General anesthetics inhibit evoked release of classic neurotransmitters. However, their actions on neuropeptide release in the central nervous system have not been well characterized.
The effects of representative intravenous and volatile anesthetics were studied on the release of sulfated cholecystokinin 8 (CCK8s), a representative excitatory neuropeptide, from isolated rat cerebrocortical nerve terminals (synaptosomes). Basal, elevated KCl depolarization-evoked and veratridine-evoked release of CCK8s from synaptosomes purified from rat cerebral cortex was evaluated at 35 degrees C in the absence or presence of extracellular Ca2+. CCK8s released into the incubation medium was determined by enzyme-linked immunoassay after filtration.
Elevation of extracellular KCl concentration (to 15-30 mM) or veratridine (10-20 microm) stimulated Ca2+ -dependent CCK8s release. Basal, elevated KCl- or veratridine-evoked CCK8s release was not affected significantly by propofol (12.5-50 microm), pentobarbital (50 and 100 microm), thiopental (20 microm), etomidate (20 microm), ketamine (20 microm), isoflurane (0.6-0.8 mM), or halothane (0.6-0.8 mMm).
Clinically relevant concentrations of several classes of general anesthetics did not affect basal, KCl-evoked, or veratridine-evoked CCK8s release from isolated rat cortical nerve terminals. This is in contrast to the demonstrable effects of certain general anesthetics on the release of amino acid and catecholamine transmitters. These transmitter-specific presynaptic effects of general anesthetics suggest that anesthetic-sensitive presynaptic targets are not common to all transmitter classes.
GENERAL anesthetics have both presynaptic and postsynaptic effects on synaptic transmission; they produce agent-specific effects on neurotransmitter release and the responses of neurons to neurotransmitters. 1Most studies of the presynaptic actions of general anesthetics have focused on the classic transmitters: the amino acids (glutamate, γ-aminobutyric acid [GABA]) and acetylcholine. There is considerable evidence that the evoked release of these transmitters is depressed by both intravenous and volatile anesthetics, 1–4which suggests that anesthetics target a process common to the release of all transmitters. However, few studies have analyzed the presynaptic actions of general anesthetics on peptidergic neurotransmission, 5,6largely due to the technical difficulties involved in quantifying peptide release. 7Given the conserved molecular mechanisms which are thought to control neurosecretion, 7–9we hypothesized that general anesthetics should also inhibit neuropeptide release.
Cholecystokinin (CCK) was chosen as a representative neuropeptide for analysis since its release from central nervous system (CNS) nerve terminals has been studied in detail. Cholecystokinin is an 8-residue peptide (CCK8) found throughout the CNS. It is involved in many important functions, including memory, pain, appetite, and anxiety. 10–13CCK8 is largely colocalized with classic transmitters in CNS terminals, where it is stored in morphologically distinct, large dense core vesicles. These vesicles are present throughout the presynaptic terminal and not at the active zones where classic transmitters contained in small synaptic vesicles are secreted. Sulfated CCK8 (CCK8s) is the predominant form in the CNS. 14There are two CCK receptor subtypes in brain: CCK2 receptors mediate anxiety, panic attacks, satiety and pain, while the function of CCK1 receptors, which have limited distribution within the CNS, is poorly understood. 10,12,15
The mechanism of CCK8s release has been studied extensively in vivo 11,16,17and in vitro . 13,18–20CCK8s release requires membrane depolarization and is dependent on extracellular Ca2+. CCK8s coexists in brain areas and neurons that also contain other neurotransmitters, including catecholamines, acetylcholine, GABA, and glutamate. 13,21,22These neurotransmitters can modulate CCK8s release from nerve endings, while CCK8s can modulate release of these neurotransmitters. 13,15The coexistence of CCK8s with other neurotransmitters and its potential for modulation of transmission by other neurotransmitters implicated in anesthetic actions 1–3suggest a potential role for CCK8s in general anesthesia. This study was designed to assess the direct presynaptic actions of representative general anesthetics on CCK8s release from nerve terminals isolated from rat cerebral cortex using a sensitive enzyme-linked immunoassay.
