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CARDIOVASCULAR
Department of Anesthesiology, Gunma University Graduate School of Medicine, Maebashi, Japan
Received May 25, 2004; accepted August 5, 2004.
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Abstract |
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). Perivascular nerve stimulation in mesenteric vascular beds induces neurogenic vasodilation; this effect is mediated by nonadrenergic, noncholinergic (NANC) nerves (Kawasaki et al., 1988). Primary afferent neurons, originating from the dorsal root ganglia, provide a perivascular network of fibers around the arterial system throughout the body. Stimulation of these fibers causes NANC vasodilation via the release of calcitonin gene-related peptide (CGRP) (Bell and McDermott, 1996).
CGRP is a 37-amino acid peptide that is generated by alternative RNA splicing processes during calcitonin gene expression in rat and human tissues (Bell and McDermott, 1996). CGRP binds to a specific cell surface receptor, a seven-transmembrane domain receptor protein that belongs to a subgroup of the G protein-coupled receptor family (McLatchie et al., 1998). The receptor is primarily coupled to the activation of adenylate cyclase, which produces cAMP, leading to smooth muscle relaxation (Bell and McDermott, 1996). CGRP is widely distributed in perivascular nerves throughout the cardiovascular system. It has a potent vasodilatory effect and positive inotropic and chronotropic effects in rat and human (Bell and McDermott, 1996). Intravenous administration of CGRP causes a significant decrease in the mean arterial blood pressure (MAP), total peripheral resistance, and sustained tachycardia in rats and humans (Bell and McDermott, 1996). CGRP is implicated in the regulation of circulatory homeostasis and pathophysiology of various diseases. For example, CGRP is known as a potent vasodilator in brain vessels and has been implicated in the pathogenesis of migraines (Moreno et al., 2002). Increased plasma levels of CGRP have been demonstrated in endotoxemia, congestive heart failure (Bell and McDermott, 1996), hepatic cirrhosis (Henriksen et al., 2000), and during cardio-pulmonary bypass (Terazawa et al., 2003). The development of autonomic dysfunction in a mouse model of spinal cord injury is associated with sprouting of CGRP fibers (Jacob et al., 2001). These data suggest that CGRP is responsible for the hemodynamic alterations in these disorders. Because general anesthetics are often used for patients with such disorders, it is important to elucidate the effects of general anesthetics on the CGRP-induced hemodynamic response for better hemodynamic management of complicated patients.
The pithed rat model is used to investigate cardiovascular responses mediated by autonomic outflow from the spinal cord in vivo (Gillespie and Muir, 1967). In pithed animals, there is no intrinsic sympathetic nerve activity or centrally mediated compensatory reflex (Gray et al., 1990). The depressor response during spinal cord stimulation in the pithed rat is attributed to endogenous CGRP released from CGRP-containing nerves (Taguchi et al., 1992). Volatile anesthetics inhibit NANC depressor responses through inhibition of the CGRP depressor effect, not through inhibition of CGRP release after NANC nerve stimulation in the pithed rat model (Yoshikawa et al., 2003), suggesting that volatile anesthetics inhibit vascular responses to CGRP. Anesthetic actions on cardiac output (CO), systemic vascular resistance (SVR), and CGRP receptor-mediated cellular responses, however, have not yet been studied.
The purpose of the present study was to investigate the effects of volatile anesthetics on 1) CGRP-induced vasodilation by evaluating the changes in hemodynamic parameters, including MAP, CO, and SVR in vivo, and 2) the CGRP receptor-mediated signal transduction pathway in vitro. In the in vivo study, we used a pithed rat model to evaluate the direct vascular effect of drugs without interference of centrally mediated circulatory reflexes. In the in vitro study, we used SK-N-MC human neuroblastoma cells as a model system to study the CGRP receptor and its downstream pathways (Van Valen et al., 1990; Muff et al., 1992; Poyner et al., 1998; Choksi et al., 2002).
