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Am J Physiol Lung Cell Mol Physiol 290: L723-L729, 2006. First published November 11, 2005; doi:10.1152/ajplung.00485.2004
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Inhibition of phosphodiesterase 1 augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs with acute pulmonary hypertension

Oleg V. Evgenov,1 Cornelius J. Busch,1,2 Natalia V. Evgenov,3 Rong Liu,1 Bodil Petersen,1 George E. Falkowski,1 Beata Petho,4 Ádám Vas,4 Kenneth D. Bloch,1,2 Warren M. Zapol,1 and Fumito Ichinose1,2

1Department of Anesthesia and Critical Care, 2Cardiovascular Research Center, and 3Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts; and 4Gedeon Richter Ltd., Budapest, Hungary

Submitted 30 December 2004 ; accepted in final form 7 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Phosphodiesterase 1 (PDE1) modulates vascular tone and the development of tolerance to nitric oxide (NO)-releasing drugs in the systemic circulation. Any role of PDE1 in the pulmonary circulation remains largely uncertain. We measured the expression of genes encoding PDE1 isozymes in the pulmonary vasculature and examined whether or not selective inhibition of PDE1 by vinpocetine attenuates pulmonary hypertension and augments the pulmonary vasodilator response to inhaled NO in lambs. Using RT-PCR, we detected PDE1A, PDE1B, and PDE1C mRNAs in pulmonary arteries and veins isolated from healthy lambs. In 13 lambs, the thromboxane A2 analog U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mmHg. Four animals received an intravenous infusion of vinpocetine at incremental doses of 0.3, 1, and 3 mg·kg–1·h–1. In nine lambs, inhaled NO was administered in a random order at 2, 5, 10, and 20 ppm before and after an intravenous infusion of 1 mg·kg–1·h–1 vinpocetine. Administration of vinpocetine did not alter pulmonary and systemic hemodynamics or transpulmonary cGMP or cAMP release. Inhaled NO selectively reduced mean pulmonary arterial pressure, pulmonary capillary pressure, and pulmonary vascular resistance index, while increasing transpulmonary cGMP release. The addition of vinpocetine enhanced pulmonary vasodilation and transpulmonary cGMP release induced by NO breathing without causing systemic vasodilation but did not prolong the duration of pulmonary vasodilation after NO inhalation was discontinued. Our findings demonstrate that selective inhibition of PDE1 augments the therapeutic efficacy of inhaled NO in an ovine model of acute chemically induced pulmonary hypertension.

guanosine 3',5'-cyclic monophosphate; vinpocetine; sheep

PULMONARY HYPERTENSION (PH) is a life-threatening condition that is characterized by an increased pulmonary arterial pressure due to an elevated pulmonary vascular resistance. Impaired production of endogenous vasodilators, such as nitric oxide (NO) and prostacyclin, and excessive release of vasoconstrictors, such as thromboxane A2 and endothelin, play an important role in the pathophysiology of PH (15, 24). Treatment of PH with oral or intravenous vasodilating agents including NO-releasing drugs and analogs of prostacyclin can be associated with potentially catastrophic systemic arterial hypotension. In contrast to systemically administered vasodilators, inhaling low concentrations of NO produces pulmonary vasodilation in well-ventilated lung regions without causing systemic arterial hypotension because NO is rapidly bound by hemoglobin upon entry into the intravascular space (19, 31).

