Opioid Effects of Racemic Ketamine on the Excitability of Sciatic and Fibers of the Frog

Jong Hwa LEE*and George B. FRANK Departmentof Pharmacology,The Universityof Alberta, Edmonton,Alberta, Canada, T6G 2H7 AcceptedJuly 7, 1989

Abstract-The effects of ±ketamine were tested on the excitability of frog sciatic using a sucrose gap apparatus and skeletal muscle fibers using intracellular microelectrodes. When applied extracellularly by perfusion, ketamine depressed the of sciatic nerves in a dose-dependent manner. This depression was partially antagonized by the simultaneous treatment with a small concentration of naloxone. However, when the ketamine was applied intracellularly by placing it in a compartment with a cut end of the nerve, only very small and inconsistent decreases were produced. Ketamine also blocked excitability in skeletal muscle by depressing the sodium conductance (gNa). This also could be partly an tagonized by the addition of a small concentration of naloxone to the solution bathing the muscle. These results support previous findings by other workers that ketamine has a stereospecific opioid agonist effect in addition to its other actions.

The 'dissociative' anesthetic ketamine and in skeletal muscle (15, 16). In squid giant which is related to the psychostimulant , ketamine is more effective in depressing phencyclidine produces many pharmacolo the transient sodium current when applied gical actions and has effects at many different intracellularly (12). In and skeletal types of drug receptors (1, 2). One of these is muscle fibers, opiate receptors are located on a stereospecific effect at opiate receptors or near the intracellular openings of the related to the analgesic effect of ketamine sodium channels (17). This suggests the (3-5). This effect is antagonized by naloxone, possibility that ketamine effects on these and the (+) isomer of ketamine is more active tissues might be due, at least in part, to these than the (-) isomer in producing this effect. stereospecific opiate receptors. However, in It is generally thought that ketamine produces the only study which tested for this possible this effect by acting at the opiate sigma re effect (13), both drugs were used in high con ceptor subtype (2-6). There are also reports centrations; i.e., 1-2x10-3 M ketamine and that naloxone reduced the duration of 1-2.5x10-5 M naloxone. Since high con ketamine anesthesia (7) and antagonized a centrations of many drugs, including opioids, ketamine produced increase in brain dop produce nonspecific local anesthetic-like ef amine metabolism (8, 9). However, there are fects (18, 19) and high naloxone concentra some negative reports (9, 10) and suggestions tions tend to have opiod agonist (17, 19, 20, that ketamine effects are 'naloxone-insensi 21), it is not surprising that Artem and tive' (2). Rydqvist observed only additive effects. In Ketamine has been shown to suppress ac the present study when tested using lower tion potential production in axons (11-14) concentrations of both drugs, naloxone was found to partially antagonize the depression * Present address: Department of Pharmacy , Sam of the action potential produced by ketamine yook University, Dobonggu Kongreungdong 223, in frog's sciatic nerve axons and skeletal Seoul, Korea muscle fibers. with each drug condition using Student's t Materials and Methods test, and P<0.05 was taken as the level of The experiments were carried out on significance. isolated sciatic nerves and sartorius muscles The action potentials were recorded be from the leopard frog Rana pipiens at room tween two compartments separated by a temperature (18-21 °C). sucrose gap. The stimulating voltage was ap Experiments on frog sciatic nerve fibers: plied between the fifth and the third chambers The nerves were desheathed under a dis of the bath, and the membrane potentials secting microscope and split longitudinally were recorded between the first and the third into two bundles from each nerve. For stabili chambers. The stimulating voltage was set to zation of the split nerves, they were allowed to produce a maximal compound action potential rest in a bath for 1 hr. The desheathed nerves using single rectangular pulses of supramaxi were then placed in a sucrose gap apparatus mal strength and 0.01-0.05 msec in duration. similar to the one used by Hunter and Frank The action potentials were recorded using (22) and by Frank and Sudha (23). a digital oscilloscope (Nicolet 4094) and then The nerve bundles were pulled through stored on a floppy disk by the disk