Materials and Methods
These studies were approved by Weill Medical College of Cornell University Institutional Animal Care and Use Committee (New York, New York).
CCK8s was obtained from RBI (Natick, MA); Percoll was obtained from Pharmacia (Uppsala, Sweden); isoflurane was obtained from Abbott Laboratories (North Chicago, IL); and halothane (thymol-free) was obtained from Halocarbon Products (River Edge, NJ). Propofol was purchased from Aldrich Chemicals (Milwaukee, WI) or was a gift from AstraZeneca Pharmaceuticals (Wilmington, DE), and etomidate was a gift from Janssen Biotech n.v. (Olen, Belgium). Pentobarbital, thiopental, ketamine, polymeric L-glutamic acid (poly-Glu), 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride, horseradish peroxidase (HRP)–labeled goat antirabbit IgG, rabbit anti-CCK8s IgG, bacitracin, Tween 20, veratridine, tetrodotoxin, ω-conotoxin MVIIC, dimethyl sulfoxide (DMSO), o -phenylenediamine, tablets for preparation of sodium carbonate-bicarbonate buffer pH 9.6, and HRP substrate solution (phosphate-citrate buffer with sodium perborate) were from Sigma Chemical Co. (St. Louis, MO). ω-Agatoxin IVA and ω-conotoxin GVIA were obtained from Alamone Labs (Jerusalem, Israel). All other chemicals were of analytical grade. Spectra/Por 12,000–14,000 dialysis membrane was obtained from Spectrum (Houston, TX); bovine serum albumin (fraction 5) was obtained from J.T. Baker (Phillipsburg, NJ); Nunc-immuno MaxiSorp flat-bottom modules (No. 469949), CoStar Spin-X centrifuge tube filters with 0.45-μm cellulose acetate membrane (Corning No. 8163), CoStar 96-well microtiter plates (No. 3797), and 0.4-ml polypropylene microcentrifuge tubes (No. 20170-326) were obtained from VWR Scientific Products (Bridgeport, NJ); and multiplates for filtration (No. MAFBNOB10) were obtained from Millipore (Bedford, MA).
Preparation of CCK8s-polyglutamic Acid
CCK8s was coupled to poly-Glu to form CCK8s-polyglutamic acid (CCK8s-poly-Glu). 23A solution of poly-Glu (1.4 mg) in 1.3 ml water was added to a solution of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (3.9 mg) in 0.39 ml water and stirred. A solution of CCK8s (0.2 mg) in 0.2 ml water was added over 45 s with mixing and left for 10 min at room temperature. The pH was adjusted to 6.0 with 0.27 ml sodium phosphate buffer, 1 m (pH 5.0), and the solution was stirred for 1 h at room temperature. The solution was diluted to 4.4 ml and dialyzed against 2 l water for 5 days at 4°C with a daily change of the dialysate. The dialyzed solution of CCK8s-poly-Glu conjugate was divided into aliquots, lyophilized, and stored at −70°C.
Preparation of Synaptosomes
Purified synaptosomes were prepared from rat cerebral cortex by the Percoll gradient method 24as described. 25The synaptosome fraction was washed from Percoll with HEPES-buffered medium (HBM) composed of the following: 130 mm NaCl, 3 mm KCl, 5 mm NaHCO3, 1 mm MgCl2, 1.2 mm Na2HPO4, 10 mm d-glucose, and 20 mm HEPES, titrated to pH 7.4 with Tris base. Protein content was measured using the Bio-Rad Protein Assay Kit (Hercules, CA) based on the method of Bradford 26using bovine serum albumin as a standard. Synaptosomes were stored as a pellet on ice and used within 5 h of preparation.
