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Differential Pharmacologic Sensitivities of Phosphodiesterase-3 Inhibitors Among Human Isolated Gastroepiploic, Internal Mammary, and Radial Arteries

Onomoto, Masanori, MD*; Tsuneyoshi, Isao, MD*; Yonetani, Arata, MD*; Suehiro, Shoich, MD; Matsumoto, Kazuhisa, MD; Sakata, Ryuzo, MD; Kanmura, Yuichi, MD*

doi: 10.1213/01.ane.0000172114.30383.23
Cardiovascular Anesthesia: Research Report
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Systematic investigations of the actions of phosphodiesterase (PDE)-3 inhibitors on different human vascular tissues have not been performed. We investigated the effects of specific PDE-3 inhibitors (olprinone, milrinone, and amrinone) on contracted human gastroepiploic arteries (n = 70), internal mammary arteries (n = 72), and radial arteries (n = 70) harvested from a total of 134 patients, all of whom were undergoing coronary artery bypass surgery. Each of these PDE-3 inhibitors dose-dependently diminished the contractile responses to 10−6 mol/L norepinephrine and to either 10−9 or 10−8 mol/L of the thromboxane A2 analog U46619. In inducing vasorelaxations, these inhibitors were significantly more potent in norepinephrine-contracted rings than in those contracted with U46619. Further, at concentrations similar to the maximum therapeutic plasma concentrations (10−7 mol/L olprinone; 10−6 mol/L milrinone; 10−5 mol/L amrinone) olprinone and milrinone were more potent at inducing relaxations than amrinone in gastroepiploic arteries and radial arteries, whereas in internal mammary arteries milrinone was more potent than the others. These results suggest different activities for the three PDE-3 inhibitors among human arteries located in different regions and may be informative about the effectiveness of these inhibitors in preventing spasms in the various arterial grafts used in revascularization.

IMPLICATIONS: Because three phosphodiesterase-3 inhibitors (milrinone, olprinone, and amrinone) differed in their vasodilator potencies (against the contractile response to either norepinephrine or a thromboxane A2 analog) among human arteries removed from different parts of the body, their vascular relaxation profiles should be considered before they are used clinically.

*Department of Anesthesiology and Critical Care Medicine, †Second Department of Surgery, Kagoshima University School of Medicine, Kagoshima, Japan

Accepted for publication May 11, 2005.

Address correspondence and reprint requests to Masanori Onomoto, MD, Department of Anesthesiology and Critical Care Medicine, Kagoshima University School of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. Address electronic mail to tsune@m.kufm.kagoshima-u.ac.jp.

Cyclic nucleotide phosphodiesterases (PDEs) play major roles in the regulation of the intracellular concentrations of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) (1). At least 11 PDE isoforms have been identified on the basis of primary protein and cDNA sequence information (2). The members of the PDE-3 family, by virtue of their high affinities for cAMP, are the isozymes primarily responsible for cellular cAMP hydrolysis, with a resultant decrease in the intracellular level of cAMP. Four types of PDE-3 isoforms have been identified in cardiac and vascular myocytes (3). Since selective pharmacological inhibitors of PDE-3 (such as milrinone, amrinone, and olprinone) first became available, their therapeutic potential has been documented in animal studies and clinically (4–8).

Despite the widespread use of PDE-3 inhibitors in the treatment of cardiovascular disorders, only limited information is available concerning their mechanisms of action in human vascular smooth muscle. Some studies have revealed that PDE-3 inhibitors cause potent, endothelium-independent dilation of human arteries via inhibition of the PDE-3 activities present within the vascular smooth muscle (9–12). However, no published study has simultaneously investigated the efficacies of these PDE-3 inhibitors in blood vessels from different parts of the body. There are differences among different vascular tissues in terms of contractility and the vascular relaxations induced by drugs (13), indicating that they are not uniform in function. We therefore decided to investigate whether the profiles of these agents differed among different isolated arterial preparations. We used three human arteries (the human radial artery [RA], internal mammary artery [IMA], and gastroepiploic artery [GEA]) obtained from patients undergoing coronary artery bypass grafting (CABG). Accordingly, the results obtained in the present study may reflect the functional status of these vessels when PDE-3 inhibitors are administered in vivo.

