Introduction

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Urodilatin is an A-type natriuretic peptide, discovered in 1988 (1), that is synthesized in kidney distal tubules and interacts with the tubular cells located downstream in a paracrine fashion (2). Urodilatin is involved in the regulation of body fluid volume and electrolyte balance. Other members of the natriuretic peptide family are atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide. ANP and BNP, potent natriuretic agents as well, are predominantly synthesized in the heart, where up to 95% of cardiac ANP secretion derives from the atria and 60% of BNP release comes from the ventricles (2).

All these peptides stimulate particulate guanylate cyclase-linked receptors, to raise intracellular cyclic guanosine monophosphate (cGMP) levels as underlying mechanisms of their biologic actions (5). This type of second messenger signaling well explains that all peptides of the natriuretic family possess strong vasodilatory potency. Such vasoactivity has been demonstrated for several vascular beds, but is of particular relevance for the pulmonary circulation: cardiac ANP and BNP synthesis is upregulated in the right heart tissue under conditions of pulmonary hypertension, with afterload-related distension of moycardial cells representing the responsible trigger mechanism, to effect relaxation of the lung vessels as a negative feedback mechanism (6, 7). Beyond such short-term effects, substantial evidence suggests that ANP mitigates chronic vascular remodeling and cardiac hypertrophic response to hypoxia, thereby protecting against the development of cor pulmonale. Given such an efficacy profile, infusion of exogenous ANP was used to attenuate pulmonary hypertension and right ventricular hypertrophy in experimental models (8- 10) and under clinical conditions such as chronic obstructive lung disease (11, 12). Such an approach is, however, hampered by the fact that in addition to the pulmonary vasodilation, decrease in systemic vascular resistance is provoked by ANP infusion, and that due to the antagonisms of hypoxic pulmonary vasoconstriction, worsening of ventilation-perfusion matching may occur, as similarly observed in response to other intravascularly applied vasodilators (13, 14).

In line with this use of ANP for pharmacologic purposes, BNP was recently demonstrated to antagonize the acute lung vasoconstrictor response to alveolar hypoxia and to inhibit remodeling of the pulmonary vasculature occurring during 2 wk of hypoxia in rats (15). In the dose range used in that study, BNP turned out to be even more potent than ANP in the pulmonary circulation. To the best of our knowledge, the vasodilatory effect of urodilatin in the lung vasculature has hitherto never been addressed in detail. Such an approach is of interest because urodilatin induces a stronger natriuresis and diuresis than do equimolar doses of ANP (16). It is therefore favored in clinical trials for treatment of acute renal failure (17, 18), congestive heart failure (19), and asthma (20, 21), but might also possess potential utility as a pharmacologic agent in the treatment of pulmonary hypertension. In the present study in intact rabbits, we established stable pulmonary hypertension by infusion of the thromboxane mimetic U46619 to characterize the vasorelaxant properties of urodilatin in the pulmonary circulation as compared with the systemic circulation. In addition, coadministration of the phosphodiesterase (PDE) 5 inhibitor dipyridamole was undertaken, via intravascular and via inhalative routes, for augmentation of the urodilatin; effect due to stabilization of its second messenger, cGMP. In essence, marked lung vasodilator potency was noted for infused urodilatin; however, this peptide reduced the pulmonary and the systemic vascular resistance to similar extents in both the absence and presence of dipyridamole.

	Materials and Methods

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Materials

The thromboxane-A₂ mimetic U46619 (9,11-dideoxy-11,9-epoxy-methano-prostaglandin f 2) was supplied by Paesel-Lorei (Frankfurt, Germany). Urodilatin was manufactured as previously described (1). Dipyridamole was purchased from Berlin-Chemie (Berlin, Germany). All other chemicals and drug supplies were from standard commercial sources. cGMP levels were determined using a radioimmunoassay (Immunotech, Marseille, France). The cGMP concentrations in blood were expressed as picomoles per milliliter.