Because the vasodilator effects of NO are largely mediated via cGMP-dependent mechanisms (19, 34), it has been hypothesized that inhibition of the cGMP-metabolizing phosphodiesterases (PDEs) would augment the ability of inhaled NO (iNO) to dilate the pulmonary vasculature by further increasing cGMP levels in pulmonary vascular smooth muscle cells (15). In the lungs, at least six cGMP-metabolizing PDE families (PDE 1, 2, 3, 5, 9, and 10) have been identified (8, 28, 29, 33, 35, 37). We have previously reported that zaprinast, an inhibitor of several cGMP-metabolizing PDEs, potentiates and markedly prolongs pulmonary vasodilation induced by iNO when administered in lambs with chemically induced PH (16, 17). Subsequently, oral administration of the more potent, clinically approved PDE5 inhibitor sildenafil has been shown to produce selective pulmonary vasodilation in experimental models, as well as in patients with PH (21, 25, 26, 39). In addition, sildenafil augmented the pulmonary vasodilator response to iNO, when administered in an aerosolized form in lambs with PH (18), suggesting an important modulatory role of PDE5 on pulmonary vascular tone. However, the specific roles of other cGMP-metabolizing PDEs in modulating the pulmonary vasodilator response to iNO remain largely unexplored.

Recent evidence suggests that PDE1 contributes to the regulation of systemic vascular tone (32, 38). Vinpocetine [14-ethoxycarbonyl-(3{alpha},16{alpha}-ethyl)-14,15-eburnamenine], a synthetic alkaloid derivative, is one of the most selective PDE1 inhibitors currently available (1, 12, 38). Vinpocetine has been recently shown to limit nitroglycerin-induced tolerance in isolated rat aortas, as well as to attenuate hypoxic pulmonary vasoconstriction in an in situ perfused rat lung preparation (20, 28). On the basis of these observations, we hypothesized that PDE1 is expressed in the ovine pulmonary vasculature and that systemic administration of vinpocetine would produce pulmonary vasodilation and also enhance vasodilator responsiveness to iNO in lambs with acute chemically induced PH. We found that three PDE1 isozymes (PDE1A, PDE1B, and PDE1C) are expressed in pulmonary arteries and veins isolated from healthy lambs. We also demonstrated that administration of vinpocetine alone did not attenuate PH but augmented the pulmonary vasodilation induced by iNO.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
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 REFERENCES
 
This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and was approved by the Subcommittee on Research Animal Care of the Massachusetts General Hospital.

Isolation of pulmonary vessels and analysis of PDE1 isozyme expression. Four healthy lambs were killed with an intravenous injection of 100 mg/kg pentobarbital (Abbott Laboratories, North Chicago, IL). The chest cavity was immediately exposed through a midline sternotomy, and the lungs and heart were carefully removed en bloc and placed in ice-cold phosphate-buffered saline solution (MP Biomedicals, Aurora, OH). The main and fourth-generation pulmonary arteries and the main and fourth-generation pulmonary veins were dissected free, cleaned of the surrounding parenchyma, and rapidly frozen in liquid N2 before being homogenized. Total RNA was extracted from the main pulmonary artery with the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). cDNA was generated with reverse transcriptase and random primers (Promega, Madison, WI) and was amplified by PCR and primers designed to detect PDE1A (5'-CGGGATCCAARTTYATGATHTAYGA-3', 5'-CGAATTCTGYTCRTARTCRTGDAT-3'), PDE1B(5'-CGGGATCCAARAARATGTGGATHAA-3', 5'-CGAATTCAAYTTNGGYTTYTCYTC-3'), and PDE1C (5'-CGGGATCCAARGARATHGARGARTT-3', 5'-CGAATTCTGNACNGCRTGNACDAT-3'). To detect cDNAs encoding ovine PDE1 isozymes, we synthesized degenerate primers corresponding to amino acid sequences conserved in the mouse, rat, and human genome. The PCR products were purified and ligated into pUC19 (EcoRI/BamHI). The identity of the cDNAs was verified by direct sequencing.