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Materials and Methods |
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; Yoshikawa et al., 1998; 2003). Male Wistar rats weighing 300 to 360 g were anesthetized with isoflurane in oxygen. The animals were ventilated artificially using a Harvard respirator (Harvard Apparatus Inc., Holliston, MA) via a tracheal cannula at a rate of 50 to 60 breaths · min–1 with a stroke volume of 1 ml · 100 g b.wt.–1. Respiration rate was adjusted to maintain a PaCO2 of 35 to 40 mm Hg. A polyethylene (PE-50) catheter was placed in the left carotid artery to measure MAP. Both vagus nerves were severed in the neck. Each rat was pithed by inserting a stainless steel rod (diameter, 1.5 mm) through the right orbit and foramen magnum down the spinal canal to its sacral terminus. This procedure destroys the entire central nervous system. Isoflurane anesthesia was discontinued immediately after pithing. Following a median sternotomy, a flow probe (Transonic Systems Inc., Ithaca, NY) was placed around the ascending aorta to measure aortic blood flow, i.e., CO. The right jugular vein was cannulated with PE-50 for the administration of norepinephrine. A 24-gauge Teflon cannula (Angiocath; Becton Dickinson Medical Systems, Sandy, UT) was placed in the tail vein for the administration of CGRP. Body temperature was maintained between 36° and 37°C using a heated blanket placed beneath the animal and controlled by a rectal thermistor probe (CMA/150; CMA/Microdialysis, Solna, Sweden).
Experimental Measurements
MAP was recorded with a Gould (Cleveland, OH) pressure transducer connected to the left carotid artery via a PE-50 catheter. Aortic blood flow was measured using a volume flowmeter (Transonic Systems Inc.). CO and SVR were normalized by the body weight using the following formula: CO = ml · min–1 · kg b.wt.–1 and SVR (mm Hg · ml–1 · min–1 · kg b.wt.–1) = MAP/CO. SVR was determined when MAP and CO reached their minimal values.
Cell Culture
The SK-N-MC cell line was obtained from the American Type Culture Collection (Manassas, VA). The cells were grown in minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C, 95% humidity, and 5% CO2. For cAMP assays, the cells were seeded on 100-mm culture dishes and grown to confluence. For binding studies, the cells were seeded on 24-well plates at a density of approximately 5 x 104 cells/well and grown to confluence.
Assay of cAMP Production
Equilibration with Volatile Anesthetics. Preincubation with volatile anesthetics was performed as described by Sill et al. (1993) with minor modifications. Serum-free MEM without cells at 37°C (4 ml) was aerated in semiclosed glass vials (5 ml) for 20 min with 95% O2 and 5% CO2 gas mixture at 50 ml/min to which volatile anesthetic was added using a calibrated anesthetic vaporizer. After a 20-min equilibration with anesthetics, the gas mixture containing an appropriate concentration of anesthetic was passed continuously over the medium to minimize the loss of anesthetic. The cells (5 x 106 cells/ml) were gently added to the medium at 37°C and preincubated with anesthetic for 20 min before the addition of CGRP or forskolin, a direct adenylate cyclase activator, or cholera toxin, a Gs protein activator. Gas chromatographic analysis (GC-7A; Shimadzu, Kyoto, Japan) of the media at the end of the protocol indicated that exposure to 1% (v/v) sevoflurane, 2% sevoflurane, 4% sevoflurane, 0.5% isoflurane, 1% isoflurane, and 2% isoflurane corresponded to actual concentrations of 0.969 ± 0.116, 2.181 ± 0.211, 3.991 ± 0.119, 0.482 ± 0.015, 1.086 ± 0.154, and 1.978 ± 0.155% (v/v), respectively. Cell suspension for control experiments was prepared in the same manner but without anesthetics.
Measurement of cAMP. Suspensions of SK-N-MC cells were used for determining cAMP production. Incubation was terminated by adding 400 µl of 1 N HCl to a 4-ml cell suspension and strong vortexing at the end of each exposure protocol. The mixture was frozen quickly in liquid nitrogen and stored at –70°C until cAMP determination. After thawing, the cell debris in the mixture was pelleted by centrifugation (13,000g for 10 min at 4°C), and the supernatant was transferred to a test tube for the cAMP assay. A cAMP 125I-radioimmunoassay kit (YAMASA Corporation, Choshi, Japan) was used to determine the cAMP levels. All assay procedures were performed according to the manufacturer's instructions. Data were normalized for protein content using Bradford's method (Bradford, 1976).