Surgical Preparation

Rabbits were anesthetized with xylazine/ketamine and anticoagulated with 200 U/kg heparin as previously described (22). They were intubated via tracheal incision and ventilated with a FiO₂ of 0.5 using a volume-controlled respirator (cat ventilator; Hugo Sachs Elektronik, March Hugstetten, Germany). Respiratory rate was set at 40 breaths/min and tidal volume at 8 ml/kg to set arterial PCO₂ in a physiologic range of 35 to 45 mm Hg. A positive end expiratory pressure of 0.5 mm Hg was used throughout. A balloon-tipped pulmonary artery catheter (Berman Angiographic Balloon Catheter, 4 Fr; Arrow, Reading, PA) was inserted into the pulmonary artery through the right external jugular vein. Another catheter was inserted into the left arteria carotis and connected to a pressure transducer for arterial pressure monitoring.

Hemodynamics

Mean pulmonary artery pressure (PAP) and mean systemic arterial pressure (SAP) were continuously recorded by use of fluid-filled pressure transducers (Braun, Combitrans, Germany) referencing the level of left atrium to zero. Pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) were computed using the standard formulas. Cardiac output (CO) was calculated by the Fick principle. The oxygen uptake was measured online using a Labotect O₂-Controller (Labotect, Goettingen, Germany). Blood samples (1 ml) were maintained on ice until analyzed for PO₂, pH, and PCO₂ (ABL330; Radiometer-Copenhagen, Copenhagen, Denmark). Hemoglobin and oxygen saturation were measured using an OSM2 Hemoximeter (Radiometer-Copenhagen).

Aerosolization

For aerosolization of dipyridamole, an ultrasonic nebulizer (Pulmo Sonic 5500; DeVilbiss Medizinische Produkte GmbH, Langen, Germany) was placed in the inspiratory limb of the ventilator tubing as previously described (23). The ultrasonic device produced an aerosol with a mass median aerodynamic diameter of 4.5 µm and a geometric standard deviation of 2.5, as measured with a laser-diffractometer (HELOS; Sympatec, Clausthal-Zellerfeld, Germany). The nebulized mass was calculated by weighing the nebulizer before and after aerosolization. The lung deposition of the nebulized mass was determined by laser photometric technique (24).

Experimental Protocols

After surgical preparation, a mean dosage of 1.3 ± 0.9 µg/kg min U46619 was intravenously infused to increase PAP from ~ 13 to ~ 28 mm Hg within 20 min. The individually titrated dose was then continuously applied, and stable pulmonary hypertension was established by this technique (22). Immediately after tracheotomy, animals received 20 ml sterile Krebs-Henseleit buffer per hour.

Dose-response curves were established for intravenous urodilatin and dipyridamole and inhaled dipyridamole. In these experiments, urodilatin was applied as a short-term infusion (10 min) in the following doses: 3, 30, 300, and 900 ng/kg min after adjustment of stable pulmonary hypertension. Doses were infused in random order and after each infusion animals were allowed to recover for at least 30 min. The PDE inhibitor dipyridamole was either infused (2, 10, 20, and 100 µg/kg min) or nebulized (0.1, 1, 10, and 80 µg/kg min) to establish dose-response curves and to determine a subthreshold dose, which per se did not affect hemodynamics or gas exchange. Dipyridamole was applied in incremental doses that were either infused (10 min) or nebulized (10 min). In the definite experiments, animals received short-term infusion (10 min) of urodilatin (30 ng/kg min) with or without a preceding subthreshold dose of either intravenous (1 µg/kg min) or inhaled (50 ng/kg min) dipyridamole. The PDE inhibitor was applied for 10 min, 15 min before urodilatin infusion. Blood gases, 2 ml samples for cGMP measurements and hemodynamics, were determined at baseline conditions, at U46619-induced pulmonary hypertension, immediately after short-term urodilatin infusion and 40 and 70 min after the beginning of infusion. Urine flow of the animals was measured by placing a catheter in the bladder and continuous weighing of the reservoir.

Data Analysis

Data are shown as means ± standard error of the mean (SEM). Differences between means of several groups were compared by analysis of variance and Student-Newman-Keuls test for multiple comparisons with P < 0.05 regarded as significant. Differences between means of two groups were analyzed with an unpaired t test.