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Methods

With approval from the human ethics committee of Kagoshima University hospital and informed consent from all patients who donated grafts, human RA, GEA, and IMA were harvested from 72, 70, and 72 patients, respectively. All were undergoing CABG surgery, and the perioperative drug therapy was as follows: 37 patients were receiving β-adrenoceptor blockers, 107 were taking nitrites, 98 were receiving calcium antagonists, 59 were taking potassium channel openers, and 41 were taking angiotensin-converting enzyme inhibitors or an angiotensin II receptor antagonist. In each patient, the optimal length of the heparin-treated RA, GEA, or IMA pedicle required for grafting onto one of the coronary arteries was dissected out. The discarded distal end of each arterial graft was immediately stored in oxygenated Krebs buffer maintained at 4°C. Within 2 h of resection, the distal portion of each artery was isolated from the sample in a dissecting chamber filled with Krebs solution. Fat and connective tissue were carefully removed under a binocular microscope, and 1–3 vascular rings (length, 2–2.5 mm; inner diameter, 500–700 μm) were prepared from each artery for tension recording. Rings cut from a single graft artery were not used in duplicate in the same experimental protocol.

Organ chamber experiments: The mechanical activity of each ring was measured using a strain gauge (UL-100GR; Minebea, Tokyo) in a tissue bath (volume, 1.0 mL) filled with Krebs solution continuously bubbled with 95% O2:5% CO2. The temperature of the solution was maintained at 37°C. The resting tension was set at 20 mN, a value shown by the length-tension relationship to allow a maximal active tension to be induced by norepinephrine (NE) (10−6 mol/L). During a 2-h equilibrium period, Krebs solution was continuously infused at a rate of 2 mL/min by a pump (Perista pump SJ-1211; ATTO, Tokyo) from one end of the bath and simultaneously aspirated from the other. During the experiment, the infusion rate was increased to 10 mL/min so that the bath solution was exchanged quickly for new solution. In an initial series of experiments, control contractile responses were induced by adding the α-adrenergic agonist NE (10−6 mol/L) to the Krebs solution for 7 min every 30 min. Preliminary experiments showed that the responses reached maximal levels within 30 s after application of NE and that a 23-min interval was sufficient for the tension to return to the control level (13,14).

When a stable level of contraction to NE (10−6 mol/L) was obtained, acetylcholine (ACh) was applied at a concentration of 10−6 mol/L. The presence of a functional endothelium was assessed by the presence of a relaxation response to ACh. In endothelium-intact rings if the relaxation induced by ACh proved to be smaller than 30% of the initial maximal contraction, the segment was discarded, as the endothelium was considered to be damaged. Furthermore, if our efforts to remove the endothelium failed (i.e., if an ACh-induced relaxation of more than 5% was observed after attempted denudation), we discarded that ring.

The control contractile response in each experiment was determined as the maximum amplitude of the response to either NE or the thromboxane A2 analog U46619 (normalized as a relative tension of 1.0 in each ring). In the present study, we tested the effects of PDE-3 inhibitors and forskolin on the contractions induced by NE; the concentration-response relationship for these agents was determined by adding them to a 10−6 mol/L NE-containing solution for 7 min every 30 min. Similar experiments were performed in separate groups of U46619-contracted rings. However, as demonstrated by other investigators (12,15), these three arterial segments exhibited larger contractions in the presence of U46619 than with NE. In addition, U46619 was more potent at inducing contraction in RA than in either GEA or IMA. Therefore, we attempted to obtain equal constrictions with NE and U46619 in the three types of arteries; to this end, RA was contracted with 10−9 mol/L U46619, whereas GEA and IMA were each contracted with 10−8 mol/L U46619 (Fig. 1).