Binding Studies
Equilibration with Volatile Anesthetics. Binding studies were performed at 37°C in an air-tight glass chamber (50 liters) under a continuous flow of a 95% O2 and 5% CO2 gas mixture at 3 liters/min, supplemented with 4% sevoflurane or 2% isoflurane using a calibrated anesthetic vaporizer. The anesthetic concentrations in the chamber were monitored continuously with a calibrated gas analyzer (Capnomac Ultima; Datex-Ohmeda, Helsinki, Finland). Eagle's MEM was equilibrated by continuous bubbling with the corresponding concentration of anesthetic for 20 min at 37°C in semiclosed glass vials. Following the preparation, a gas mixture containing the anesthetic was passed continuously over the medium to minimize the loss of anesthetic. Then 1% bovine serum albumin was added to the medium and incubated with anesthetic for 30 min at 37°C. The solution equilibrated with anesthetic was used as the binding buffer. The anesthetic concentrations in the buffer were measured by gas chromatography (GC-7A). Control experiments were performed in the same manner without anesthetic.
Analysis of Binding Data. Saturation binding studies were performed using (2-[125I]iodohystidyl10) CGRP (2000 Ci/mmol; Amersham Biosciences, Ltd., Little Chalfont, Buckinghamshire, UK) at concentrations ranging from 5 to 200 pM per well in the absence (total binding) or presence (nonspecific binding) of excessive unlabeled human 
CGRP (1 µM) (Peptide Institute, Inc., Osaka, Japan). Specific binding was determined as the difference of total binding and nonspecific binding. Scatchard analysis was used to determine receptor density (Bmax) and the ligand dissociation constant (Kd) (Scatchard, 1949).
Experimental Protocols
Protocol 1: The Effects of Volatile Anesthetics on Hemodynamic Changes Induced by CGRP in Pithed Rat. Pithed rats were allowed to stabilize for 30 min before any experimental intervention. MAP and CO were maintained at approximately 100 mm Hg and 50 ml · min–1 by continuous infusion of norepinephrine (2 to 3 µg · kg–1 · min–1) throughout the protocol. Rats were randomly assigned to anesthetic groups (n = 7, each) and no anesthetic 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 anesthetic groups, a volatile anesthetic (2% sevoflurane, 4% sevoflurane, 1% isoflurane, or 2% isoflurane) was administered for 30 min 1 h after pithing. Concentrations of inspired anesthetics were monitored continuously with a calibrated gas monitor (Capnomac Ultima). In the no-anesthetic group, no volatile anesthetic was administered. Rat CGRP (0.1, 0.3, 1.0, and 3.0 µg · kg–1; Peptide Institute, Inc.) was administered intravenously for 30 s in 0.1 ml of saline.
Protocol 2: The Effects of Volatile Anesthetics on cAMP Production in SK-N-MC Cells. SK-N-MC cells were preincubated for 20 min with or without anesthetics in media containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich, St. Louis, MO) to limit cAMP breakdown. Human CGRP (10–7 M), forskolin (10–6.5 M; Sigma-Aldrich), or cholera toxin (10–9 M; Wako Pure Chemicals, Osaka, Japan) was added to the cells. Cell suspensions were incubated for 15 min (the appropriate times for cholera toxin) at 37°C with or without anesthetics, then used for cAMP assay. CGRP induced maximal cAMP production at 10–7 M in SK-N-MC cells, and 10–6.5 M forskolin and 10–7 M CGRP induced comparable cAMP production in our preliminary studies. The dose of cholera toxin and the incubation time were determined as described previously (Orlandi et al., 1993). Incubation with 10–9 M cholera toxin for 2 h induced maximal cAMP production. The amount was comparable with that of 10–7 M CGRP or 10–6.5 M forskolin in the current study.