	Results

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Baseline and U46619-Induced Pulmonary Hypertension

As shown in Table 1, hemodynamic data and blood gases of the animals were in physiologic ranges. After a stable steady state of 30 min, the infusion of 1.4 ± 0.6 µg/kg min U46619 resulted in pulmonary hypertension, which was characterized by an increase of PAP from 13.9 ± 0.4 to 27.8 ± 0.7 mm Hg (all groups, P < 0.01). No changes were measured in pulmonary artery occlusion pressure (PAOP), heart rate, or ventilation pressure. Mean SAP and cardiac output did not change significantly. There were no substantial alterations in blood gases compared with control animals.

Dose-Effect Curves of Urodilatin and Dipyridamole

As shown in Figure 1, urodilatin effected dose-dependent reduction of the elevated PAP in animals with U46619-elicited pulmonary hypertension. All doses in the range of 3 to 900 ng/kg min intravenous urodilatin decreased PAP. Concomitantly, there was a dose-dependent decrease of SAP. Intravenous dipyridamole showed similar results (dose range 2 to 80 µg/kg min; Figure 2); there was a dose-dependent decrease in PAP and SAP. In contrast, nebulized dipyridamole (0.1 and 1 µg/kg min) reversed elevated PAP in a pulmonary-selective manner. Systemic pressure drop was noted in higher concentrations (10 and 80 µg/kg min). A half-maximal effective concentration of 1.5 µg/kg min was calculated. For the intravenous route of application (dipyridamole and urodilatin) the data of doses in the range of the maximal effective concentration dose could not be evaluated due to hemodynamic instability. Subthreshold doses of dipyridamole (intravenous: 1 µg/kg min; aerosolized: 50 ng/kg min) with no effects on plasma cGMP level, hemodynamics, or blood gases were used in the combination experiments.

View larger version (15K): [in this window] [in a new window]   Figure 1.   Dose-effect curve of intravenous urodilatin on U46619-elicited pulmonary hypertension. PAP and SAP are given (means ± SEM of six independent experiments each). After stable pulmonary hypertension, urodilatin was applied as a short-term infusion (10 min) in the following doses: 3, 30, and 300 ng/kg min. The doses were applied in random order and after each infusion the animals were allowed to recover for at least 30 min.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Dose-effect curves of intravenous and inhaled dipyridamole on U46619-elicited pulmonary hypertension. PAP is given (mean ± SEM of six independent experiments each). After stable pressure plateau, dipyridamole was applied in different doses as a short-term infusion (10 min) or short-term inhalation (10 min) in incremental doses (intravenous: 2, 10, and 20 µg/kg min; inhaled: 0.1, 1, and 10 µg/kg min).

Infusion of Urodilatin

Short-term infusion of urodilatin at a dose of 30 ng/kg min for 10 min resulted in a decrease of PAP from 27.3 ± 1.0 to 24.7 ± 1.1 mm Hg (P < 0.05). The vasodilatory effect started within 2 min after onset of infusion and was accompanied by significant peripheral vasodilation, as documented by the significant decrease in the SAP values (Table 1). A minor (not significant) decrease in PAOP was noted. After termination of urodilatin infusion, PAP started to rise and baseline values were reached within 40 min (Figure 3). The urodilatin-elicited decrease in PAP and SAP was accompanied by an increase in cardiac output, from 390 ± 15 to 438 ± 16 ml/min (not significant). PVR, expressed as percent of resistance values under stable U46619 infusion, decreased to 83.0 ± 2.2% in response to the short-term urodilatin infusion (Figure 4). This vasodilatory response was accompanied by an increase of plasma cGMP levels from 14.5 ± 5.9 to 37.6 ± 1.2 nmol/l (Figure 5). No significant changes in blood gases, heart rate, or ventilation pressure occurred. These effects were further accompanied by a marked diuresis. As compared with control animals, a significant increase in urine excretion was measured (control, 17.1 ± 2.4 ml/h; urodilatin, 35.6 ± 3.6 ml/h; P < 0.01).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Influence of urodilatin infusion and its combination with subthreshold doses of intravenous and inhaled dipyridamole on U46619-elicited pulmonary hypertension. PAP (in % of U46619- induced increase) is given (mean ± SEM of six independent experiments each; SEM bars are not visible when falling into symbol). Interventions are indicated by the horizontal bars. Uro i.v., urodilatin infusion (30 ng/kg min); Dipyr i.v., dipyridamole infusion (1 µg/kg min); Dipyr aer., dipyridamole inhalation (50 ng/kg min). *P < 0.05 as compared with urodilatin infusion.