Figure 1.

Figure 1.

To assess clinical significance, we performed additional experiments using concentrations of these PDE-3 inhibitors similar to the maximum serum concentrations achieved in clinical settings (10−7 mol/L olprinone, 10−6 mol/L milrinone, and 10−5 mol/L amrinone). To compare their effects under the same experimental conditions, these drugs were applied to the same rings with or without a functional endothelium (Fig. 2). To exclude any residual effects of these PDE-3 inhibitors, subsequent experiments were performed 30 min after a given PDE-3-inhibitor trial. If the control contraction to NE or U46619 before each PDE-3-inhibitor application was smaller than the initial control contraction, we stopped the experiment and the data were omitted.

Figure 2.

Figure 2.

The Krebs solution had the following composition (in mM): Na+ 137.4, HCO3 15.5, K+ 5.9, Ca2+ 2.6, H2PO4 1.2, Mg2+ 1.2, Cl 134.4, glucose 11.5. All solutions were bubbled with 95% O2:5% CO2 throughout the experiment, and the pH was adjusted to 7.3-7.4 (37°C).

Milrinone, amrinone, and olprinone were obtained from Yamanouchi Pharmaceutical (Tokyo, Japan), Meiji Seika (Tokyo, Japan), and Eisai (Tokyo, Japan), respectively. NE, forskolin, and ACh were all obtained from Sigma Chemical (St. Louis, MO).

Results are expressed as mean ± sd (n = number of patients from whom blood vessels were obtained). Statistical analysis was performed by a two-factor analysis of variance for repeated measures, followed by Scheffé’s test for multiple comparisons, including those among the different vessel-types (Figs. 3 and 4) or by a two-tailed, unpaired Student’s t-test (Figs. 1–4). Probability values <0.05 were considered significant.

Figure 3.

Figure 3.

Figure 4.

Figure 4.

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Results

All preparations were exposed to NE and ACh before the PDE-3-inhibitor experiments. In rings with intact endothelium, 10−6 mol/L ACh relaxed the 10−6 mol/L NE-induced contraction to 49.7% ± 15.9% (n = 177) of the control maximal contraction. In rings considered to be properly endothelium-denuded, an ACh-induced relaxation was not observed (96.7% ± 5.4%, n = 49).

Arterial segments were contracted with the specific agonists before the vasodilator responses to PDE-3 inhibitors and forskolin were examined. As shown in Figure 1, there was no significant difference in the contractile tension between these agonists in any vessel type. Moreover, there was no difference between the maximum contractile force obtained with endothelium and that obtained without endothelium for a given artery in response to a given drug. These results are in reasonably good agreement with those obtained in other investigations of the contractile functions of human arterial rings (12,15).

As demonstrated in Figure 3, milrinone, olprinone, and amrinone all relaxed the NE-induced contractions, but with various potencies, in the three vessel types with intact endothelium. Statistical analysis (two-factor factorial analysis of variance) showed that for NE-induced contractions: a) olprinone was significantly more potent in either GEA or RA than in IMA, b) milrinone was significantly more potent in IMA than in either GEA or RA, and c) amrinone induced only small relaxations in the three vessels (at the concentrations tested). In addition, the vascular relaxations to these PDE-3 inhibitors were significantly larger in rings contracted with NE than in rings contracted with U46619 (Fig. 3). As shown in Figure 2, olprinone and milrinone at clinically relevant concentrations were significantly more potent as vasodilators than amrinone in RA contracted with NE, although there was no such significant difference in rings contracted with U46619. The vascular relaxation profiles of these two drugs were qualitatively similar in GEA, except that in GEA olprinone and milrinone were significantly more potent than amrinone at inducing relaxations in both NE-contracted rings and in U46619-contracted rings. However, in IMA milrinone was more potent as a vasodilator than either olprinone or amrinone (both in NE-contracted and U46619-contracted rings). Results similar to those described above were also obtained in arteries without a functional endothelium (Fig. 2). In contrast, the vasodilator responses to the cAMP-increasing agent forskolin were similar among all arterial types and, moreover, were independent of the specific agonist used to induce contraction (Fig. 4).