Filtration Release Assay
The effects of intravenous anesthetics on CCK8s release were assayed using a multifiltration system (Millipore Multiscreen Assay System; Millipore, Bedford, MA) by minor modifications of the procedure of Walaas. 27All buffers contained 0.5 mg/ml bacitracin to minimize degradation of CCK8s. 28Dilutions from stock solutions of propofol, thiopental, and etomidate in DMSO were made into HBM immediately prior to assays. DMSO as a vehicle at a final concentration of up to 0.5% (v/v) did not affect CCK8s release in control experiments (data not shown). Stock solutions of pentobarbital and ketamine were prepared in water and diluted into HBM.
Synaptosomes (120 μg protein in 60 μl HBM) plus any drugs for preincubation, and HBM (60 μl) containing drugs and/or the indicated secretogogues (15 or 30 mm final KCl; 10 or 20 μm veratridine) were preincubated in wells of separate plates (96-well microtiter plates, CoStar, No. 3797; VWR Scientific Products) with shaking for 5 min at 35°C. Reactions were initiated by transfer of synaptosomal aliquots to HBM aliquots (usually in triplicate or quadruplicate). After 5 min, reactions were stopped by transfer of synaptosomal suspensions to a third 96-well plate containing 15 μl EGTA, 100 mm, in HBM (final concentration approximately 10 mm). Synaptosomes were separated from incubation medium rapidly by vacuum filtration of samples through Millipore multiscreen filtration 96-well plates.
Centrifugation Release Assay
The effects of volatile anesthetics on CCK8s release were assayed using a centrifugation assay that allowed incubations in closed containers with minimal head space to reduce anesthetic losses. This assay was also used in control experiments to determine the effects of Ca2+channel blockers on CCK8s release. Volatile anesthetics were prepared as saturated solutions in HBM (10–12 mm) at room temperature. Required volumes were diluted into 0.4-ml polypropylene microcentrifuge tubes containing synaptosomes. Synaptosomes (100–150μg in 330 μl HBM) were preincubated for 5 min at 35°C with or without volatile anesthetics in capped tubes. Ca2+channel blockers prepared in HBM were added to synaptosomes in 1.5-ml polypropylene microcentrifuge tubes and preincubated at 35°C for 5 min. Concentrated KCl or veratridine (20 μl) was added through the cap with a Hamilton microsyringe, and tubes were sealed quickly with several layers of Parafilm (American National Can Co., Menasha, WI).
After incubation for 5 min at 35°C with mixing, reactions were stopped with EGTA (final concentration 10 mm). Synaptosomes separated from incubation solution rapidly from low-speed centrifugation for 30 s in CoStar Spin-X centrifuge tube filters (VWR Scientific Products) with an additional GF/B glass fiber filter layer.
CCK8s Quantification by Antigen Competition Enzyme-linked Immunoassay
CCK8s was quantified by enzyme-linked immunoassay 23,29with minor modifications.
The solutions used were as follows: (1) antigen-coating solution, 50 mm sodium carbonate–bicarbonate buffer, pH 9.6; (2) blocking solution, 20 mm Tris HCl, pH 7.4, 0.1% (w/v) BSA, 0.05% (v/v) Tween 20; (3) washing solution, blocking solution plus 0.15 mm NaCl; (4) substrate solution, 0.1 m phosphate–citrate buffer, pH 5.0, 0.03% (w/v) sodium perborate; and (5) stop solution, 4 N H2SO4.