recorder each hole of the four rubber membranes in a (Nicolet XF-44). The records were later an five-chambered sucrose gap apparatus. The alyzed using a Hewlett Packard 9816 com experiments by the sucrose gap method were puter, and individual action potentials were designed in two different ways in order to drawn with a 701 5B Hewlett Packard X-Y compare the difference in drug application on Recorder. the effects of drugs on the action potentials of Experiments on frog skeletal muscle fibers: the frog sciatic nerves. Resting membrane and action potentials of For single sucrose gap experiments (in the frog's sartorius muscle fibers were re tracellular application), after setting the nerve corded intracellularly using conventional glass bundle in the bath, the central (the third) capillary microelectrodes (10-40 M ohm chamber was perfused with frog Ringer's resistance) filled with 3 M KCI. A small pro solution at the rate of 1 ml/min, and one portion of the muscle fibers were electrically adjacent chamber (the second) was perfused stimulated by an extracellular, bipolar with isotonic sucrose solution (214 mM) at platinum-filled pore electrode placed on the the rate of 1 ml/min. IsoKCI (123 mM) or surface of the muscle 5-10 mm from the drug in IsoKCI was applied to the chamber recording electrode. The bipolar stimulating (the first chamber), and the other chambers electrode was constructed with 0.4 mm (the fourth and fifth) were filled with frog platinum wires placed in polyethylene tubing Ringer's solution. and cemented together so that the ends of the For double sucrose gap experiments (ex vvires which rested on the surface of the tracellular application), the second and the muscle were 1.5 mm apart. In most instances, fourth chambers were perfused with isotonic the muscle fibers were stimulated initially sucrose solution (214 mM), the central with supramaximal 0.5 msec pulses delivered chamber was perfused with frog Ringer's through a stimulus isolator unit (WP Instru solution, and the other chambers (both ends) ments, model 305). Most of the techniques were filled with frog Ringer's solution. Drugs employed have been described in detail in in frog Ringer's solution were perfused into previous papers (19, 24). the central chamber via a threeway stopcock The experiments were performed using after a stabilization period for the nerve bundle either a Nicolet 3091 Digital Oscilloscope or a in the bath. Philips PM3305 Digital Oscilloscope. The The nerve bundle was stimulated, and the records were stored on floppy disks and later action potentials were recorded at 2, 5, 10, 20, analyzed, and pictures of action potentials 30 and 60 min for the first hour and ever 30 were drawn using a Compaq Deskpro 286 min thereafter. The nerves were exposed to computer and a Roland DG PR-1212A printer. drugs and tested for 4 hr. The means of the All programs for transmitting, storing and effects recorded at each time were compared analyzing data were written in Turbo Pascal. Resting and action potentials were recorded poor after a 4-hr drug exposure even usually from six to eight or more surface fibers with low concentrations. As can be seen in over short time periods (about 5 min). After Fig. 3, stable, equilibrium drug effects had not control readings were obtained, the tissues were exposed to the drug at time 0 and re cordings of the electrical responses obtained about every 30 min thereafter. The drugs were dissolved in frog Ringer's solution for intracellular microelectrode ex periments. Solutions and drugs: The composition of the frog Ringer's solution was as follows: 111.87 mM NaCI, 2.47 mM KCI, 1.08 mM CaCI2, 0.087 mM NaH2PO4, 2.38 mM NaHC03 and 11.1 mM dextrose. For skeletal muscle experiments, d-tubocurarine (0.1 mg/ Fig. 1. Effects of ketamine applied extracellularly by ml) was added to the Ringer's solution. perfusion on action potentials of a frog's sciatic nerve. The isotonic sucrose solution contained 214 In A and B are two separate experiments using two mM sucrose, and the IsoKCI solution con small bundles of nerve fibers removed from a single tained 123 mM KCI. The drugs used in this sciatic nerve. The ketamine concentration was 10-2 experiment were ketamine HCI solution M in A and 2.5x 10-4 M in B. Duration of drug exposure listed on the line below the records. (Ketalar®, Parke Davis & Company) or the salt (RBI Research Biochemicals, Inc.) and naloxone HCI (Endo Laboratories). All the solutions were adjusted to pH 7.1 7.2 for sciatic nerve experiments and 7.2-7.4 for skeletal muscle experiments.