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Discussion

We found that the three PDE-3 inhibitors tested (milrinone, olprinone, and amrinone) differed in their potencies as vasodilators (against the contractile response to either NE or a thromboxane A2 analog) among human arteries removed from different parts of the body. Moreover, these three drugs differed among themselves in their potencies against the contractile response to a given spasmogen. The significance of the present study is that it not only systematically investigated the actions of PDE-3 inhibitors on human vascular tissues sourced from different regions but it also concomitantly provided useful information as to the possible effectiveness of these inhibitors in preventing spasms of several of the arterial grafts used in human CABG surgery.

In the present study, milrinone, olprinone, and amrinone all induced concentration-dependent relaxations in NE-constricted human arterial rings (Fig. 3). However, the responses to these PDE-3 inhibitors differed considerably among rings from the three arteries when they were constricted by NE. These results were supported by the observation that the responses to the PDE-3 inhibitors also differed among the three arteries when they were constricted by U46619, although the relaxations were less potent than those seen in NE-contracted rings.

Histologic studies have revealed major differences among the various types of human arteries in the structure of the smooth muscle, such as the elastic lamellae (16). However, we found that forskolin, an adenylate cyclase stimulator producing an increase in the intracellular level of cAMP, was equipotent against both NE-induced and U46619-induced contractions regardless of the source of the artery. Although these findings may simply relate to the potency and ability of forskolin to maximally activate cAMP in vascular smooth muscle, irrespective of the potency of the contraction, these results could be interpreted as suggesting that the mechanisms underlying the present differential vasodilator actions of PDE-3 inhibitors depend on the existence in the myoplasm of different signals upstream of cAMP-mediated signaling.

Early investigators suggested that at least 4 of the 11 recognized PDE isozyme families exist within human vascular smooth muscle: namely, the Ca2+-calmodulin-dependent family (PDE1), cAMP-specific PDE (PDE-3), the cAMP-specific family selectively inhibited by rolipram (PDE4), and the cGMP-specific family selectively inhibited by zaprinast (PDE5) (17). In vascular myocytes at least three isoforms of PDE-3 (PDE-3A-118, PDE-3A-94, and PDE-3B-137) have been identified by alternative transcriptional, translational or post-translational processing (3). Several investigators have reported that PDE-3A-118 has sites for phosphorylation and activation by protein kinase A, but not by protein kinase B. PDE-3A-94 appears to lack sites for phosphorylation and activation by either protein kinase A or protein kinase B, whereas PDE-3A-137 has sites for both (3). It has also been reported that these PDE-3 isozymes differ in their cellular and intracellular distributions in vascular myocytes (3).

PDE-3 inhibitors, including milrinone and amrinone, produce vasodilation via inhibition of PDE-3 isozymes and an accumulation of cAMP with an associated decrease in the intracellular calcium level in vascular smooth muscle. A newly developed PDE-3 inhibitor, olprinone, 1,2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a] pyridine-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate, has been used in patients with acute heart failure, and is also reported to selectively inhibit PDE-3 enzymes (11). Therefore, the observed differences in vascular relaxation potencies among the PDE-3 inhibitors could result primarily from differences in affinities for the PDE-3 isoforms found within vascular myocytes. However, previous studies have demonstrated some overlaps in the potencies and selectivities of PDE-3 inhibitors among the PDE isozymes isolated from vascular smooth muscle (5,6,18). For example, in guinea pig and canine aortas, milrinone exerts an inhibitory effect on PDE1 that, although weak, is still 5 to 9 times more potent than that of amrinone (6,18). As PDE1 preferentially hydrolyzes cGMP, the additive inhibition of cGMP hydrolysis by milrinone may, in part, account for its vascular effects in the arteries studied in our experiments.

