Alfentanil is one-eighth as potent as fentanyl in terms of the opioid dosage required to reduce the minimum alveolar concentration of isoflurane by 50%, and is a short-acting fentanyl congener on account of its short terminal elimination half-life (70–112 min) and rapid onset of action . Because of these pharmacokinetic profiles, alfentanil has been used to induce anaesthesia as well as to maintain brief anaesthesia [2,3]. Although alfentanil (20 μg kg−1 min−1) produces relative minor changes in the haemodynamic variable , high doses (120, 175 μg kg−1) of alfentanil cause a significant decrease in the mean blood pressure (BP) [4,5]. The direct effect of anaesthetics on the blood vessels is very difficult to determine using in vivo studies because of the concomitant changes in myocardial contractility, preload and central sympathetic activity induced by anaesthetics .
Alfentanil, when given as a loading dose (45 μg kg−1) followed by a continuous infusion (3 μg kg−1 min−1), decreases the BP in dogs but does not alter blood flow to the heart, brain, muscle and skin . A very high dose (500 μg kg−1) of alfentanil was reported to produce vasodilatation in an almost anaesthetic-free isolated canine hindlimb . Alfentanil relaxes the rat aorta by direct action on the vascular smooth muscle independent of the endothelium . However, there are no reports of an associated cellular mechanism that is responsible for the vasodilatation induced by alfentanil in isolated blood vessels. Therefore, this study investigated the effect of alfentanil on phenylephrine-induced contractions in an isolated rat aorta in vitro and examined the associated cellular mechanism in the vascular smooth muscle.
The Institutional Animal Care and Use Committee of Gyeongsang National University Hospital approved all the experimental procedures and protocols. Male Sprague–Dawley rats weighing 250–350 g each were anaesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg kg−1). The descending thoracic aorta was dissected free, and the surrounding connective tissue and fat were removed under a microscope while the blood vessel was bathed in Krebs solution of the following composition: 118 mmol NaCl, 4.7 mmol KCl, 1.2 mmol MgSO4, 1.2 mmol KH2PO4, 2.4 mmol CaCl2, 25 mmol NaHCO3, 11 mmol glucose and 0.03 mmol EDTA. The aorta was then cut into 2.5 mm rings, and these rings were suspended on Grass isometric transducers (FT-03, Grass Instrument, Quincy, MA, USA) at 2.0 g resting tension in 10 mL temperature-controlled baths (37°C) containing Krebs solution that was continuously gassed with 95% O2 and 5% CO2. The rings were equilibrated at a 2.0 g resting tension for 120 min, during which time the bathing solution was changed every 15 min. Only one concentration–response curve elicited by the contractile agonist (phenylephrine), potassium chloride (KCl) and calcium (Ca2+) was made for each ring in all experiments. In all aortic rings, the endothelium was removed intentionally by inserting a 25-G needle tip into the lumen of the ring and gently rolling the ring for a few seconds. The contractile response induced by isotonic 60 mmol KCl was measured in all the aortic rings.
After complete washout of isotonic 60 mmol KCl from the organ bath and the return of isometric tension to baseline values, a cumulative concentration–response curve to contractile agents (phenylephrine, KCl and calcium chloride) was performed in each ring. The aim of the first series of in vitro experiments was to determine the effect of alfentanil on the contractile responses induced by the α-1 adrenoceptor agonist phenylephrine in endothelium-denuded rings. Alfentanil was added directly to the organ bath 20 min before a cumulative phenylephrine-induced contraction. The effect of alfentanil on the concentration–response curve for phenylephrine (10−9–10−5 mol) in the endothelium-denuded rings was assessed by comparing the contractile response in the presence or absence of alfentanil (10−6, 5 × 10−5 and 10−4 mol).
In the second series of experiments to investigate the participation of opioid receptors in the alfentanil-induced attenuation of the contractile response induced by phenylephrine, the phenylephrine (10−9–10−5 mol) concentration–response curve was assessed 20 min after the non-specific opioid receptor antagonist naloxone, 10−6 mol, was added to the bath, either alone or after combined pretreatment with alfentanil (5 × 10−5 mol).