View larger version (18K): [in this window] [in a new window]   Figure 4.   Influence of urodilatin infusion and its combination with subthreshold doses of intravenous and inhaled dipyridamole on PVR and SVR. PVR (in % of U46619-induced increase) and SVR (in %) are given at the end of the short-term infusion of 30 ng/kg min of urodilatin (means ± SEM of six independent experiments each). Dipyridamole was applied 15 min before urodilatin as a short-term infusion (10 min) or inhalation (10 min). Uro i.v., urodilatin infusion (30 ng/kg min); Dipyr i.v., dipyridamole infusion (1 µg/kg min); Dipyr aer., dipyridamole inhalation (50 ng/kg min). *P < 0.05 as compared with control; P < 0.05 as compared with urodilatin infusion.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Influence of urodilatin infusion and its combination with subthreshold doses of dipyridamole on plasma cGMP level. cGMP level is given (mean ± SEM of six independent experiments each). Urodilatin infusion (30 ng/kg min) and dipyridamole application are indicated by the horizontal bars. Dipyridamole was applied 15 min before urodilatin as a short-term infusion (10 min) or inhalation (10 min). Uro i.v., urodilatin infusion (30 ng/kg min); Dipyr i.v., dipyridamole infusion (1 µg/kg min); Dipyr aer., dipyridamole inhalation (50 ng/kg min). **P < 0.01 compared with urodilatin infusion only.

Combination of Infused Urodilatin and Infused/Inhaled Dipyridamole

Subthreshold doses of infused and inhaled dipyridamole were derived from the dose-effect curves. No changes in hemodynamics and blood gases were provoked by short-term infusion (10 min) or inhalation (10 min) of the subthreshold dipyridamole doses in animals with U46619-elicited pulmonary hypertension. In the presence of intravenous dipyridamole (1 µg/kg min), the urodilatin-induced PVR decrease was significantly augmented as compared with sole urodilatin administration (decrease to 72.7 ± 4.8 versus 83.0 ± 2.2%; P < 0.05; Figure 4). PAP drop in response to urodilatin was significantly amplified (Figure 3) but, as in the urodilatin group, preinfusion values were reached 40 min after infusion. Concomitantly, a significant increase of plasma cGMP levels was measured under conditions of combined application of intravenous dipyridamole and urodilatin (Figure 5). Compared with control, diuresis was increased (32.7 ± 5.1 ml/h; P < 0.05). The combination of inhaled dipyridamole (50 ng/kg min) with intravenous urodilatin resulted in a significant decrease of PAP (Table 1 and Figure 3) and PVR (Figure 4), accompanied by a decrease in SAP and enhancement of diuresis (39.5 ± 6.1 ml/h). The hemodynamic changes evoked by dipyridamole inhalation/urodilatin infusion did not, however, significantly surpass those of sole urodilatin infusion. This was also true for heart rate and ventilation pressure.

	Discussion

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In the present study, continuous infusion of U46619 was used to establish stable pulmonary hypertension, with approximate doubling of the baseline values of PAP. As previously described, the PAP response evoked by the stable thromboxane analogue is predominantly attributable to an increase in precapillary vascular resistance, with near-constancy of capillary hydrostatic pressure (25, 26), thus preventing lung edema formation. Accordingly, the wet-to-dry ratios of the lungs undergoing the U46619 infusion regimen were in the normal range at the end of experiments (data not given in detail), and gas exchange conditions did not change in response to the vasoconstrictor administration, as documented by the measurement of arterial PO₂ and PCO₂ values. The wedge pressure did not change significantly, which is well in line with an investigation from Kleen and colleagues, who measured PAOP in sheep with U46619-induced pulmonary hypertension (25). The enhanced afterload of the right ventricle due to increased PVR did not result in a significant decrease in cardiac output.