The maximum therapeutic plasma olprinone concentration obtained from a pharmacokinetic study of acute heart failure was almost 20 ng/mL (≈ 1 × 10−7 mol/L) (19), suggesting that at that concentration, this compound would also bring about vasodilation of the arteries tested. Similarly, the concentrations of milrinone that produced significant relaxations of the arteries used in our study were comparable to the clinically effective plasma concentrations of this drug (100-400 ng/mL, approximately equal to 0.5 − 2 × 10−6 mol/L) (20). Meanwhile, the rate of amrinone infusion required to increase cardiac output is typically between 5 and 10 μg·kg−1·min−1, giving plasma concentrations of 1 − 7 × 10−6 mol/L.(21) When we compared the vascular relaxations induced by these PDE-3 inhibitors at concentrations similar to these maximum therapeutic plasma concentrations (10−7 mol/L olprinone, 10−6 mol/L milrinone and 10−5 mol/L amrinone), milrinone and olprinone produced similar relaxing effects (each of which was more potent than that seen with amrinone) in both RA and GEA, whereas in IMA milrinone was more potent than either olprinone or amrinone. Our finding that the direct effects of amrinone on human arterial smooth muscle are relatively weak is consistent with previous observations made using human IMA.(10) Based on the present results, olprinone and milrinone, but not amrinone, would be predicted to be potent vasodilators in the arterial grafts used in revascularization. In addition, olprinone and milrinone might be useful for the treatment of cardiac failure patients exhibiting high vascular resistance, as GEA and RA are postulated to play major roles in the maintenance of peripheral vascular resistance. Furthermore, amrinone might be hemodynamically more beneficial for cardiac insufficiency in patients with a low vascular resistance, such as septic patients.

As shown in Figure 2, removal of the endothelium had little or no influence on the relaxations induced in human RA, GEA, and IMA by the three PDE-3 inhibitors. Thus, these PDE-3 inhibitors are unlikely to have effects on the endothelium in the arteries examined. Indeed, Lugnier and Schini (22) reported finding no PDE-3 isozymes within endothelial cells, making it unlikely that the endothelium is important for the relaxant actions of PDE-3 inhibitors. Therefore, these drugs, with their endothelium-independent vasodilator effects, might be useful in the treatment of any excess vasoconstriction resulting from an endothelial dysfunction caused by atherosclerosis or a surgical procedure (11).

We chose NE and U46619 as the standard vasoconstrictors to induce arterial spasms in vitro because NE is the major sympathomimetic amine at vascular nerve endings and because high levels of thromboxane A2 are produced during CABG surgery (23,24). In addition, an important reason for studying U46619 is the pathologic role played by platelets and thromboxane generation in injury and potential spasm (24). Recently, Jhaveri et al. (25) reported differences among arterial grafts in the effects of PDE-3 inhibitors against the responses to different spasmogens (e.g., NE, phenylephrine, epinephrine, endothelin, U46619). In their experiments on small porcine pulmonary arteries, the vasorelaxation induced by milrinone was significantly greater in rings contracted with NE or epinephrine than in those contracted with phenylephrine or U46619. Likewise, in our experiments the vasorelaxations induced by the three PDE-3 inhibitors were larger in NE-constricted rings than in U46619-constricted rings. Overall, our findings are consistent with those of Jhaveri et al. and collectively these results suggest that PDE-3 inhibitors may act synergistically with β-adrenoceptor agonists to cause vasodilation, the interaction leading to increased cAMP production and degradation. However, further pharmacologic and biochemical experiments will be needed to clarify the situation.

In conclusion, our results indicate a central role for PDE-3 inhibition and cAMP modulation in human arteries from different regions (RA, GEA, and IMA). RA, GEA, and IMA have all been widely used to provide arterial grafts for CABG surgery. The results of the present study provide valuable additional information about the pharmacological actions of PDE-3 inhibitors in human vascular smooth muscle, and suggest their potential as therapeutic options against perioperative spasm in arterial coronary bypass grafts.

We thank Dr. R. J. Timms for his help in preparing the manuscript.

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