Subsequently, PDE1A, PDE1B, PDE1C, and 18S ribosomal RNA transcript levels were determined in the total RNA extracted from the main and fourth-generation pulmonary arteries and the main and fourth-generation pulmonary veins by real-time quantitative PCR with an ABI Prism 7000 (Applied Biosystems, Foster City, CA). PDE1 isozyme cDNA were detected by using the following primer sets with SYBR Green reagent: PDE1A (5'-GACATCAGCTGCATGAATCAA-3', 5'-TCTTGCCTAATTGCCTTTGC-3'), PDE1B (5'-TCTGCTCGTAGTCGTGGATG-3', 5'-ACTGCCTGTCGGAGATTGAG-3'), and PDE1C (5'-CACAGAGGCTGCATATTCCA-3', 5'-GAGAGAGGGGAGGCTTCAGT-3'). To detect 18S ribosomal RNA, we used a TaqMan primer set supplied by Applied Biosystems. Changes in the expression of PDE1 genes normalized to 18S rRNA levels were determined by the relative cycle threshold method (11, 13).

Surgical preparation. Thirteen lambs weighing 21.4 (2.8) kg [means (SD)] were anesthetized with an intramuscular injection of 15 mg/kg ketamine hydrochloride (Fort Dodge Animal Health, Fort Dodge, IA) and 0.5 mg/kg xylazine (Lloyd Laboratories, Shenandoah, IA). A tracheotomy was performed, and an 8.0-mm cuffed tracheostomy tube (SIMS Portex, Keene, NH) was inserted. The lungs were ventilated at 15 breaths/min, 15 ml/kg tidal volume, and an inspired fraction of oxygen (FIO2) of 0.5 by a volume-controlled ventilator (model 613; Harvard Apparatus, South Natick, MA). Anesthesia was maintained with inhalation of 1.2–1.5% isoflurane (Baxter Healthcare, Deerfield, IL) and intravenous injections of 1 mg/kg morphine (Elkins-Sinn, Cherry Hill, NJ). Through a left thoracotomy in the forth intercostal space, a polyvinyl chloride catheter [1.5-mm inner diameter (ID)] was implanted into the left atrium. An 8-Fr introducer (Cordis, Miami, FL) was placed in the left external jugular vein, and a 7-Fr flow-directed thermal dilution catheter (Edwards Lifesciences, Irvine, CA) was introduced into the pulmonary artery. In addition, a polyvinyl chloride catheter (1.5-mm ID) was inserted into the left common carotid artery (6). The catheters were continuously flushed with a saline solution containing 2 IU/ml heparin (Abbott Laboratories, North Chicago, IL).

After emergence from general anesthesia, the animals were housed in a large-animal mobile restraint unit (Lomir, Malone, NY) and allowed at least 3 h of recovery. The study commenced if the following baseline exclusion criteria did not occur: a peripheral white blood cell count <4,000 or >12,000 mm–3, mean pulmonary arterial pressure >20 mmHg, or a body temperature measured by the pulmonary artery catheter >40°C (16, 17).

Hemodynamic measurements. Mean arterial pressure (MAP), mean pulmonary arterial pressure (PAP), and mean left atrial pressure (LAP) were measured continuously, and pulmonary arterial occlusion pressure (PAOP) was measured intermittently with calibrated pressure transducers (Maxxim Medical, Athens, TX) zero-referenced at the midchest. The pressure signals were amplified (model 7700; Hewlett Packard, Palo Alto, CA) and recorded using a DT-220 converter and a WinDaq 2.25 data acquisition program (DATAQ Instruments, Akron, OH). Heart rate was obtained from the phasic arterial pressure trace. Effective pulmonary capillary pressure (Pc) was derived from the PAOP tracing according to a technique described by Holloway et al. (14). In brief, when the balloon of the pulmonary artery thermal dilution catheter was rapidly inflated, the pressure at the distal port initially declined rapidly and then more slowly to approach PAOP. Pc was estimated visually as the inflection point between the rapid and the slow exponential components of the PAOP tracing. Cardiac output was determined by a cardiac output computer (SAT-2, Edwards Lifesciences) as the average of four measurements after injection of 10 ml of ice-cold isotonic saline. Body surface area was calculated as (body wt in kg)0.67 x 0.084. Cardiac index (CI) was calculated as cardiac output divided by body surface area, systemic vascular resistance index (SVRI) as MAP/CI x 80, and pulmonary vascular resistance index (PVRI) as (PAP – LAP)/CI x 80 (6).

