, coincident with the vasodilatory response in isolated perfused lungs. Furthermore, in PASMCs pretreated with NECA for 45 min, washed, and rechallenged with NECA, no increase in cAMP was observed. These findings demonstrate that Ado receptor desensitization occurs, likely due to reduced activation of the Ado receptor-coupled G_s-adenylyl cyclase stimulation of cAMP.

NECA-induced desensitization was not limited to the Ado receptor. In PASMC culture treated with NECA for 45 min followed by Iso, cAMP accumulation was significantly reduced compared with PASMCs treated with Iso alone. A plausible explanation for the observed decrease in cAMP accumulation in NECA-desensitized PASMCs treated with Iso is ₂-adrenergic receptor phosphorylation by protein kinase A (PKA; see Ref. 5). NECA-stimulated increase in cAMP production acutely activates PKA, which phosphorylates the ₂-adrenergic receptor and uncouples the receptor from G_s protein. PKA activation by NECA-generated increases in intracellular cAMP may be the mechanism responsible for ₂-adrenergic receptor phosphorylation and desensitization in this study. Penn et al. (21) have recently reported that agents which stimulate cAMP production such as forskolin and PGE₂ activate PKA, which results in ₂-adrenergic receptor desensitization in human airway smooth muscle cell culture. This cannot be excluded as a possible mechanism for the observed decrease in cAMP accumulation with Iso in PASMC culture. Data derived from NECA-desensitized PASMCs treated with cholera toxin (irreversibly couples the A₂ adenosine receptor to G_s) revealed a decrease in stimulation of cAMP accumulation when compared with cAMP accumulation in PASMCs treated with cholera toxin alone. This observation coupled with the decreased cAMP accumulation after Iso in NECA-desensitized PASMCs suggests that a common mechanism of desensitization is likely that involves G_s-adenylyl cyclase signaling. Potential mechanisms of G_s involvement may include dissociation of ligand-receptor complex from G_s, impaired G_s binding to and activation of adenylyl cyclase, and downregulation of G_s. Downregulation of G_s protein as a mechanism of A_2a-adenosine-receptor desensitization has been described in rat pheochromocytoma PC-12 cells when exposed to the selective A_2a-adenosine agonist CGS-21680 for 12-20 h (2).

Desensitization of the A₂-adenosine receptor has been characterized in smooth muscle of porcine coronary artery ring (15), dithiothreitol, MF-2 cells (22), and rat pheochromocytoma PC-12 cells (2, 14). To date, studies characterizing A₂-adenosine-receptor desensitization have evaluated the A_2a-adenosine receptor (2, 14, 22). There is a paucity of studies characterizing A_2b-adenosine-receptor desensitization. Reported mechanisms of A_2a-adenosine receptor-desensitization include inhibition of adenylyl cyclase, downregulation of G_s protein, and activation of phosphodiesterase. Interestingly, no studies demonstrate desensitization due to a change in receptor number or affinity. Common to all the reported studies, A₂-adenosine-receptor desensitization occurs rapidly. Makujina and Mustafa (15) demonstrated that NECA and CGS-21680 caused rapid desensitization of A₂-adenosine-receptor-mediated vasodilation in precontracted porcine coronary artery rings. Tissues pretreated with NECA for 30 min exhibited a blunted relaxation response to Ado and NECA but not to other vasodilators such as Iso, forskolin, and sodium nitropusside (15). This led to the conclusion that, in porcine coronary artery smooth muscle, A₂-adenosine-receptor desensitization is homologous. Similar observations were made in the isolated lung in this study, suggesting a homologous pattern of rapid desensitization to NECA manifested by blunted NECA-induced vasodilation while maintaining Iso and Ind vasodilation in NECA-desensitized lungs. Despite evidence in the pulmonary circulation that prolonged exposure to NECA functionally induces homologous desensitization, data from in vitro studies indicated that NECA desensitization of PASMCs was heterologous. Prolonged NECA exposure eliminated NECA-induced increases in cAMP and also diminished Iso-stimulated rises in cAMP. Why the patterns of desensitization differed between PASMC culture and isolated lung cannot be answered from this study but suggest Iso-induced vasodilation is mediated only in part via G_s-adenyl cyclase-cAMP signaling. We could speculate that the preservation of Iso-induced vasodilation in the lung reflects the capacity of the many different cells to make cAMP. Although this is true, vasodilation is a reflection of cAMP accumulation in smooth muscle cells and not the amount of cAMP in the circulation. The most plausible explanation for the reported differences observed in this study is perhaps related to the modulation of vascular smooth muscle K⁺ channels by Ado and -adrenoreceptor agonists. Several investigators (24, 26) have reported that Ado, calcitonin gene-related peptide, and -adrenoreceptor agonists activate the ATP-sensitive K⁺ channel through a cAMP-dependent protein kinase, which results in vasodilation. In addition, Iso has been shown to also activate the Ca²⁺-activated K⁺ channels (BK_Ca) through G_s independent of phosphorylation by PKA (23). It is possible that vasodilation in the isolated lung was preserved through activation of the BK_Ca and explains why Iso-induced cAMP accumulation in NECA-desensitized PASMC culture was blunted while Iso-induced vasodilation was preserved. Further studies will be necessary to address this question.

Because the A₂-adenosine receptor has two subtypes, A_2a and A_2b, their relative individual roles in desensitization were assessed. NECA binds the A_2a receptor with high affinity and the A_2b receptor with low affinity (1, 9, 11). The concentration of NECA utilized in this study exceeds the previously reported dissociation constant for NECA binding to the low-affinity A_2b-adenosine receptor in rat striatum (286 nM; see Ref. 9) and human peripheral lung (200 nM; see Ref. 11). Thus it could not be used to distinguish which A₂-adenosine-receptor subtype(s) is involved in the process of Ado agonist desensitization. CGS-21680 is now the current ligand of choice for the characterization of the high-affinity A_2a receptor (10). In this study, pretreatment with CGS-21680C (10 µM) neither caused desensitization to NECA-induced vasodilation in the isolated lung nor caused a significant accumulation of cAMP in PASMC culture. We previously reported (8) that pretreatment with CGS-21680C (1 mM) was required to significantly promote desensitization to NECA-induced vasodilation in the isolated lung. The requirement of a millimolar concentration of CGS-21680C to desensitize the lung to NECA and its lack of effect at a micromolor concentration on cAMP accumulation in PASMC culture strongly supports that the low-affinity A_2b receptor is the predominant receptor involved in Ado-mediated vasodilation and subsequent desensitization in the rat pulmonary circulation. This differs from the observation in the porcine coronary artery ring where a desensitization to CGS-21680 parallels that observed with NECA (15). This difference in response to CGS-21680 suggests that the presence of the A_2a-adenosine receptor may either be species specific and/or vary according to the vascular bed studied, i.e., pulmonary vs. coronary. Using immunohistochemical staining, we have been able to demonstrate the presence of both the A_2a- adenosine receptor and A_2b-adenosine receptor in rat pulmonary vascular beds (data not shown).

In summary, this study demonstrates that the A_2b- adenosine receptor is involved in Ado-mediated vasodilation through increasing intracellular cAMP and that relatively prolonged agonist exposure results in desensitization via G_s-adenylyl cyclase coupling. The findings in this study provide a functional model for expanding our understanding of the various effects of Ado on pulmonary vascular hemodynamics and desensitization.