Adrenomedullin (AM) is implicated in the regulation of circulatory homeostasis and the pathophysiology of various diseases. For example, the plasma AM levels are increased in septic shock , hepatic cirrhosis  and during cardiopulmonary bypass , correlating with relaxation of vascular tone in these conditions. Plasma AM levels are also elevated in congestive heart failure , myocardial infarction  and chronic renal failure , seeming to function as a counter-regulator in these conditions. Since these patients frequently need to undergo surgery under general anaesthesia, it is important to elucidate the effects of general anaesthetics on the AM-induced haemodynamic responses for better haemodynamic management of complicated cases.
AM is a potent vasodilatory peptide that was first isolated from human pheochromocytoma cells . Human adrenomedullin (hAM) is a 52-amino acid peptide with a disulphide bond forming a ring structure, which has some homology with calcitonin gene-related peptide (CGRP) . Rat AM has 50 amino acids, with two deletions and six substitutions, compared with hAM , and is more potent than hAM in decreasing systemic arterial pressure . The initial mechanism of action of AM and CGRP is, in most cases, via guanine nucleotide guanosine 5′-triphosphate-binding protein (G-protein)-coupled receptor activation of stimulatory G-protein (Gs) and adenylate cyclase, which produces cyclic adenosine monophosphate (cAMP) . CGRP receptors might, at least in part, mediate the vasodilator response to AM in some vascular beds, whereas the hypotensive effect of AM on mean arterial pressure (MAP) in anaesthetized rats is not mediated via CGRP receptors . Intravenous (i.v.) infusion of AM results in potent and sustained hypotension, which is mediated mainly by nitric oxide generation . The precise mechanisms via which AM induces vascular relaxation are, however, not fully understood.
We previously reported that volatile anaesthetics inhibit the CGRP depressor effect, but not through inhibition of CGRP release in the pithed rat model . Furthermore, sevoflurane and isoflurane inhibit CGRP-induced vasodilation at the site between the CGRP receptor and adenylate cyclase activation. The inhibitory site of volatile anaesthetics on the CGRP receptor-mediated response involves Gs protein . There is an overall similarity in the mechanisms of AM-induced and CGRP-induced haemodynamic responses. Therefore, we hypothesized that volatile anaesthetics might also affect AM-induced haemodynamic responses. These results might support the notion that one of the general mechanisms of action of volatile anaesthetics is the inhibition of G-protein-coupled receptor-mediated responses.
The purpose of the present study was to investigate the effects of volatile anaesthetics on AM-induced haemodynamic responses by evaluating the changes in haemodynamic parameters, including MAP, cardiac output (CO), stroke volume (SV) and systemic vascular resistance (SVR) in pithed rats. In pithed animals, there are no centrally mediated compensatory reflexes or intrinsic sympathetic nerve activity . Therefore, we used a pithed rat model to evaluate the direct cardiovascular effect of drugs without interference from centrally mediated circulatory reflexes.
All surgical procedures and experimental protocols were approved by the Gunma University Institutional Animal Care and Use Committee. Surgical procedures were performed as described previously [13,14,16,17]. Male Wistar rats weighing 300–350 g were anaesthetized with isoflurane in oxygen. The animals were artificially ventilated using a Harvard respirator (Harvard Apparatus, South Natick, MA, USA) via a tracheal cannula at a rate of 50–60 breaths min−1 with an SV of 1 mL 100 g−1 body weight. Respiratory rate was adjusted to maintain a PaCO2 of 35–40 mmHg. A polyethylene (PE 50) catheter was placed in the left carotid artery to measure MAP. Both vagus nerves were severed at the neck. The rats were pithed by inserting a stainless-steel rod (1.5 mm in diameter) through the right orbit and foramen magnum down the spinal canal to its sacral terminus. This pithing procedure destroys the entire central nervous system, enabling evaluation of the direct cardiovascular effect of drugs without interference from centrally mediated circulatory reflexes. Isoflurane anaesthesia was discontinued immediately after pithing.