Delivery of NO. The tracheostomy tube was connected to a circuit consisting of a 5-l reservoir bag and a two-way nonrebreathing valve (Hans Rudolph, Kansas City, MO) to separate inspired from expired gas. Using volumetrically calibrated flowmeters, we mixed oxygen and air to produce FIO2 at 0.6, which was continuously monitored (Oxygen Monitor 5590; Hudson, Temecula, CA). NO gas (800 ppm in nitrogen; INO Therapeutics, Clinton, NJ) was introduced into the inspiratory limb of the breathing circuit immediately before the reservoir bag (5, 17). The concentration of NO was continuously measured with a calibrated chemiluminescence analyzer (Sievers 280; Ionics Instruments, Boulder, CO) on the inspiratory limb of the two-way valve. Exhaled gases were scavenged via a Venturi exhalation trap maintained at negative atmospheric pressure by the central vacuum system. The NO concentration in ambient air was ≤4 ppb.

Experimental protocol. During the experiments, the lambs were awake and breathed spontaneously while receiving an intravenous infusion of lactated Ringer solution (10 ml·kg–1·h–1). All measurements and samples were obtained at baseline and before and at the end of each treatment.

Following baseline measurements, we intravenously administered the stable endoperoxide analog of thromboxane A2 U-46619 (Cayman Chemical, Ann Arbor, MI), dissolved in lactated Ringer solution, at a rate of 1.6 (0.4) µg·kg–1·min–1 to increase PAP to ~35 mmHg (5). After a 30-min stabilization period, four lambs received an intravenous infusion of vinpocetine (Gedeon Richter, Budapest, Hungary) in sequential incremental doses of 0.3, 1, and 3 mg·kg–1·h–1, which were administered for 1 h each. Of note, the maximal dose of vinpocetine was selected based on our pilot experiments, where bolus intravenous injection of the drug at a higher dose (5 mg/kg over 5 min) caused signs of acute toxicity, including convulsions, profound tachycardia, and hypertension.

In nine additional lambs with U-46619-induced acute PH, NO gas was administered via inhalation in a random sequence at 2, 5, 10, or 20 ppm. At each dose level, NO was inhaled for 10 min followed by a 15-min NO-free period. All hemodynamic parameters returned to pretreatment values during the latter period. A loading intravenous dose of 3 mg/kg vinpocetine was then administered over 5 min, followed by a continuous intravenous infusion of 1 mg·kg–1·h–1. After a 30-min stabilization period, NO was inhaled again at the same concentrations and in the same order as was given before vinpocetine administration. After the final measurements, the animals were killed with an intravenous injection of 100 mg/kg pentobarbital.

Blood samples and analysis of blood gases. Blood samples were simultaneously obtained from the carotid and pulmonary arteries and analyzed for pH, PO2, and PCO2 with a blood gas analyzer (Rapidlab 840; Chiron Diagnostics, Medfield, MA). Oxygen saturation and the concentrations of hemoglobin and methemoglobin were determined by an OSM3 Hemoximeter (Radiometer, Copenhagen, Denmark). Barometric pressure was noted daily. Using the alveolar gas equation, we calculated alveolar oxygen tension, assuming a respiratory quotient of 0.8. Assuming a hemoglobin oxygen binding capacity of 1.31 ml/g and pulmonary end-capillary oxygen saturation of 100%, we calculated oxygen content of arterial blood, pulmonary end-capillary blood, and mixed venous blood. Venous admixture (Qs/Qt) was calculated with a standard equation (23). In addition, arterial and mixed venous blood was collected in sample tubes containing a buffered sodium citrate solution and 3-isobutyl-1-methyl-xanthine (Sigma-Aldrich, St. Louis, MO) at final concentration of 0.5 mM. The mixture was centrifuged at 4°C (2,000 g for 10 min). The plasma supernatants were stored at –70°C.