A third series of experiments was carried out to determine the effect of alfentanil on the contractile response induced by KCl. The effect of alfentanil was assessed by comparing the KCl dose (10–60 mmol)–response curves obtained in the presence or absence of alfentanil (10−6, 5 × 10−5 and 10−4 mol). Alfentanil was added directly to the organ bath 20 min before the KCl-induced contraction.
The aim of the fourth series of experiments was to determine the involvement of the l-type calcium channels in the alfentanil-induced attenuation of the contractile response induced by phenylephrine. For the endothelium-denuded rings pretreated with the l-type calcium-channel blocker verapamil (10−5 mol), the effect of alfentanil (5 × 10−5 mol) on the concentration–response curve for phenylephrine was assessed by comparing the contractile response in the presence or absence of alfentanil (5 × 10−5 mol). The incubation period for verapamil (10−5 mol) and alfentanil (5 × 10−5 mol) or verapamil (10−5 mol) alone was 20 min before the phenylephrine-induced contraction.
The fifth series of experiments examined the role of a decrease in the calcium influx from the extracellular to the intracellular space during the alfentanil-induced attenuation of the phenylephrine-induced contractile response. The denuded aortic rings were exposed to a calcium-free Krebs solution containing 2 mmol of ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) for 10 min. The solution was then replaced with a calcium-free isotonic depolarizing solution containing a high KCl concentration (100 mmol KCl). Fifteen minutes before the calcium (Ca2+)-induced contraction, alfentanil was added directly to the calcium-free isotonic depolarizing solution containing a high KCl concentration (100 mmol). Finally, calcium was added cumulatively to achieve a final bath concentration ranging from 0.5 to 2.5 mmol . The effect of alfentanil on the concentration–response curve for calcium was examined by comparing the contractile response induced by the addition of calcium in the presence or absence of alfentanil (5 × 10−5 and 10−4 mol).
The final series of experiments investigated the effect of verapamil (10−5 mol) on the contractile responses in endothelium-denuded rings induced by phenylephrine. Verapamil was added directly to the organ bath 20 min before the cumulative phenylephrine-induced contraction. The effect of verapamil on the concentration–response curve for phenylephrine (10−9–10−5 mol) was assessed by comparing the contractile response in the presence or absence of verapamil (10−5 mol).
Drugs and solutions
The following drugs used in this study were of the highest purity commercially available: phenylephrine HCl, verapamil hydrochloride, EGTA (Sigma Chemical, St. Louis, MO, USA), alfentanil hydrochloride (Keukdong Pharm Co. Ltd., Seoul, Republic of Korea). Alfentanil was dissolved and diluted in distilled water, and tested at several concentrations (10−6, 5 × 10−5 and 10−4 mol). All drug concentrations are expressed as the final molar concentration in the organ bath. All other drugs were dissolved and diluted in distilled water.
The values are expressed as the mean ± standard deviation (SD). The contractile responses to phenylephrine, KCl and calcium chloride are expressed as the percentage of the maximum contraction to the isotonic 60 mmol KCl. The logarithm of the drug concentration (ED50) eliciting 50% of the maximal contractile response was calculated using non-linear regression analysis by fitting the concentration–response relation for phenylephrine to a sigmoidal curve using commercially available software (Prism version 3.0; Graph Pad Software, San Diego, CA, USA). The maximal contractile response induced by the contractile agents was measured as a percentage of the maximum contraction to isotonic 60 mmol KCl. Statistical analysis was performed using a t-test for paired samples or one-way analysis of variance followed by Tukey’s multiple comparison test. P < 0.05 was considered significant. N refers to the number of rats whose descending thoracic aortic rings were used in each protocol.
