PDE1 or PDE5 inhibition augments NO‐dependent hypoxic constriction of porcine coronary artery via elevating inosine 3′,5′‐cyclic monophosphate level

Abstract Hypoxic coronary vasospasm may lead to myocardial ischaemia and cardiac dysfunction. Inosine 3′,5′‐cyclic monophosphate (cIMP) is a putative second messenger to mediate this pathological process. Nevertheless, it remains unclear as to whether levels of cIMP can be regulated in living tissue such as coronary artery and if so, what is the consequence of this regulation on hypoxia‐induced vasoconstriction. In the present study, we found that cIMP was a key determinant of hypoxia‐induced constriction but not that of the subsequent relaxation response in porcine coronary arteries. Subsequently, coronary arteries were treated with various phosphodiesterase (PDE) inhibitors to identify PDE types that are capable of regulating cIMP levels. We found that inhibition of PDE1 and PDE5 substantially elevated cIMP content in endothelium‐denuded coronary artery supplemented with exogenous purified cIMP. However, cGMP levels were far lower than their levels in intact coronary arteries and lower than cIMP levels measured in endothelium‐denuded coronary arteries supplemented with exogenous cIMP. The increased cIMP levels induced by PDE1 or PDE5 inhibition further led to augmented hypoxic constriction without apparently affecting the relaxation response. In intact coronary artery, PDE1 or PDE5 inhibition up‐regulated cIMP levels under hypoxic condition. Concomitantly, cGMP level increased to a comparable level. Nevertheless, the hypoxia‐mediated constriction was enhanced in this situation that was largely compromised by an even stronger inhibition of PDEs. Taken together, these data suggest that cIMP levels in coronary arteries are regulated by PDE1 and PDE5, whose inhibition at a certain level leads to increased cIMP content and enhanced hypoxic constriction.


| INTRODUC TI ON
Coronary artery spasm (CAS) refers to transient coronary artery constriction that may lead to incomplete or complete blood vessel closure and myocardial ischaemia. 1,2 Clinically, CAS may elicit angina, arrhythmia, myocardial infarction and even death. The pathogenesis of CAS includes dysfunction of vascular endothelium, hypersensitivity of vascular smooth cell, oxidative stress, chronic inflammation and increased autonomic nerve activity. 1,2 In addition, hypoxia-induced constriction of coronary arteries (also known as hypoxic coronary vasospasm) is a potential candidate for the mechanism of CAS. Impairment or dysfunction of endothelium remarkably potentiates the acute hypoxic constriction of canine coronary artery, substantially decreasing the blood supply to cardiac muscle. 3 As in the case for example in sleep apnoea, the hypoxic constriction that is repeated consistently even after several episodes of exposure to hypoxia could be a vital risking factor in patients (especially for those with a previous history of coronary disease) exposed to repeated episodes of reduced oxygen in the blood. [4][5][6] Hypoxic constriction of coronary arteries is independent of vasoconstrictors 3,7-9 ; however, it entails endothelial released nitric oxide (NO) (considered a classical vasodilator). [7][8][9][10][11][12] The removal of endothelium substantially blunts this hypoxic response, while the addition of NO restores this reaction. 9,10 Apart from NO, the hypoxic response relies on the activation of its downstream signal, soluble guanylyl cyclase (sGC). 9,13 Normally, NO activates sGC to elevate intracellular guanosine 3′,5′-cyclic monophosphate (cGMP) levels that further induce vasodilation. 13,14 However, the reported evidence does not support the notion that this hypoxic response requires cGMP. 9,10 Although cGMP is a well-established second messenger that is formed by catalysation of sGC, 13,15 it has been postulated that there may be other cGMP-like molecules produced via sGC activation with signal-transducing functions. [16][17][18] This hypothesis is supported by findings of a study using purified recombinant human sGC in which the enzyme catalysed the formation of cGMP and that of adenosine 3′,5′-cyclic monophosphate (cAMP), inosine 3′,5′-cyclic monophosphate (cIMP), cytidine 3′,5′-cyclic monophosphate, uridine 3′,5′-cyclic monophosphate and xanthosine 3′,5′-cyclic monophosphate. 19 Except for cGMP, the maximal rate formation of cIMP is substantially greater than that of other nucleosides. 19,20 In a previous study, we found that the activation of the NO-sGC axis led to increased levels of cIMP (proposed as a novel second messenger 21 ), that further activated Rho kinase, inhibited myosin light chain phosphatase activation and induced constriction in porcine coronary artery. 9 Studies using purified recombinant human phosphodiesterases (PDEs) and 3′,5′-cyclic nucleotide salts showed that cIMP is hydrolysed by a number of types of PDEs as in the case of cGMP. 22,23 However, it remains unclear as to whether this regulating mode is the same in living tissues, and if so, what are the consequences for hypoxic constriction. To address this question, we investigated the effects of various PDE inhibitors on cIMP levels and hypoxia-induced constriction in porcine coronary arteries with or without endothelium.