Transpulmonary cGMP and cAMP release. Arterial and mixed venous plasma concentrations of cGMP or cAMP were determined by radioimmunoassay (BT-340 and BT-300; Biomedical Technologies, Stoughton, MA) as previously described (16). The quantity of cGMP or cAMP released by the lung per minute during each treatment was calculated as the product of cardiac output times the difference between the arterial and mixed venous plasma cGMP concentrations (5).

Measurements of plasma concentrations of vinpocetine. An internal standard working solution of apovincaminic acid methyl ester was added to plasma samples. The samples were extracted with hexane and centrifuged (1,000 g for 20 min). The organic layer was transferred into a glass tube, and the solvent was evaporated with a stream of N2 at 40°C in a water bath. For gas chromatography-mass spectrography (GC-MS), the dry residue was dissolved in tert-butyl-methyl-ether, and the analysis was performed by using a Trace DSQ GC-MS (Thermo Finnigan, San Jose, CA) and an HP-5 analytical column (12 m x 0.2 mm x 0.33 µm; Hewlett-Packard, Palo Alto, CA). Helium was used as carrier gas. Injections were performed in split mode with the split ratio of 1:20. The molecular ions at mass-to-charge ratio (m/z) 321.1 and 307.1 for vinpocetine and internal standard, respectively, were monitored in single ion monitoring mode. The lower limit for quantification of vinpocetine was 5 ng/ml.

Data analysis. Values are expressed as means (SD). We determined the half-time (T1/2) of the decay of pulmonary vasodilation by measuring the elapsed time from the termination of NO inhalation to the time when PAP returned to a value halfway between the values recorded at the end of NO inhalation and the baseline PH level (5, 17, 18). Statistical comparisons were made by repeated-measures ANOVA with a post hoc Student-Newman-Keuls test for multiple comparisons (SigmaStat 3.0; Systat Software, Richmond, CA). A value of P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Using RNA extracted from ovine pulmonary vessels, we amplified mRNA specifying PDE1A, PDE1B, and PDE1C isozymes by RT-PCR (data from the main pulmonary arteries are shown in Fig. 1). The translated sequences of the PCR products were compared with amino acid sequences of human PDE1A (accession number P54750), PDE1B (accession number Q01064), and PDE1C (accession number Q64338) by BLASTx. Ovine PDE1A, PDE1B, and PDE1C were found to be 97, 95, and 92% homologous to human PDE1A, PDE1B, and PDE1C, respectively. By using quantitative PCR, we detected no significant differences in the levels of mRNAs specifying PDE1A, PDE1B, and PDE1C between the main pulmonary arteries, the fourth-generation pulmonary arteries, the main pulmonary veins, and the fourth-generation pulmonary veins (data not shown).


Figure 1
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  		Fig. 1. RT-PCR amplification of phosphodiesterase (PDE) 1A, PDE1B, and PDE1C mRNAs in RNA extracted from the main pulmonary artery of healthy sheep. BP, base pairs; M, marker.

 
As shown in Table 1, administration of incremental doses of vinpocetine alone did not alter pulmonary or systemic hemodynamics, pulmonary gas exchange, or transpulmonary cGMP or cAMP release in lambs with acute PH. The plasma concentration of vinpocetine increased step-wise in accordance with the increments of the vinpocetine infusion rate.


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  		Table 1. Effects of incremental doses of intravenously infused vinpocetine in lambs with U-46619-induced acute pulmonary hypertension

 
As illustrated in Fig. 2 and Table 2, administration of iNO produced dose-dependent reductions in PAP and PVRI starting from the dose of 5 ppm (P < 0.05). In addition, iNO reduced Pc and increased transpulmonary cGMP release (Fig. 3), when inhaled at 10 and 20 ppm (P < 0.05). However, systemic hemodynamics (heart rate and LAP are not shown) and transpulmonary cAMP release were unaffected by breathing NO gas. At 20 ppm of iNO, Qs/Qt decreased below that of the baseline PH value, whereas arterial oxygenation correspondingly increased (P < 0.05). After iNO was discontinued, the mean T1/2 of the decay of pulmonary vasodilation was ~30 s for all iNO doses.