Following a median sternotomy, a flow probe (Transonic Systems Inc., Ithaca, NY, USA) was placed around the ascending aorta to measure aortic blood flow, i.e. CO. The right jugular vein was cannulated with a PE-50 catheter for the administration of norepinephrine. A 24-G Teflon cannula (Angiocath; Desert Medical, Sandy, UT, USA) was placed in the tail vein for the administration of AM. Body temperature was maintained between 36°C and 37°C using a heated blanket placed beneath the animal and controlled by a rectal thermistor probe (CMA/150; Stockholm, Sweden).
MAP was recorded with a Gould (Cleveland, OH, USA) pressure transducer connected to the left carotid artery via a PE-50 catheter. Aortic blood flow was measured using a volume flow meter (Transonic Systems Inc., Ithaca, NY, USA). CO, SV and SVR were normalized by the body weight using the following formulae: CO = mL min−1 kg−1 body weight; SV (mL beat−1 kg−1 body weight) = CO/heart rate (HR); and SVR (mmHg mL−1 min−1 kg−1 body weight) = MAP/CO. SVR was determined when MAP and CO reached their minimal values.
Pithed rats were allowed to stabilize for 30 min before any experimental intervention. MAP and CO were maintained at approximately 100 mmHg and 50 mL min−1, respectively, by continuous infusion of norepinephrine (2–3 μg kg−1 min−1) throughout the protocol. Rats were randomly assigned to anaesthetic groups (n = 7, each) or the no-anaesthetic group (n = 7) and there was a 1-h waiting period after the termination of isoflurane administration to avoid the effects of isoflurane. In the anaesthetic groups, 1 h after pithing, a volatile anaesthetic (1% isoflurane or 2% isoflurane) in oxygen was administered for 30 min. Concentrations of inspired anaesthetics were monitored continuously with a calibrated gas monitor (Capnomac Ultima; Datex, Helsinki, Finland). In the no-anaesthetic group, no volatile anaesthetic was administered. Rat AM (1, 3, 10 and 30 μg kg−1; Peptide Institute, Inc., Osaka, Japan) was administered i.v. for 30 s in 0.1 mL of saline.
Statistical comparisons within groups were assessed using analysis of variance. When differences were significant, multiple intergroup comparisons were performed with Scheffe's test. All values are expressed as mean ± SD. A P value of less than 0.05 was considered statistically significant.
Baseline values of haemodynamic parameters and the infusion rates of norepinephrine before the administration of AM are shown in Table 1. There were no significant differences in these data among groups.
I.v. administered AM induced a biphasic response in MAP, a transient increase followed by a persistent decrease. MAP and CO changed similarly. MAP and CO maximally increased immediately after injection of AM (early phase) and reached the minimal value approximately 1 min after the injection of AM (late phase). SVR decreased in each phase after the administration of AM. HR did not change significantly during the response. Changes in SV were similar to those in CO. Injection of saline had no effect.
The effects of isoflurane on AM-induced changes in haemodynamic parameters in the early phase are shown in Figure 1. MAP, CO and SV increased immediately after the administration of AM. SVR slightly decreased. Isoflurane inhibited these AM-induced increases in MAP in a concentration-dependent manner (at 2%, P < 0.01, n = 7), whereas increases in CO and SV and decreases in SVR were not significantly inhibited by isoflurane, there being no significant difference between groups (Fig. 1).
The effects of isoflurane on AM-induced changes of the haemodynamic parameters in the late phase are shown in Figure 2. AM induced a dose-dependent decrease in MAP, CO, SV and SVR. Isoflurane inhibited the AM-induced decrease in MAP (at 2%, P < 0.05, n = 7) and SVR (at 1%, P < 0.05, n = 7 and at 2%, P < 0.01, n = 7) in a concentration-dependent manner, while the decrease in CO and SV were not significantly inhibited by isoflurane and did not differ significantly among groups (Fig. 2).
We found that isoflurane, at clinically relevant concentrations, inhibited the AM-induced initial increase in MAP and the later decrease in MAP and SVR in a dose-dependent manner in pithed rats. Our data suggest that isoflurane inhibits AM-induced vasodilatory and positive inotropic effects in pithed rats. Therefore, isoflurane might inhibit the AM receptor-mediated response, which is a common pathway for these actions. Furthermore, these results might support the notion that one of the general mechanisms of action of volatile anaesthetics is the inhibition of G-protein-coupled receptor-mediated responses.