Protocol 3: The Effects of Volatile Anesthetics on Ligand-Receptor Binding. Binding studies were performed as described by Muff et al. (1992) with minor modifications. Confluent cell monolayers (approximately 2 x 105 cells per well) grown on 24-well plates were washed once with binding buffer (MEM supplemented with 1% bovine serum albumin). They were then incubated with binding buffer equilibrated with volatile anesthetic in the experimental chamber, saturated with the anesthetic, and incubated for 20 min at 37°C. 125I-CGRP (with or without unlabeled CGRP) was added to each well and incubated for 15 min at 37°C in the chamber. Unbound label was then removed by aspiration of binding buffer. Cells were washed once with 500 µl of ice-cold binding buffer and subsequently lysed with 500 µl of 0.5% sodium dodecyl sulfate. Lysates were then counted for radioactivity in a gamma counter. The procedures in the experimental chamber were performed using manipulation arms that were tightly sealed to the side of the chamber. The total incubation volume was 300 µl. The binding buffer contained 30 µl of 125I-CGRP solution.
Statistical Analysis. 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 the mean ± S.D. A p value of less than 0.05 was considered statistically significant.
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Results |
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The effects of sevoflurane on CGRP-induced changes of the hemodynamic parameters are shown in Fig. 1. Sevoflurane significantly inhibited CGRP-induced increases in MAP (p < 0.01, n = 7). On the other hand, CGRP induced a dose-dependent decrease in MAP, CO, and SVR. Sevoflurane concentration dependently inhibited the CGRP-induced decrease in MAP and SVR (p < 0.05, n = 7). The decrease in CO did not differ significantly among groups. Heart rate did not change significantly after administration of CGRP in either group. Isoflurane also inhibited CGRP-induced increases in MAP (p < 0.05, n = 7) and dose dependently inhibited CGRP-induced decreases in MAP and SVR (p < 0.05, n = 7) without effects on CO reduction and HR (data not shown).
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The Effects of Volatile Anesthetics on CGRP-, Forskolin-, or Cholera Toxin-Induced cAMP Production in SK-N-MC Cells. Sevoflurane did not alter basal cAMP accumulation (Fig. 2A). The effect of sevoflurane on CGRP-induced cAMP production is shown in Fig. 2B. CGRP (10–7 M) increased cAMP production in SK-N-MC cells (20-fold increase over basal). Preincubation of cells with sevoflurane concentration dependently inhibited CGRP-induced cAMP production. Sevoflurane (4%) significantly inhibited CGRP-induced cAMP production to 35% of no anesthetic (p < 0.01, n = 6). Forskolin (10–6.5 M) increased cAMP production in SK-N-MC cells (25-fold increase over basal), whereas preincubation with sevoflurane did not influence the forskolin-induced increase in cAMP (Fig. 2C). Preincubation of cells with isoflurane also caused a concentration-dependent reduction in CGRP-induced cAMP production. Isoflurane (2% or 4%) significantly inhibited CGRP-induced cAMP production to 46% of no anesthetic (p < 0.05, n = 6) or to 27% (p < 0.01, n = 6), respectively, whereas preincubation with isoflurane did not alter forskolin-induced cAMP production (data not shown).
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The effects of sevoflurane on time-dependent cholera toxin-induced cAMP production in SK-N-MC cells were shown in Fig. 3 (inset). After a lag of 15 min, cholera toxin (10–9 M) exhibited time-dependent increases in cAMP production in each group. Sevoflurane (4%) significantly inhibited cholera toxin-induced cAMP production at 90 and 120 min [Fig. 3 (inset); p < 0.05, n = 6]. Dose dependence of sevoflurane on cholera toxin-induced cAMP production was shown in Fig. 3. Sevoflurane did not affect basal cAMP production (data not shown). Cholera toxin (10–9 M) increased cAMP production in SK-N-MC cells (30-fold increase over basal). Sevoflurane dose dependently inhibited cholera toxin-induced cAMP production. Sevoflurane (4%) significantly inhibited cholera toxin-induced cAMP production to 70% of no anesthetic (p < 0.05, n = 6). Isoflurane dose dependently inhibited cholera toxin-induced cAMP production. Although isoflurane (2%) did not significantly inhibit cholera toxin-induced cAMP production (83% of no anesthetic; p = 0.10), isoflurane (4%) significantly inhibited it to 58% of no anesthetic (p < 0.01, n = 6).