Figure 2
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  		Fig. 2. Changes in mean pulmonary arterial pressure (PAP), pulmonary capillary pressure (Pc), and pulmonary vascular resistance index (PVRI) during inhaled nitric oxide (iNO) administered alone (iNO group) or in combination with intravenously infused vinpocetine (V + iNO group) in lambs with U-46619-induced acute pulmonary hypertension (PH). U-46619 was infused intravenously to increase PAP to ~35 mmHg. NO gas was administered via inhalation in a random sequence at 2, 5, 10, or 20 ppm for 10 min followed by a 15-min NO-free period. Thereafter, the animals received intravenous infusion of vinpocetine, and iNO was administered again. Values are means (SD), n = 9; *P < 0.05 vs. baseline pulmonary hypertension; {dagger}P < 0.05 vs. the corresponding dose of iNO alone.

 

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  		Table 2. Effects of inhaled nitric oxide administered alone or in combination with intravenously infused vinpocetine in lambs with U-46619-induced acute pulmonary hypertension

 

Figure 3
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  		Fig. 3. Transpulmonary cGMP release following NO inhaled alone (iNO group) or in combination with intravenously infused vinpocetine (V + iNO group) in lambs with U-46619-induced acute PH. Values are means (SD), n = 6; *P < 0.05 vs. baseline pulmonary hypertension; {dagger}P < 0.05 vs. the corresponding dose of iNO alone. For further information, see legend to Fig. 1.

 
As shown in Fig. 2 and Table 2, the concomitant infusion of vinpocetine markedly enhanced the reductions in PAP and Pc induced by 2, 5, and 10 ppm of iNO (P < 0.05). In parallel, the combined administration of vinpocetine and iNO at all doses decreased PVRI below that obtained during iNO alone (P < 0.05). The combination of vinpocetine and iNO administered at 10 and 20 ppm also increased CI (Table 2, P < 0.05 vs. baseline PH), whereas other hemodynamic variables remained unchanged. Furthermore, transpulmonary cGMP release was greater during 5, 10, and 20 ppm of iNO administered in combination with vinpocetine than during iNO alone (Fig. 3, P < 0.05), whereas no significant changes in transpulmonary cAMP release occurred (Table 2). The vinpocetine infusion enabled iNO to reduce Qs/Qt starting from the dose of 2 ppm, as well as to improve PaO2/FIO2 starting from the dose of 5 ppm (Table 2, P < 0.05 vs. baseline PH). However, vinpocetine did not change T1/2 of the decay of pulmonary vasodilation after iNO was discontinued, as shown in Table 2. The mean plasma concentration of vinpocetine remained stable throughout this part of the study. The mean arterial concentration of methemoglobin did not exceed 1% during iNO administered alone or in combination with vinpocetine (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
Our study demonstrates that the PDE1A, PDE1B, and PDE1C isozymes are expressed in ovine pulmonary vasculature. Selective inhibition of the PDE1 family by intravenously administered vinpocetine does not attenuate acute chemically induced PH in awake lambs; however, it augments the pulmonary vasodilator effect of iNO. Similarly, vinpocetine alone does not modulate transpulmonary cGMP release, whereas the iNO-induced increase in transpulmonary cGMP release is augmented by concomitant infusion of this drug.

In mammalian tissues, PDE1 is expressed as three isozymes named PDE1A, PDE1B, and PDE1C (3). It is generally agreed that PDE1A and PDE1B selectively hydrolyze cGMP, while PDE1C hydrolyses cGMP and cAMP with equal affinity (32, 35). In the present investigation, we detected the expression of PDE1A, PDE1B, and PDE1C genes in the main pulmonary arteries, the fourth-generation pulmonary arteries, the main pulmonary veins, and the fourth-generation pulmonary veins isolated from healthy lambs. Our findings add to previous reports demonstrating the expression of the PDE1 isozymes in normal rabbit and bovine lungs, as well as in rat and human pulmonary arteries (28, 29, 35, 36).