When infusing incremental doses of urodilatin, a dose-dependent attenuation of the U46619-elicited pulmonary hypertension was noted. The natriuretic peptide did not, however, possess selectivity for the pulmonary circulation, as the SAP declined in a corresponding fashion. Inasmuch as the cardiac output showed even a moderate increase in response to the urodilatin, the drops in PAP and SAP were both clearly attributable to vasorelaxation in both the lung and the systemic vasculature, and in fact both the PVR and the SVR values declined in parallel upon increase of the urodilatin dosage. The pulmonary vasodilatory efficacy of urodilatin has been previously noted in platelet-activating factor-induced pulmonary hypertension of isolated rat lungs (27).

When performing short-term (10 min) infusion periods with low-dose urodilatin, the vasodilator response evoked by this peptide was rapidly lost: within 30 min after termination of infusion, both the PAP and the SAP values returned to pre-urodilatin baseline values. This finding is of interest, because the catabolism of urodilatin differs from that of ANP and BNP. The latter natriuretic peptides are inactivated by neutral endopeptidases (28), and inhibitors of these enzymes have been employed to amplify and prolong the efficacy of ANP and BNP (10, 29). In contrast, the major inactivating mechanism of urodilatin is not the degradation by endopeptidases, but the clearance through C-type receptors (30). This major difference in catabolism depends on an NH₂-terminal extension of four amino acids in the urodilatin molecule. Nevertheless, rapid loss of the vasodilator capacity of urodilatin was noted in the rabbits after terminating the infusion of this peptide, suggesting effective clearance via the C-type receptors in this species.

The biologic effects of the natriuretic peptides are exerted via enhanced cGMP generation, and increased circulating levels of this cyclic nucleotide were indeed detected in the rabbits undergoing urodilatin infusion, even when performed for only a 10-min period, as shown in Figure 4. Thus, blocking of the catabolism of this second messenger may offer amplification and prolongation of the pulmonary vasodilatory effects of urodilatin. The hydrolysis of the cyclic nucleotides proceeds via a group of PDE isoenzymes (31), and the presence of the isoenzymes 1, 3, 4, and 5 has been demonstrated for the lung parenchyma (35). PDE3 and PDE4 preferentially hydrolyze cyclic adenosine monophosphate, whereas PDE5 possesses a high affinity for cGMP (31). In fact, virtually all of the cGMP hydrolyzing capacity of the lung tissue was previously shown to be attributable to PDE5 (36), thus rendering this enzyme a suitable target when aiming to amplify the effects of the urodilatin-cGMP axis in the lung vasculature. Dipyridamole is an agent with well-established PDE5 inhibitory capacity, with half-maximal inhibitory concentration values of 0.9 µM being reported for PDE5 activity in smooth muscle-cells (37, 38). We first established dose-effect curves for dipyridamole, using both the intravascular and the inhalative routes of application. The rationale for the latter approach is the previous finding that in addition to the gaseous nitric oxide (NO), aerosolized agents such as the prostaglandins I₂ and E₁ may effect selective vasodilation in the pulmonary circulation due to predominant regional efficacy (14, 39). Moreover, in a very recent study in isolated perfused rabbit lungs, alveolar aerosol delivery of PDE3/4 inhibitors at doses that per se exerted no effect on the pulmonary hemodynamics amplified the vasodilator response of the pulmonary vasculature to prostacyclin admixed to the perfusion fluid of the isolated organs (42).

