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).
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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