Vinpocetine has been shown to produce selective inhibition of the PDE1 isozymes in various tissues with IC50 ranging from {approx}10 to 50 µM (1, 12, 20, 22, 40). In preconstricted isolated rabbit aortic strips and in isolated perfused rat lungs exposed to hypoxia, vinpocetine produced vasorelaxation and increased cGMP but not cAMP levels (1, 12, 28). However, in the present in vivo study, intravenous infusion of vinpocetine at incremental doses up to 3 mg·kg–1·h–1 produced no effect on acute pulmonary vasoconstriction induced by U-46619. In addition, no changes were observed in transpulmonary cGMP or cAMP production and systemic hemodynamics. In contrast, zaprinast, which inhibits PDE1 with an IC50 similar to vinpocetine (1, 12, 22, 40), selectively decreased PAP and PVRI and increased transpulmonary cGMP release when administered intravenously as either a bolus (0.5–5 mg/kg over 5 min) or a continuous infusion (6 mg·kg–1·h–1) or after inhalation in an aerosolized form (an effective dose of {approx}3–4 mg·kg–1·h–1) in the same ovine model of PH (4, 16, 17). The difference in the pulmonary responses to vinpocetine and zaprinast may be attributed to the fact that zaprinast also inhibits PDE5 (IC50 <1 µM) and PDE9 (IC50 = 35 µM) (1, 2, 7). Furthermore, sildenafil, a more potent and selective PDE5 inhibitor (IC50 {approx} 3.5 nM for PDE5; IC50 {approx} 280 nM for PDE1) (2), produced marked pulmonary vasodilation when administered in animal models (18, 39) and patients with PH (21, 25, 26). Thus the absence of a pulmonary vasodilator response to vinpocetine alone suggests that in chemically induced acute PH in sheep, the PDE1 family plays a relatively modest role in the modulation of the pulmonary vascular tone compared with PDE5.

In the present investigation, iNO administered alone produced selective pulmonary vasodilation at the doses starting from 5 ppm and significantly increased pulmonary cGMP production at doses starting from 10 ppm. These results are in a good agreement with previous experimental and clinical studies of iNO therapy in various forms of PH (5, 10, 16–19, 39). When iNO was combined with intravenously infused vinpocetine, the reductions in PAP, Pc, and PVRI were all markedly augmented, particularly at low doses of iNO (2–10 ppm). In addition to a further decrease of PAP, CI increased during coadministration of vinpocetine and iNO, indicating an additional dilation of the pulmonary vasculature. This augmentation of the iNO-induced pulmonary vasodilation occurred in parallel with an additional rise in transpulmonary cGMP release. Interestingly, the cGMP-specific PDE activity in isolated ovine pulmonary veins has been demonstrated to be more sensitive to inhibition by zaprinast compared with pulmonary arteries (27). In the present study, we found no significant differences in the longitudinal expression of the PDE1 isozymes in the pulmonary vasculature. Therefore, the increase in transpulmonary cGMP release following combined administration of iNO and vinpocetine is unlikely to be attributed to a relatively larger accumulation of cGMP in pulmonary veins than in pulmonary arteries. Nevertheless, it is conceivable that vasodilation observed in our model of PH mainly occurred in pulmonary veins because thromboxane A2 or its analog U-46619 are potent constrictors of pulmonary veins in a number of species including dog, sheep, and human (9).

Because of its ability to improve ventilation-perfusion matching, iNO is used to enhance systemic oxygenation in newborn and adult patients with PH or acute hypoxic respiratory failure (19). In contrast, systemic administration of vasodilators can lead to worsening of preexisting mismatching of ventilation and perfusion and subsequent deterioration of arterial oxygenation (30). Although the present lamb model is not designed to produce marked abnormalities of pulmonary gas exchange, it is characterized by a moderately increased Qs/Qt and decreased arterial oxygenation. We observed that iNO administered at 20 ppm decreased Qs/Qt and correspondingly increased PaO2/FIO2, findings that concur with our previous investigations in the same model (5, 18). Importantly, the intravenous infusion of vinpocetine enabled lower doses of iNO (2–10 ppm) to reduce venous admixture and thereby improve arterial oxygenation. These results suggest that by reducing pulmonary cGMP degradation vinpocetine augments the iNO-induced increase of blood flow in better-ventilated lung areas.