| Vessel tension studies
Coronary artery rings (length: 3-5 mm) were suspended in organ chambers filled with 10 mL of Krebs solution maintained at 37 ± 1°C and aerated with 95% O 2 and 5% CO 2 (pH = 7.4). Each ring was suspended by two stirrups passing through its lumen; one stirrup was anchored to the bottom of organ chamber and the other connected to a strain gauge (Power-Lab/8sp, ADI Instrument, Australia) for measuring isometric force.

| In vitro incubation study
For the cIMP incubation study, endothelium-denuded coronary artery rings were suspended in tubes filled with 5 mL of Krebs solution, maintained at 37 ± 1°C and aerated with 95% O 2 and 5% CO 2

| UPLC/MS/MS method for the quantification of cIMP and cGMP
After incubation, the frozen coronary artery tissue (40-100 mg) was homogenized and dissolved in 100% methanol containing 10 ng/mL tenofovir as an internal standard (IS) and centrifuged at 12 000 g for 20 minutes (4°C). The supernatants were collected and dried using Termovap Sample Concentrator. Subsequently, the powder was dissolved in 120 μL pure water and filtered with a 0.22 μm filter for ultra-performance liquid chromatography (UPLC)-MS/MS analysis.
The detection and quantification of cIMP and cGMP levels in porcine coronary arteries were performed using an ACQUITYI-Class UPLC system equipped with a XEVO TQS-micro triple quadrupole mass spectrometry (Waters Corp., Milford, MA, USA). 9,24 After the injection of 2 μL volume, analysts were separated using an ACQUITY UPLC ® BEH C18 (2.1 × 50 mm, 1.7 μm) column at 40°C. The gradient mobile phases consisted of solvent A (Milli-Q pure water containing 0.01% formic acid and 0.05% ammonia) and solvent B (acetonitrile containing 0.01% formic acid). The initial gradient containing 97% solvent A and 3% solvent B was maintained for 1 minute, and the fraction of solvent B was then raised to 15% in 1 minute, held for 2 minutes, restored to starting conditions in 1 minute and held for 1 minute. The flow rate was 0.4 mL/min.

| Statistical analyses
Data were expressed as mean ± SEM. Hypoxia-induced contractions were expressed as percentage of the reference contraction to U-46619 (taken as 100%) in coronary arteries. Student's unpaired t tests were used to compare two groups. In the comparison of mean values of more than two groups, one-way analysis of variance (ANOVA) test with Tukey's multiple comparisons test was used.
Two-way ANOVA test with Sidak's multiple comparisons test was used to compare two or more than two groups when the time course of treatment was investigated. P-values (two tailed) less than .05 were considered statistically significant. N represents the number replicated in corresponding experiment.

| Inhibition of PDE1 or PDE5 elevates cIMP levels in endothelium-denuded porcine coronary artery treated with exogenous cIMP
Although studies using purified PDEs and 3′,5′-cyclic nucleotides has shown that cIMP level is hydrolysed by several types of PDE, 22 Figure 3A,B,D) and lower than cIMP levels measured in endothelium-denuded coronary artery (supplemented with exogenous cIMP) ( Figure 3A,C). Taken together, these data suggest that cIMP levels in coronary artery are regulated by PDE1 or PDE5, whose inhibition in turn induces significant increases in intracellular cIMP levels.

| Inhibition of PDE1 or PDE5 augments cIMPmediated hypoxic vasoconstriction in endotheliumdenuded porcine coronary artery
We previously showed that the strength of hypoxic constriction in porcine coronary artery is determined by relative quantities of cIMP in the tissue. 9 Given that the inhibition of PDEs including PDE1 and

| Inhibition of PDE1 or PDE5 potentiates cIMP levels and associated vasoconstriction in intact porcine coronary arteries under hypoxic conditions
To explore the effect of PDE inhibition on cIMP levels and vessel tension under hypoxic condition in intact porcine coronary artery (with endothelium), these PDE inhibitors were applied prior to exposure to hypoxia. In these conditions, IBMX (2 × 10 −5 mol/L) induced a robust elevation of cIMP levels compared to pure hypoxia treatment ( Figure 5A,B). Consistent with this finding, the increase of cIMP content also occurred following 8-methoxymethyl-IBMX ( Figure 5C Therefore, we further explored whether inhibition of PDE1 and PDE5 would induce enhanced constricting responses in intact coronary arteries exposed to hypoxia. Unlike endothelium-denuded coronary arteries exposed to NLA, which completely abolished intramuscular