CCK8s-poly-Glu was dissolved in antigen-coating solution to 2 μg/100 ml (calculated for CCK8s content), and 200 μl of this solution was placed in each well of a Nunc-immuno MaxiSorb module (VWR Scientific Products) mounted in a frame. The plate with lid was incubated for 2 h at room temperature. The plate was rinsed twice with 200 μl blocking solution, filled again with 375 μl of this solution and left for at least 1 h at room temperature, and then rinsed thrice with 200 μl washing solution, which was completely decanted on a paper towel. Samples (60–120 μl synaptosome incubation medium) containing CCK8s were added to wells. Standard dilutions of pure CCK8s in duplicate in the same buffer were added to separate wells as standards. Concentrated washing solution was added to each well to achieve final concentrations of 0.15 m NaCl, 0.1% BSA, and 0.05% (v/v) Tween 20 as in the washing solution. An appropriate dilution of anti-CCK8s antibody in washing solution was added, and the covered plate was incubated overnight (12–16 h) at 4°C. Wells were washed five times over 30 min with washing solution, following which 200 μl HRP-conjugated goat antirabbit IgG (1:1000 dilution in washing solution) was added to each well. Covered plates were incubated for 2 h at 35°C. Wells were rinsed five times with washing solution, two times with 20 mm Tris HCl, pH 7.4, and two times with substrate solution. The HRP reaction was initiated by addition of 200 μl o -phenylenediamine (final concentration 0.4 mg/ml) in substrate solution to each well. Plates were incubated at room temperature for 30–45 min in the dark, and reactions were stopped by addition of 50 μl H2SO4, 4 N. Absorbance at 492 nm was measured using a Bio-Rad Microplate Reader (model 3550). Release of CCK8s is expressed as fmol CCKs/mg synaptosomal protein for 10 min, which includes 5 min of preincubation and 5 min of stimulation. In figures 1–3, CCK8s release was normalized to the mean basal release of CCK8s in HBM in the absence of added CaCl2(400–600 fmol/mg synaptosomal protein).
Analysis of Anesthetic Concentrations
Free propofol concentrations in assay mixtures were determined by high-performance liquid chromatography 30; the concentrations of propofol refer to the measured free concentrations. Initial and final volatile anesthetic concentrations in assay mixtures were determined by gas chromatography following extraction into n-heptane. 31The extract (5 μl) was injected into a gas chromatograph (GC-8A; Shimadzu Corp., Kyoto, Japan) equipped with a thermal conductivity detector. Separation was achieved on a 1.8 m/6 mm ID glass column packed with Porapack Q (Supelco, Bellefonte, PA). The column temperature was 210°C, the injector temperature was 230°C, and carrier gas (He) flow was 40 ml/min. Volatile anesthetic concentrations refer to the average measured concentrations; there were no significant differences between the initial and final concentrations.
Values are expressed as mean ± SD. Statistical significance was assessed by two-tailed Student t test or analysis of variance (ANOVA) with the Newman-Keuls multiple range test or Dunnett multiple comparison test using GraphPad Prism, version 2.01 (GraphPad Software, Inc., San Diego, CA);P < 0.05 was considered statistically significant.
Characterization of CCK8s Release
Content of CCK8s in synaptosomes prepared from rat cerebral cortex was 6.7 ± 1.9 pmol/mg synaptosomal protein (n = 12). Basal (spontaneous) release in the absence of CaCl2was 400–600 fmol/mg synaptosomal protein over 10 min. Basal release was not increased significantly by exposure to 1.3 mm CaCl2(fig. 1A). The efflux of CCK8s evoked by elevated KCl or veratridine was concentration dependent and Ca2+dependent. Release was increased threefold to fourfold by 30 mm KCl or by 20 μm veratridine. In control experiments, known P/Q-type Ca2+channel antagonists inhibited KCl-evoked release, while an N-type Ca2+channel antagonist was ineffective (fig. 1B). The specific Na+channel antagonist tetrodotoxin completely inhibited veratridine-evoked release (see below).
Effects of Intravenous Anesthetics on CCK8s Release
Propofol (12.5–50 μm), pentobarbital (50–100 μm), thiopental (20 μm), etomidate (20 μm), or ketamine (20 μm) did not affect basal (fig. 2), 30 mm KCl-evoked (fig. 3), or 20 μm veratridine-evoked (fig. 4) release of CCK8s. Similar results were obtained whether synaptosomes were preincubated with the anesthetic for 5 min before addition of secretogogue, or anesthetic and secretogogue were added simultaneously (data not shown). Potentiation or inhibition of release was not observed when submaximal stimuli (15 mm KCl or 10 μm veratridine) were used (data not shown).