Results Effects on frog sciatic nerve: Before testing the effects of ketamine on frog sciatic nerve action potentials, control tests lasting 4 hr without drug were conduced using both the single gap method (4 experiments) and the double sucrose gap method (9 experiments). In these control experiments, only small de creases in the action potential amplitude oc curred over the 4 hr. These averaged 9.3± 2.74% for the double and 7.5±3.24% for the Fig. 2. Dose-response curves for the effects of single sucrose gap method. The initial size of ketamine on the size of sciatic nerve action potentials. the action potentials recorded averaged Results obtained at the end of 4-hr drug exposure. (mean±S.E.) 71.7±3.0 mV for the double •, extracellular drug application. Number of separate sucrose gap and 58.4±3.0 for the single experiments to obtain each mean and standard error sucrose gap. of the mean were with increasing concentrations: The effects of two concentrations of 5, 6, 5, 5, 3; and 0, intracellular drug application. Number of experiments per point were: 3, 3, 3, 4, 4. ketamine applied extracellularly on sciatic The means±S.E. of the control action potential nerve action potentials are shown in Fig. 1. amplitudes for the extracellular drug applications The higher concentration not only depressed were 68.8±3.6 mV, 63.4±7.5 mV, 55.9±6.8 mV, the amplitude more quickly but it also pro 55.7±4.4 mV and 48.0±7.5 mV; and for the intra longed the action potential duration. Upon cellular drug applications, they were 56.4±4.0 mV, drug removal, recovery was good if the drug 59.8±2.5 mV, 68.2±6.8 mV, 56.2±6.6 mV and exposure lasted one hour or less, but it was 63.1 ±5.5 mV. response curves presented in Fig. 2. There was a dramatic difference in the depression pro duced by ketamine depending upon the method of drug application. When applied extracellularly, a good dose-response relation and at the highest concentration, complete inexcitability was produced by ketamine. However, when applied intracellularly by the single sucorse gap method, ketamine pro duced only small inconsistent responses. In the three tests at 10-2 M ketamine (double sucrose gap), one nerve bundle was completely blocked by 60 min, and the other two were completely blocked by the next 120 min observation. This finding plus the change in the shape of the action potential (Fig. 1) and the divergence of this point from the ex pected s-shaped dose-response curve suggest that another mechanism producing the com plete block is occurring at this higher concen tration. However, this possibility was not further investigated in the present study. Using these results, it was decided to test for opioid effects using extracellular applica tion of 2.5 and 5 X10-4 M ketamine. Since we had previously found that 10-8 M naloxone did not reduce the action potential in frog's Fig. 3. Naloxone antagonism of extracellularly sciatic nerve fiber bundles, but had a good applied ketamine depression of sciatic nerve action opioid antagonist effect (22, 23), this con potentials. Means±standard errors. A, filled symbols, centration was used. As can be seen from the 2.5 x 10'4 M ketamine (n=6); open symbols, results of these experiments presented in Fig. ketamine+10'8 M naloxone (n=8). B, filled symbols, 3, naloxone antagonized the depression pro 5x10'4 M ketamine (n=5); open symbols, ketamine +10-8 M naloxone (n=3). The control action poten duced by both concentrations. In both ex tial sizes (mean±S.E.) were for A, without naloxone: periments, the change produced by naloxone 63.4±7.5; with naloxone: 67.4±4.8; and for B, was statistically significant from 120 min on. without naloxone: 55.77±4.4; with naloxone: Effects on sartorius muscle action poten 61.1 ±6.1. tials: The effects of ketamine were examined in 27 separate experiments using concentrations from 10-4 to 10-3 M. The changes produced been achieved by 4 hr which was the after a relatively short drug exposure (15 to 40 length of the experiments. There was some min) to 4X10-4 M ketamine can be seen in continued decline in the action potential Fig. 4A. Ketamine caused a decrease in the amplitude in the control experiments, but action potential amplitude, maximum rate of this was not large enough to account for the rise, and maximum rate of fall and an increase decline in the presence of ketamine. For ex in the initial amplitude of the negative after ample, in the control experiments with the potential. As previously reported by Marwaha double sucrose gap method, the action poten (15), we observed no consistent effect on the tial decreased 3.8% between 180 and 240 resting potential; in some experiments, it went min, but with ketamine in the two experiments up a few mV and in the others, it decreased a in Fig. 3, it decreased 9.4% in A and 8.2% in B. few mV (<10 mV). The results obtained after 4-hr drug ex With 10-3 M ketamine (n=1), the muscle posures were used to construct the dose became inexcitable by 30 min of exposure to the drug. With 6x10-4 M (n=2) and 7x10-4 M (n=3), the muscles became inexcitable usually in 60 to 90 min. With 10-4 M (n=1) and 3x10-4 M (n=1), the muscles did not become inexcitable for up to 5 hr of con tinuous drug exposure. With 5 x 10-4 M ketamine (n=12), the results were incon sistent, being variable depending upon the time of year and the supply of frogs. Consistent results were obtained by using 4x10-4 M ketamine and frogs received in a single shipment from a supplier. It was pointed out some time ago that the maximum rate of rise of the action