In the endothelium-denuded rings, the low dose of alfentanil (10−6 mol) had no effect on the phenylephrine-induced contraction, whereas the high dose (5 × 10−5, 10−4 mol) significantly increased the ED50 (P < 0.05) of phenylephrine compared with the rings untreated with alfentanil (Fig. 1). In the endothelium-denuded rings pretreated with naloxone (10−6 mol), 5 × 10−5 mol alfentanil significantly increased the ED50 (P < 0.05) of phenylephrine compared with the rings untreated with alfentanil (Fig. 2).
The low dose of alfentanil (10−6 mol) had no significant effect on the KCl-induced contraction compared with the rings untreated with alfentanil, whereas the high dose (5 × 10−5, 10−4 mol) significantly attenuated (P < 0.01) the KCl-induced contraction in a dose-dependent manner (Fig. 3).
Alfentanil had no effect on the phenylephrine-induced contraction in endothelium-denuded rings pretreated with 10−5 mol verapamil compared with the rings untreated with alfentanil (Fig. 4).
In the calcium-free isotonic depolarizing solution containing 100 mmol KCl, the high dose of alfentanil (5 × 10−5, 10−4 mol) significantly attenuated (P < 0.001) the contraction induced by the cumulative addition of calcium (0.5–2.5 mmol) in a dose-dependent manner (Fig. 5).
Verapamil (10−5 mol) significantly increased the ED50 (P < 0.001) of phenylephrine and decreased phenylephrine-induced maximal contraction (P = 0.004) compared with the verapamil-untreated rings (Fig. 6).
This study is the first to demonstrate that a supraclinical dose of alfentanil (5 × 10−5 mol) attenuates the phenylephrine-induced contraction via an inhibitory effect on calcium influx by blocking the l-type calcium channel in the rat aortic vascular smooth muscle. This attenuation was found to be independent of opioid receptor activation.
Two types of stimulants are widely used in the vascular smooth muscle to increase the cytosolic Ca2+ level ([Ca2+]i): high-K+-induced membrane depolarization and the activation of the receptor by contractile agonists such as phenylephrine and 5-hydroxytryptamine . High K+ induces membrane depolarization, which in turn opens the voltage-dependent Ca2+ channels . High-K+-induced contraction is totally abolished by agents blocking the Ca2+ channels including verapamil and nifedipine . In contrast, contractile agonists including phenylephrine and 5-hydroxytryptamine cause the release of Ca2+ from the sarcoplasmic reticulum, which induces initial transient contractions, and subsequently open the receptor-operated Ca2+ channels to elicit a sustained contraction . The contractile agonist-induced contraction is less sensitive to the Ca2+-channel blocker than the high-K+-induced contraction . A previous study suggested that the norepinephrine-induced increase in [Ca2+]i is due to Ca2+ influx through both the l-type and non-l-type calcium channels . Norepinephrine induces a contraction of the rat aortic ring by activating the release of intracellular calcium and the subsequent calcium influx through the voltage-dependent calcium channels and the receptor-operated calcium channels . In this in vitro study, verapamil (10−5 mol) attenuated the phenylephrine-induced contraction, which is in agreement with previous reports [12–14], Overall, the verapamil-induced attenuation of the contractile response stimulated by phenylephrine suggests that the calcium influx for the phenylephrine-induced contraction in rat aorta occurs through the l-type calcium channels and the receptor-operated calcium channels insensitive to verapamil. In accordance with a previous report , alfentanil attenuated the phenylephrine-induced contractile response in the endothelium-denuded rings with or without naloxone. Pretreatment with naloxone partially inhibits the relaxant response to morphine in the small mesenteric artery of the rat . However, the fact that alfentanil attenuated the phenylephrine-induced contraction in the rings pretreated with 10−6 mol naloxone suggests that the alfentanil-induced attenuation is caused by direct action on the pathway for phenylephrine-induced contraction.