Effects of Volatile Anesthetics on CCK8s Release
Isoflurane or halothane at 0.6–0.8 mm (approximately 2 times minimum alveolar concentration [MAC]) did not affect basal or stimulus-evoked release of CCK-8s (figs. 5 and 6).
Neither basal nor evoked release of the excitatory neuropeptide CCK was affected by clinically relevant concentrations of a number of intravenous and volatile general anesthetics. We employed a specific and sensitive enzyme-linked immunoassay to quantify CCK release from isolated rat cortical nerve terminals (synaptosomes). Synaptosomes are pinched-off nerve terminals prepared by gentle homogenization and subcellular fractionation of nervous tissue. 32They represent the most accessible experimental system for the study of the biochemical and functional properties of presynaptic terminals. Our observations suggest that neither the ion channels that couple depolarization of CCK-containing nerve terminals to Ca2+influx nor the molecular machinery that couples this increase in Ca2+to vesicle exocytosis is sensitive to concentrations of general anesthetics, which significantly affect the release of other transmitters under comparable conditions.
In agreement with previous studies, elevated KCl or veratridine evoked CCK release in a Ca2+-dependent manner. Previous studies have analyzed CCK release from nerve terminals isolated from guinea pig 19and rat hippocampus 20and rat cerebral cortex. 33,34Verhage et al. 20reported basal release of approximately 150 fmol CCK/mg protein/3-min assay (50 fmol/mg protein/min), compared to our value of approximately 500 fmol CCK/mg protein/10-min assay (50 fmol/mg protein/min), which was increased approximately 2.3-fold by 30 mm KCl, compared to a stimulation of threefold to fourfold by 30 mm KCl in our experiments. Synaptosomes are too small for electrical field stimulation; transmitter release must be stimulated pharmacologically. We used two secretogogues that activate secretion by distinct mechanisms. Step elevations of extracellular KCl induce a “clamped” depolarization of the synaptosomal plasma membrane above the threshold for activation of voltage-gated Ca2+channels present in the presynaptic terminal. KCl-evoked release is sensitive to P/Q-type Ca2+channel blockade (Leenders et al. 34and fig. 1A) but resistant to Na+channel blockade (e.g. , with tetrodotoxin). Na+channel activation by veratridine, a neurotoxin that inhibits channel inactivation, leads to prolonged synaptosome depolarization and influx of Ca2+. Since Na+channel activation is required, release is tetrodotoxin-sensitive (figs. 4 and 6). 32CCK release activated either by elevated KCl or by veratridine was not affected by general anesthetics, which suggests that the Na+and Ca2+channel subtypes coupled to CCK release are insensitive. The interpretation of these studies must consider that artificial secretogogues are required to evoke transmitter release from isolated nerve terminals. Chemical stimulation may not accurately reproduce action potential-evoked release, which could result in underestimation of the sensitivity of neuropeptide release to anesthetics. Previous studies 34and our control studies indicate that CCK release from rat cortical nerve terminals evoked with 30 mm KCl is inhibited by the P/Q-type Ca2+channel antagonists ω-agatoxin IVA and ω-conotoxin MVIIC, and we showed that veratridine-evoked release is sensitive to the Na+channel antagonist tetrodotoxin. Together, these findings indicate that this assay is sensitive to blockade of presynaptic Ca2+and/or Na+channels. Nevertheless, it is possible that the stimuli used to evoke CCK from isolated nerve terminals do not mimic action potential–evoked release and thus do not accurately reflect their sensitivity to anesthetics. Our findings do not rule out anesthetic actions on synaptic transmission by CCK mediated by mechanisms other than direct effects on release or on transmission by peptides other than CCK, such as those demonstrated in mollusks. 35