Enhancement of iNO-induced pulmonary vasodilation and transpulmonary cGMP release has previously been reported following coadministration of zaprinast or sildenafil in the lamb model and patients with PH (16–18, 25). In addition, zaprinast markedly prolonged the duration of the pulmonary vasodilation after iNO was discontinued (16, 17). In contrast, vinpocetine had no effect on the duration of the iNO-induced pulmonary vasodilation in the present study. The latter observation suggests that other PDEs modulate the duration of iNO action. Overall, our findings indicate that the effectiveness of vinpocetine in influencing pulmonary hemodynamics, gas exchange, and hydrolysis of cGMP only becomes evident during NO inhalation, i.e., only when large amounts of cGMP accumulate in the pulmonary vasculature.

Recent evidence suggests that upregulation of PDE1A expression may be one of the key mechanisms responsible for impairing responsiveness to NO-releasing drugs, at least, in the systemic vasculature (20, 32). For example, in a nitrate-tolerant rat model induced by chronic nitroglycerin treatment, PDE1A enzyme activity, protein levels, and mRNA expression were found to be all increased. Correspondingly, vinpocetine increased the sensitivity of tolerant aortic rings to subsequent nitroglycerin exposure (20). It is well described that a significant number of patients with respiratory failure and/or PH do not respond to iNO with pulmonary vasodilation (19). Although increased expression of PDE1 isozymes has not been demonstrated in the pulmonary vasculature of patients with PH, it is conceivable that upregulation of PDE1 in the pulmonary circulation might contribute to the reduced responsiveness to iNO in such patients. If that were the case, inhibition of PDE1 might improve pulmonary vascular responsiveness to iNO. This hypothesis merits further investigation in the experimental and clinical settings.

To conclude, three PDE1 isozymes (PDE1A, PDE1B, and PDE1C) are expressed in ovine pulmonary vasculature. Selective inhibition of PDE1 by systemically administered vinpocetine does not attenuate acute PH in awake lambs. However, vinpocetine infusion augments the pulmonary vasodilatory response and transpulmonary cGMP release following NO inhalation without altering its pulmonary selectivity. Our study suggests that selective inhibition of PDE1 may represent a useful adjuvant to enhance the efficacy of iNO therapy in subjects with PH.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This study was supported in part by National Heart, Lung, and Blood Institute Grants HL-42397 (to W. M. Zapol), HL-71987 (to F. Ichinose), and HL-74352 (to K. D. Bloch), the Research Council of Norway (Grant 161151/V40 to O. V. Evgenov), and through a sponsored research agreement with INO Therapeutics Inc. (K. D. Bloch).


   DISCLOSURES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
Massachusetts General Hospital has licensed patents covering the inhalation of nitric oxide to INO Therapeutics, a division of Linde Gas Therapeutics, and Dr. Zapol receives a portion of the royalties. Dr. Zapol and Dr. Bloch are members of the Scientific Advisory Board of INO Therapeutics Inc. Dr. Petho and Dr. Vas are full-time employees of Gedeon Richter Ltd.


   ACKNOWLEDGMENTS
 
The authors thank Dr. York Zausig, Department of Anesthesiology, University of Heidelberg, Germany, for providing vinpocetine.


   FOOTNOTES
 

Address for reprint requests and other correspondence: O. V. Evgenov, Dept. of Anesthesia and Critical Care, Mass. General Hospital, 55 Fruit St., CLN 309, Boston, MA 02114 (e-mail: evgenov{at}etherdome.mgh.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

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