potential is proportional to the sodium permeability of the membrane during the rising phase of the action potential (24, 25). The effects of ketamine, 4x10-4 M, on this measurement are plotted in Fig. 5. When 10-8 M naloxone was added to the drug solution bathing the muscle, the block Fig. 4. Effects of ketamine and ketamine plus produced by ketamine was antagonized. In naloxone on intracellularly recorded action potentials another similar experiment with naloxone, the of a frog's sartorius muscle. A, control; B, 23.5 min action potential records were lost due to after putting 4x10-4 M ketamine in the bath. At 30 equipment failure, but inexcitability occurred min, 10_8 M naloxone added to the bath. C, 60 min; only after 183 min. and D, 269 min. Upper trace shows the potential of the external solution. Lower trace shows the intra Discussion cellular potential with a stimulus artifact followed after a delay by an action potential. The results obtained in the present study

Fig. 5. Naloxone antagonism of the skeletal muscle action potential depression and block produced by ketamine. Filled symbols, means±standard errors for three experiments with 4 x 10-4 M ketamine. Open symbols, three separate experiments with 10'8 M naloxone added at 30 min. Ordinate, action potential maximum rate of rise. The control mean±S.E. values for the maximum rate of rise for the three experi ments without naloxone were 531 ±38.2 V/sec (n=8), 551 ±33.8 V/sec (n=8) and 460±36.9 V/sec (n=11); and for the three with naloxone: 310±4.5 V/sec (n=6), 246±11.9 V/sec (n=8) and 493±28.2 V/sec (n=12). show that under carefully controlled con opioid agonist effects which are produced by ditions, ketamine can depress excitability by a agonist concentrations of 10-6 M or less. stereospecific opioid effect in frog sciatic This difference may be due to a lower equili nerve (Fig. 3) and skeletal muscle fibers (Fig. brium concentration of the opioid agonists at 5). This conclusion is based on the an their intracellular site of action in axons and tagonism produced by a low naloxone con skeletal muscle. However, this explanation centration, and these results are similar to the would not be in harmony with the observed findings obtained with many opioid drugs in antagonist effect of very low naloxone con axons and skeletal muscle (17). It would have centrations. It has been suggested that some been nice to compare the effects of the polypeptide other than the opioid peptides is stereoisomers of ketamine on excitability, but the natural ligand for these receptors (23). If unfortunately these were not available to us at this is the case, these receptors might well the time of this study. have a lower affinity for opioid drugs than It is clear also that ketamine is depressing possessed by 'true' opiate receptors. excitability by at least one or more other Finally, the observation that ketamine was mechanisms. This is shown by the inability of effective only when applied extracellularly by naloxone to completely antagonize the de the double sucrose gap method (Fig. 2) was pressant effect of ketamine and by the opposite to previous findings with opioid actions of high ketamine concentrations peptides (23). This would support the sug which produced a rapid block of excitability. gestion made in the latter paper that the This action was only accelerated by the ad peptides do not reach the portion of the dition of 10-8 M naloxone. These high ket axoplasm in the central chamber of the amine concentrations also modified the action sucrose gap by simple diffusion when applied potential differently (Fig. 1) and seem to to the cut end of the nerve, but they are carried diverge from its expected position on the there by a special transport system. In any dose-response curve (Fig. 2). Again this is event, it has been a general finding in our similar to the non-peptide opioid drugs which laboratory that opioid peptides are effective were shown to block excitability at higher only when applied intracellularly, and the concentrations by a local anesthetic-like non-peptide opioids are effective only when effect (17, 19, 22). The mechanism(s) for the applied extracellularly to vertebrate axons. non-opioid depressant effects of ketamine However, extracellularly applied ketamine were not investigated in this study. However, (Fig. 3) or morphine (22) requires a con earlier studies (11-14) clearly demonstrated siderably longer time than enkephalins do a local anesthetic-like effect for the higher (23) to produce their naloxone-antagonizable naloxone concentrations when added to effects on action potentials in peripheral ketamine. The large decrease in the maximum nerves. This probably results from the time rate of fall of the intracellularly recorded required for the slow diffusion of the non skeletal muscle action potential produced by peptides through the nerve membrane to reach ketamine (Fig. 4) also is consistent with a their intracellular receptor site on or near the local anesthetic effect (19). Thus we would . This is similar to the results conclude that part of the depression produced obtained in skeletal muscle with many non by ketamine is due to a local anesthetic effect. peptide opioids (17, 19, 21, 26). In another respect the results obtained with Acknowledgment: This work was supported by a ketamine were similar to the results obtained grant from the Medical Research Council of Canada. with opioid drugs on excitability in axons and skeletal muscle. 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