In agreement with a previous report , alfentanil (5 × 10−5, 10−4 mol) attenuated the KCl-induced contraction mediated through the activation of voltage-dependent calcium channels in a dose-dependent manner. Alfentanil is more potent in relaxing the rat aorta precontracted with KCl than phenylephrine . The magnitude of alfentanil (5 × 10−5, 10−4 mol)-induced attenuation was greater in the KCl dose–response curve than in the phenylephrine dose–response curve (Figs 1 and 3), which is in agreement with a previous study . This suggests that the l-type calcium channels are more sensitive to alfentanil than the receptor-operated calcium channels. The verapamil (10−5 mol) pretreatment completely abolished the alfentanil (5 × 10−5 mol)-induced attenuation of the contractile response induced by phenylephrine (Fig. 4). Therefore, alfentanil would act as a calcium-channel blocker on the vascular smooth muscle. Norepinephrine and high K+ open the same verapamil-sensitive calcium channels . Reinforced with the results from a previous in vitro protocol, alfentanil (5 × 10−5, 10−4 mol) produced a parallel rightward shift in the CaCl2 dose–response curves in a dose-dependent manner (Fig. 5). Taken together, these results indicate that alfentanil (5 × 10−5 mol) attenuates the phenylephrine-induced contractile response via an inhibitory effect on the influx of calcium from the extracellular to intracellular space through the l-type calcium channels in the vascular smooth muscle. However, further research will be needed to elucidate the effect of alfentanil on the G-protein, phospholipase C, inositol triphosphate and diacylglycerol, which are involved in the cellular signal transduction pathway for the contractile agonist-induced contraction.
In previous in vivo studies [7,8,16], high doses (500, 320 μg kg−1) of alfentanil were reported to decrease the BP with a concomitant decrease in vascular resistance. A high dose of alfentanil (plasma concentration 1.86 × 10−6 mol or 500 μg kg−1) was reported to decrease the BP and vascular resistance in an isolated canine hindlimb . This suggests that alfentanil produces vasodilatation by acting directly on the vascular smooth muscle . However, 10−6 mol alfentanil, which corresponds to the concentration (approximately 1000 ng kg−1) encountered in a clinical setting , had no effect on either the phenylephrine-induced contraction or the KCl-induced contraction. This difference can be attributed to the experimental method (in vitro vs. in vivo), different species (rat vs. dog) and different vascular beds (aorta vs. femoral artery). A supraclinical dose of alfentanil (5 × 10−5 mol) significantly attenuated both the phenylephrine-induced and KCl-induced contractions. Therefore, these results suggest that the hypotension induced by alfentanil in the previous in vivo experiments [7,8,16] is not mediated by the direct vasodilatating action of alfentanil on the vascular smooth muscle. In addition, alfentanil has no significant effect on the cardiac contractility in an isolated perfused rat heart . Fentanyl produces hypotension by inhibiting the central sympathetic outflow in intact dogs anaesthetized with enflurane [19,20]. An alfentanil dose exceeding 30 μg kg−1 completely attenuates the cardiovascular and catecholamine response to tracheal intubation . Overall, the indirect vasodilatating mechanism, which is mediated via the decreased systemic vascular resistance produced by an inhibited central sympathetic outflow induced by fentanyl congeners, might be involved in the hypotension observed in the previous in vivo experiments [4,7,8,16]. It is believed that the direct vasodilatating action of a supraclinical dose in the vascular smooth muscle might partially contribute to the severe hypotension observed in clinical settings, e.g. an alfentanil overdose. Further studies will be needed to determine the effects of alfentanil on the central sympathetic outflow in vivo. It should be noted that the clinical implications of alfentanil on regional haemodynamics should be tempered by the fact that the aorta was used in this in vitro experiment. The organ blood flow is controlled by resistance vessels containing arterioles with a diameter of 100–300 μm . Even with these limitations, these findings are expected to contribute to an understanding of the interaction between alfentanil and the calcium influx pathway associated with the phenylephrine-induced contraction on the vascular smooth muscle.
In conclusion, a supraclinical dose of alfentanil (5 × 10−5 mol) attenuates the phenylephrine-induced contraction through an inhibitory effect on the influx of calcium from the extracellular to intracellular space by blocking the l-type calcium channels in the rat aortic vascular smooth muscle. This attenuation does not occur through activation of the opioid receptor.
The study was supported by a research grant (2005) from Gyeongsang National University Hospital.
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