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Radioligand Binding Studies. Specific binding of 125I-CGRP to SK-N-MC cells was saturable with increasing concentration of 125I-CGRP (5–200 pM). The receptor density (Bmax) and the ligand dissociation constant (Kd) calculated from plots according to the method of Scatchard are shown in Table 1. There was no significant difference in Bmax and Kd among groups. Preincubation with 4% sevoflurane did not affect CGRP receptor density and affinity. Isoflurane (2%) had no effect on Bmax and Kd when compared with no anesthetic and sevoflurane.
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Discussion |
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The Effects of Volatile Anesthetics on CGRP-Induced Hemodynamic Changes in Pithed Rats. Yoshikawa et al. (2003) reported that isoflurane and halothane inhibited the NANC depressor response in pithed rats. Although isoflurane, halothane, and sevoflurane did not affect CGRP release after spinal cord stimulation, isoflurane and halothane inhibited the depressor response to exogenously administered CGRP. These results suggest that volatile anesthetics inhibit the NANC depressor response by inhibiting the CGRP depressor effect but not by inhibiting CGRP release.
In a pithed rat model, we investigated not only the effect of volatile anesthetics on CGRP-induced changes in blood pressure but also the CGRP-induced vasodilation by measuring SVR, which reflects the systemic vascular tone. Sevoflurane (4%) and isoflurane (2%) significantly inhibited the CGRP-induced decrease in SVR. Lower concentrations of volatile anesthetics (2% sevoflurane and 1% isoflurane) did not affect the decrease in SVR.
In general, there are four main mechanisms of the vasodepressor response elicited by intravenous drug administration: 1) central nervous system actions, 2) inhibition of vascular sympathetic transmission, 3) release of endothelium-derived relaxing factors, and 4) direct vasorelaxing effects (Saxena and Villalon, 1990). Both central and autonomic nervous system activity were excluded by using pithed vagotomized rats. Therefore, CGRP-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). CGRP, among the most potent vasodilating substances known, also exerts positive inotropic effects (van Rossum et al., 1997). These biologic responses are mediated by specific cell surface receptors predominantly coupled to the activation of adenylate cyclase (van Rossum et al., 1997). In the current study, 4% sevoflurane and 2% isoflurane inhibited the CGRP-induced vasodilatory effects. In addition, 4% sevoflurane and 2% isoflurane inhibited the increase in MAP after CGRP administration, suggesting that high concentrations of volatile anesthetics inhibit both vasodilatory and positive inotropic effects induced by CGRP. These data strongly suggest that volatile anesthetics inhibit the CGRP-induced response through an effect on CGRP receptor-mediated responses.
The Effects of Volatile Anesthetics on CGRP Receptor-Mediated Signal Transduction in SK-N-MC Cells. CGRP relaxes vascular smooth muscle cells via both endothelium-dependent and endothelium-independent signal transduction pathways (Bell and McDermott, 1996). These responses are mediated by the specific cell surface family B Gs protein-coupled receptor (GPCR) and the calcitonin receptor-like receptor (McLatchie et al., 1998). The receptor is predominantly coupled to the activation of adenylate cyclase, which produces cAMP, leading to vascular relaxation in both pathways. In addition, there are other mechanisms by which CGRP elevates nitric oxide directly without the involvement of adenylate cyclase or activates ATP-sensitive potassium channels (KATP) (Bell and McDermott, 1996). On the other hand, CGRP-mediated inotropic action in the myocardium is mediated through CGRP receptors (van Rossum et al., 1997). In the present study, sevoflurane and isoflurane significantly inhibited CGRP-induced cAMP production but had no effect on forskolin-induced cAMP production. Neither anesthetic had an effect on CGRP receptor binding. Furthermore, sevoflurane and isoflurane significantly inhibited cholera toxin-induced cAMP production. Therefore, sevoflurane and isoflurane inhibited CGRP receptor-mediated signal transduction at the step between the CGRP receptor and adenylate cyclase activation by interfering with the activation of Gs protein.