In general, there are four main mechanisms of the vasodepressor response elicited by i.v. drug administration: (i) central nervous system actions; (ii) inhibition of vascular sympathetic transmission; (iii) release of endothelium-derived relaxing factors; and (iv) direct vasorelaxing effects on vascular smooth muscle . Both central and autonomic nervous system activities were excluded by using pithed, vagotomized rats in this study. Therefore, AM-induced vasodilatory effects in the present study were mediated by actions on the vascular endothelium (releasing relaxing factors) and/or the vascular smooth muscle cells (producing direct relaxation) (Fig. 3).
Actually, AM is known to elicit vascular relaxation via both endothelium-dependent and endothelium-independent signal transduction pathways . These pathways are mediated by the cell surface Gs protein-coupled receptor, which is predominantly coupled to the activation of adenylate cyclase, leading to the production of cAMP . Furthermore, AM exerts positive inotropic effects. These biologic responses are mediated by the same Gs protein-coupled receptor .
In the current study, we investigated not only the effect of isoflurane on AM-induced changes in blood pressure but also AM-induced vasodilation by measuring SVR, which reflects the systemic vascular tone. Therefore, the present data suggest that isoflurane inhibited the AM-induced vasodilatory effects.
Norepinephrine administered to all the rats might have affected the inhibitory action of isoflurane on AM-induced vasodilatory effects, because the infusion rates of norepinephrine tended to increase with the increasing doses of isoflurane. In general, isoflurane has a potent, dose-dependent vasodilatory effect. Therefore, increasing doses of norepinephrine are required with the increasing doses of isoflurane to maintain vascular tone at the same level as before AM administration. Actually, there is no significant difference in the infusion rates of norepinephrine among the groups. Although AM dose-dependently exerts vasodilatory effects, isoflurane significantly inhibits the decrease in MAP and SVR induced by higher doses of AM, at similar doses of norepinephrine. Therefore, we believe that the increasing dose of norepinephrine did not affect the inhibitory actions of isoflurane.
In addition to the inhibitory action of isoflurane on AM-induced vasodilatory effects, 2% isoflurane inhibited the increase in MAP after AM administration, suggesting that high concentrations of volatile anaesthetics inhibit both vasodilatory and positive inotropic effects induced by AM. These data strongly suggest that volatile anaesthetics inhibit the AM-induced response through an effect on AM receptor-mediated responses.
Volatile anaesthetics are likely to affect several hydrophobic sites within the cell membrane, particularly membrane-binding proteins . Possible sites of action of anaesthetics in the G-protein-coupled receptor-mediated signal transduction pathway are divided into four target groups: (a) agonist-receptor binding, (b) G-protein function, (c) effector activity (adenylate cyclase) and (d) other intracellular sites (e.g. Ca2+ stores, cellular kinases) .
In the previous study, we reported that sevoflurane and isoflurane inhibited CGRP-receptor-mediated signal transduction at the site between the CGRP receptor and adenylate cyclase activation, by interfering with the activation of Gs protein . In addition, volatile anaesthetics inhibit other types of G-protein-coupled receptor-mediated signal transduction [23,24]. Though the sites of the inhibitory actions of volatile anaesthetics are not completely understood, these reports suggest that the inhibitory sites of anaesthetics are at the G-protein level. Furthermore, there is an overall similarity in the mechanisms of AM-induced and CGRP-induced haemodynamic effects. Volatile anaesthetics might inhibit AM-induced haemodynamic responses by mechanisms similar to those of CGRP. Therefore, the present data support the notion that volatile anaesthetics inhibit G-protein-coupled receptor-mediated responses. Proposed mechanisms of inhibitory actions of isoflurane are shown in Figure 3. The exact sites of inhibitory actions of volatile anaesthetics are, however, still unclear. Further studies are required to determine the exact mechanisms of the actions of anaesthetics.
AM is responsible for the haemodynamic alterations in various disorders (e.g. endotoxemia, hepatic cirrhosis and cardiopulmonary bypass). The inhibitory actions of isoflurane on AM-mediated responses might be favourable in the haemodynamic management of patients with these disorders.
In conclusion, isoflurane, at clinically relevant concentrations, inhibits AM-induced vasodilatory and positive inotropic effects in pithed rats.
This work was supported by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan, to DY (#11671478).
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