In the present study, when infused in intact rabbits, dipyridamole effected a decrease in PAP and SAP with virtually superimposable dose-inhibition curves. This finding is in line with a previous experimental study in pulmonary hypertension in which the infusion of the PDE5 inhibitor zaprinast decreased both the PAP and the SAP (43). Such an effect of PDE5 inhibition is most probably attributable to stabilization of some baseline cGMP formation, occurring at a basal rate even in the absence of exogenously applied stimuli such as inhaled NO, NO-donor agents, or urodilatin as used in the present investigation. Interestingly, when applied via the inhalative route, the absolute quantities of dipyridamole required for a significant drop in PAP ranged more than two orders of magnitude below the doses that were infused to achieve a comparable pulmonary vasorelaxant effect. As may be anticipated, no decrease in SAP was noted upon use of such low inhalative dosages. The impressive selectivity of the aerosolized dipyridamole for the pulmonary circulation may not be explained by an intrinsic property of this PDE inhibitor, as complete loss of selectivity was noted upon intravascular administration of this agent. Thus, low-dose inhaled dipyridamole apparently exerts virtually exclusive regional (pulmonary) vasodilation, with its relaxant properties being lost as a result of "dilution" of these small quantities of the agent in the circulating blood volume. This corresponds well to preceding studies in U46619-induced pulmonary hypertensive lambs, in which the nebulization of the PDE5 inhibitor zaprinast turned out to be a pulmonary selective vasodilator (44).

Aiming to test for a true synergistic effect of urodilatin and dipyridamole, subthreshold doses of the PDE5 inhibitor were chosen (both via intravenous and via inhalative routes), which per se did not evoke any change in hemodynamics and were combined with a subsequent short-term infusion of urodilatin. In the case of intravenous dipyridamole, synergism was clearly demonstrated, evident from a more prominent drop of both PAP and SAP in response to the standardized urodilatin dosage, with corresponding decrease in the PVR and SVR values. Moreover, prolongation of the vasodilatory response to the natriuretic peptide was noted, as PAP and SAP values did not fully reach the pre-urodilatin levels within 30 min after termination of urodilatin infusion. Further, the sequential administration of intravenous dipyridamole and intravenous urodilatin provoked a markedly enhanced cGMP increase, measured at the end of the 10-min urodilatin infusion period. All of these findings are well compatible with the notion that low-dose systemic dipyridamole administration for PDE5 inhibition, itself not effecting any hemodynamic alterations, sufficed to amplify the pulmonary and systemic vasodilatory response to subsequently infused urodilatin via stabilization of its second messenger, cGMP. These data do not, of course, exclude the possibility that dipyridamole might also be effective via their known interaction with adenosine receptors. However, the currently employed dose range is far below that reported for significant adenosine receptor occupancy in rabbits (45).

In contrast, sequential performance of dipyridamole aerosolization (subthreshold dose) and urodilatin infusion caused no significant enhancement of the urodilatin-elicited drops in PAP and PVR. Interestingly, circulating cGMP levels were nevertheless amplified in the animals undergoing the sequence of dipyridamole aerosolization and urodilatin infusion. This observation may suggest that: (1) most of the cGMP detected in the blood of these animals may originate from the pulmonary circulation, inasmuch as this is the only site wheredue to the mode of alveolar depositionsignificant local concentrations of dipyridamole are to expected; and (2) the low dosage of aerosolized dipyridamole in these experiments (50 ng/kg min), chosen on the basis of the present protocol to test for synergy, might be too low to translate a synergistic effect based on cGMP stabilization into a substantial amplification of the pulmonary vasodilator response to urodilatin. Further studies are clearly mandatory to address these issues in more detail, e.g., by using higher nebulized dipyridamole doses when testing for synergy with intravascular urodilatin.

In conclusion, the present study is the first to demonstrate that the A-type natriuretic peptide urodilatin possesses vasodilatory properties in the pulmonary circulation when investigated in a model of pulmonary hypertension. This agent is, however, not selective for the lung vasculature, inasmuch as the SVR declined in a corresponding fashion. Preceding infusion of the PDE5 inhibitor dipyridamole, used at a subthreshold dosage, amplified the vasodilatory response to urodilatin, most probably via inhibition of cGMP breakdown, as suggested by increased plasma levels of this cyclic nucleotide. Such synergism was less obvious when combining aerosolized dipyridamole with infused urodilatin. These findings suggest that urodilatin may be added to the list of agents being of interest for alleviation of pulmonary hypertension via the cGMP axis.