Volatile anesthetics are likely to affect several hydrophobic sites within the cell membrane, particularly membrane-binding proteins (Franks and Lieb, 1994). Possible sites of the action of anesthetics in the GPCR-mediated signal transduction pathway are divided into four target groups: 1) agonist-receptor binding, 2) G protein function, 3) effector activity (adenylate cyclase), and 4) other intracellular sites (e.g., Ca2+ stores and cellular kinases) (Lambert, 1993).
There are no previous reports demonstrating an effect of volatile anesthetics on CGRP receptor-mediated signal transduction, although several studies reported that volatile anesthetics affect the signal transduction pathway mediated by other GPCRs. For example, halothane and isoflurane attenuate isoproterenol-induced vasodilation and interfere with 
adrenoceptor-mediated responses in the rat aorta at a point between agonist-receptor binding and adenylate cyclase activation (Tanaka and Tsuchida, 1998). Pentyala et al. (1999) reported that volatile anesthetics, including sevoflurane and isoflurane at clinically relevant doses, modulate the binding of guanine nucleotides to purified G subunits in aqueous solution, inhibiting the exchange of a nonhydrolyzable analog of guanosine 5'-triphosphate (GTP
S) for bound guanosine 5'-diphosphate (GDP). This finding suggested that volatile anesthetics directly affect the guanine nucleotide binding site, including the Gs subunit. These studies are consistent with our current study and support the notion that sevoflurane inhibits the GPCR-mediated signal transduction pathway at the Gs protein level. Furthermore, cholera toxin affects Gs protein by ADP ribosylating the -subunit of Gs. ADP ribosylation of the Gs by cholera toxin stabilizes the GTP-bound conformation of Gs and decreases its intrinsic GTPase activity, resulting in the persistent activation of adenylate cyclase (Neer and Clapham, 1988). In the current study, sevoflurane and isoflurane dose dependently inhibited cholera toxin-induced cAMP production, suggesting that both anesthetics might affect the conformation and stability of the GTP-bound state of Gs and/or GTPase activity.
On the other hand, isoflurane interferes with muscarinic receptor-G protein interactions in rat brainstem (Anthony et al., 1989). Sevoflurane depresses the adrenoceptor-mediated signal transduction in rat myocardium by reducing ligand-receptor binding and disrupting the relation between the receptor and G protein (Sanuki et al., 1994). Halothane, which inhibits receptor-activated Gi-coupled pathways in airway smooth muscle cells, must functionally target a component of the G protein-coupled receptor complex other than Gi (Streiff et al., 2003). These studies suggest that volatile anesthetics might inhibit the GPCR-mediated signal transduction pathway at the receptor itself or the interaction between the receptor and G protein. In the current study, isoflurane significantly inhibited CGRP-induced cAMP production at 2%, whereas isoflurane significantly inhibited cholera toxin-induced cAMP production at 4%. Therefore, in addition to Gs protein, isoflurane might inhibit CGRP receptor-mediated signal transduction between the receptor and the Gs protein.
In the present study, we focused on CGRP actions in rat hemodynamic system. CGRP is widely distributed in the body, including the nervous and gastrointestinal systems. Further studies elucidating CGRP actions in multiple organs may be indispensable to fully understand the roles of CGRP in the body and the importance of circulating cAMP levels in the plasma.
In conclusion, sevoflurane and isoflurane inhibit CGRP-induced positive inotropic and vasodilatory effects in pithed rats. Both anesthetics inhibited CGRP-and cholera toxin-induced cAMP production. Both anesthetics did not affect forskolin-induced cAMP production and 125I-CGRP ligand-receptor binding in SK-N-MC cells. These results suggest that the inhibitory site of volatile anesthetics on the CGRP receptor-mediated signal transduction involves Gs protein.
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Footnotes |
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ABBREVIATIONS: NANC, nonadrenergic, noncholinergic; CGRP, calcitonin gene-related peptide; CO, cardiac output; GTPS, guanosine 5'-O-(3-thio)triphosphate; GPCR, G protein-coupled receptor; IBMX, 3-isobutyl-1-methylxanthine; MAP, mean arterial pressure; MEM, minimal essential medium; SVR, systemic vascular resistance.
Address correspondence to: Masataka Kuroda, Department of Anesthesiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi City 371-8511, Japan. E-mail: mkuroda{at}med.gunma-u.ac.jp
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