| Hypoxia-induced relaxation of porcine coronary artery is independent of cIMP levels
We found that hypoxia induced a substantial relaxation response after the acute vasoconstriction in porcine coronary artery. This hypoxia-induced relaxation was greater in coronary artery pretreated with nitro-l-arginine than in the control ( Figure S4A).
However, there was no significant difference of the relaxation responses in coronary arteries with or without endothelium and in endothelium-denuded coronary arteries treated with or without cIMP ( Figure S4B,C). This indicates that hypoxia-induced relaxation after vasoconstriction in porcine coronary artery is independent of cIMP levels. Consistently, we found that PDE inhibitors had little effect on hypoxia-induced relaxation in endothelium-denuded coronary arteries treated with cIMP ( Figure S5). Moreover, we found that IBMX and 8-methoxymethyl-IBMX, but not zaprinast, milrinone or rolipram, significantly abolished the relaxation response after hypoxic constriction in intact coronary artery ( Figure S6). Nevertheless, there was no difference in relaxations between arteries treated with NLA or NLA plus IBMX ( Figure S6A). Normally, the activation of sGC triggers increasing level of cGMP to regulate vessel tension. 13,14 It has been supposed that up-regulated cGMP levels are indispensable for hypoxic constriction. 7 This hypothesis was challenged by another study that found that hypoxic constriction was independent of cGMP. 10 Subsequently, we found that cIMP but not cGMP determined the hypoxic response. 9 In the present study, exogenous supply of cGMP analogs failed to restore hypoxic response inhibited by ODQ. Nevertheless, cIMP initiated robust hypoxic constriction in endothelium-denuded porcine coronary artery. UPLC/MS/MS analysis also revealed that cIMP levels were substantially augmented by hypoxia whereas there was no significant reduction in cGMP levels. These data suggest that cIMP, but not cGMP, is a key determinant of hypoxia-induced vasoconstriction in porcine coronary arteries.

| D ISCUSS I ON
As in the case of cGMP, levels of cIMP were shown to be regulated by several PDEs in studies using purified human PEDs and 3′,5′-cyclic nucleotides. 22,23 In the present study, we found that inhibition of PDE activity using a broad-spectrum inhibitor IBMX de- When pure cIMP was applied exogenously, cGMP levels were far lower than its physiological level and less than cIMP levels measured in endothelium-denuded coronary artery in the presence of NLA ( Figure 3). However, cGMP levels were comparable to those of cIMP in intact coronary arteries exposed to hypoxia (Figures 1   and 3). cGMP levels were also regulated by PDE1 and PDE5. [37][38][39] In real hypoxic conditions, we found that inhibition of PDE1 and PDE5 induced significant up-regulation of cIMP and cGMP levels in coronary arteries, as well as increased vessel tension. We also found that treatments with all PDE inhibitors induced significant reductions of vessel tension raised by U-46619 before exposure to hypoxia, suggesting that the vasodilating effects of cGMP (by the inhibitions of PDE1 and PDE5 [37][38][39] ) and cAMP (by the inhibitions of PDE3 and PDE4 37,40,41 ) are augmented ( Figure 6). In the present study, the amplitude of hypoxic constriction is independent of the degree of After hypoxia induced an acute constriction in porcine coronary artery, there was a substantial relaxation response that was also a vital factor determining the vessel tension of coronary artery. We found that there was no significant difference of the hypoxia-induced relaxation responses in coronary arteries with or without endothelium and in endothelium-denuded coronary arteries treated with or without cIMP. Moreover, PDE inhibitors had little effect on hypoxia-induced relaxations of endothelium-denuded coronary arteries treated with cIMP. Taken together, these data suggest that hypoxia-induced relaxation after vasoconstriction in porcine coronary artery is independent of cIMP levels.
In addition, we found that the hypoxia-induced relaxation was greater in arteries pre-treated with nitric oxide synthase inhibitor, nitro-l-arginine, compared to the control. We speculate that one reason that may influence this relaxation response is due to the amplitude and duration of hypoxia-induced constriction. The greater amplitude and longer duration of the constricting response remarkably delays the time to dilate coronary artery. Furthermore, the difference of hypoxia-induced relaxation between coronary artery treated with NLA and endothelium-denuded coronary artery indicates that there may be some endothelium-derived relaxing factors released by hypoxia. 42 We also found that IBMX and 8-methoxymethyl-IBMX significantly abolished relaxations after hypoxic contraction, which   [43][44][45][46][47] our data may also provide clinical references with the use of these inhibitors in patients with coronary artery disease exposed to repeated hypoxia (as in the case of sleep apnoea 4-6 ).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.