The fundamental mechanisms underlying transmitter release are largely conserved among the various classes of neurotransmitters. 7–9Vesicles are released by Ca2+-dependent fusion with the plasma membrane upon Ca2+influx utilizing the highly conserved SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF]-attachment protein [SNAP] receptor) core complex proteins syntaxin, synaptobrevin, and SNAP-25, as well as the Ca2+-binding protein synaptotagmin. Genetic evidence for anesthetic effects on SNARE proteins was provided by a recent screening of existing mutants of the nematode C. elegans for alterations in sensitivity to volatile anesthetics, which identified mutations in all three components of the core fusion complex. 36These findings imply that inhibition of transmitter release by general anesthetics, which is observed for many agents at clinical concentrations, 3should be observed for all transmitter classes, if these mechanisms are shared by all transmitter classes. The results reported here indicate that this generalization does not hold for the excitatory neuropeptide cholecystokinin. Thus, concentrations of both volatile and intravenous anesthetics which inhibit depolarization-evoked release of glutamate (e.g. , isoflurane IC50= 0.50 mm for inhibition of veratridine-evoked release in rat cortex), 25,37–39GABA, 4and acetylcholine 40from small synaptic vesicles had no significant effect on CCK release from large dense core vesicles. This difference suggests that the mechanism involved in the presynaptic actions of general anesthetics is not a common property of all transmitter classes. Since classic transmitters are often colocalized with neuropeptides in the same nerve terminals (e.g. , GABA and CCK in cerebral cortex), 7it is possible that general anesthetics selectively affect the release of the transmitters contained within small synaptic vesicles without affecting release of neuropeptides contained within dense core vesicles from the same terminals. Whether release of other neuropeptides is similarly resistant to general anesthetics will require further study.
Neurotransmitter release involves many steps, from action potential invasion of the presynaptic terminal; depolarization; ion channel gating; Ca2+influx; vesicle translocation, docking and priming; Ca2+-release coupling; vesicle fusion/exocytosis; to vesicle endocytosis. Each of these processes is modulated by repetitive activity, trophic factors, and presynaptic receptors. Certain differences in the molecular and cellular mechanisms that underlie the exocytotic release of fast transmitters (amino acids, monoamines) and slow transmitters (neuropeptides) have been described which could result in their differential sensitivities to general anesthetics. Neurons possess two types of secretory vesicles that undergo Ca2+-dependent exocytosis with distinct kinetic differences despite their overall similar biochemical compositions. 7Classic transmitters are released by fast exocytosis from small synaptic vesicles which cluster at active zones, whereas neuropeptides are released by fusion of large dense core vesicles with slower kinetics at sites away from the active zone. 9,41Fast transmitters are released at active zones by relatively high local concentrations of Ca2+(Ca2+microdomains) produced by close physical coupling to specific Ca2+channel subtypes, while neuropeptide release occurs by a more delocalized bulk phase increase in nerve terminal Ca2+as a result of repetitive, high-frequency stimulation. 19,42The apparent Ca2+sensitivity of amino acid transmitter release from small synaptic vesicles is lower than that of neuropeptide release from large dense core vesicles 19(but see Bollmann et al. 43), which suggests the involvement of distinct Ca2+sensors. Release of different classes of transmitters may also be coupled to distinct Ca2+channel subtypes; release from small synaptic vesicles was coupled to N- and P-type Ca2+channels, whereas CCK release from large dense core vesicles was coupled primarily to P/Q-type Ca2+channels. 12,34Neuropeptides are packaged into large dense core vesicles in the trans -Golgi network, whereas small synaptic vesicles are loaded by specific transporters in the terminal. Although exocytosis of both small and dense core vesicles involves the highly conserved SNARE protein machinery for fusion/exocytosis, 9,44,45these different properties are consistent with subtle differences in their regulatory components, such as differential expression of synaptotagmin isoforms 46and CAPS, 47which is expressed on large dense core, but not small synaptic, vesicles. One or more of these differences may be crucial in conferring sensitivity to general anesthetics of release of classic transmitters such as glutamate from small synaptic vesicles and insensitivity to anesthetics of the release of CCK, and possibly other transmitters, from large dense core vesicles.