mechanism of action

Phencyclidine which is chemically similar to ketamine was introduced into clinical practice in 1958. Although phencyclidine proved useful as an anaesthetic, it produced severe adverse psychological effects in the recovery period. Ketamine, then named C1581, is one of 200 phencyclidine derivatives which were investigate subsequently and proved to be the most promising. It was synthesized as Ketalar in 1962, first used on American soldiers during the Vietnam War and released for civilian use in 1970. It was originally hoped that ketamine would be used as a sole agent for anaesthesia, inducing analgesia, amnesia, loss of consciousness, and immobility. However, because of its adverse psychological effects and the availability of other induction agents, its use diminished rapidly. Recently, the availability of S-(þ)-ketamine has regenerated interest in its clinical use, because it has greater potency and fewer side effects. Pharmacology The ketamine molecule [2-(O-chlorophenyl)-2-methylamino cylohexanone] has a molecular weight of 238. The racemic mixture is prepared in a slightly acidic solution (pH 3.5 –5.5), is freely water-soluble, and has a pK a of 7.5. There is a chiral centre with two optical iso-mers (enantiomers) (Fig. 1). Ketamine has a high lipid solubility (5–10 times that of thio-pental) and crosses the blood-brain barrier faster. It undergoes demethylation and hydro-xylation of the cyclohexanone ring. The metab-olites are conjugated and excreted in the urine. Norketamine has 20–30% of the activity of the parent compound. 1 Its other pharmacokinetic attributes are detailed in Table 1. Ketamine acts on the central nervous system (CNS) and has local anaesthetic properties. Its effects are mediated primarily by non-competitive antagonism at the N-methyl-D-aspartate (NMDA) receptor Ca 2þ channel pore. NMDA channel block appears to be the primary mechanism of the anaesthetic and analgesic action of ketamine (at the CNS and also at spinal cord receptors). In addition, it reduces the presynaptic release of glutamate. The S (þ) enantiomer has a three-to four-fold greater affinity for the NMDA receptor than the R(2) form. 2 Other mechanisms of action of ketamine include interaction with opioid receptors, with a preference for mu and kappa receptors; this interaction with opioid receptors is complex. The affinity of ketamine for these receptors is 10 times less than that for the NMDA channel, and it has been confirmed in humans that naloxone does not antagomize the analgesic effects of ketamine. There is also evidence that ketamine has an antagonistic interaction with monoaminergic, muscarinic, and nicotinic receptors. Indeed, …

tion,3 o14,18 or organelle-surface-membrane fusion. 9 These postulates have been strengthened by the finding that chemotactic factors enhance the transmembrane fluxes and exchangeable intracellular pool of Ca2+ 13,16,17,19,25,2, Recently, chemotactic factors have been shown to stimulate the aggregation of rabbit peritoneal 27-29 and human blood 30 '31 PMNs. In this report we examine the influence of factors which modulate intracellular Ca2+ levels on human PMN aggregation.

Chemotactc Factor
The synthetic chemotactic tripeptide formvl-methionvl-leucvl-phen-lalanine (FM LP) was obtained and used as previouslv described." '4 Reagents Cvtochalasin B (Aldrich Chemical Company, Mlilwaukee, Wis.) and FMlLP were dissolved in dimethylsulfoxide. In the final concentrations used in this study (0.029c% or less), the solvent did not influence PMN function. The bivalent cation ionophore A23187 was a generous gift of Dr. Robert Hamill of the Eli Lilly Company, Indianapolis, Indiana. The buffer was a modified Hanks' balanced salt solution containing (mM): NaCl, 130; KCI, 5.5; Na2HPO4, 0.6; NaH2PO4, 0.6; glucose, 10; and tris, 25. For some experiments the phosphate salts were omitted from the buffer. Where indicated, La3+, Mg2+, or Ca2+ were added to the buffers in the form of chloride salts. Chemicals were of reagent grade or better, and buffers were adjusted to pH 7.4 before use.

Neutrophils
Normal human whole blood was centrifuged over Ficoll-Hv-paque discontinuous gradients sl to obtain leukocvte populations containing greater than 96%c PMNs.

Aggregation
PMNs were freed from contaminating enthrocvtes by hvpotonic lysis and then washed and suspended (4600 PMN/;l) in the appropriate buffer. One milliliter of the suspension was placed in a plastic vial and stirred continuously with a magnetic bar. For each experiment, an aggregating substance was added directly to the cell suspension and 25-Ml samples were taken at Y4, ½, 1, 2, 4, 8, and 15 minutes thereafter. Samples were immediatelv diluted in 10 ml of Isoton solution (Coulter Electronics, Hialeah, Fla.) and analyzed with a Coulter Counter, Model ZBI, equipped with a Volume Channelvser II. For each sample, the counter was set to enumerate the concentration of particles greater than 60 fl (called T) and greater than 520 fl (called A). Since human PMNs are approximately 330 fl, T is the total particle concentration and A is the large or aggregated particle concentration. When PMNs aggregate, T falls while A rises. To quantitate these changes, the percentage of large particles (100 X A/T) for each sample and the aggregation index (the mean of the percentage of large particles found ½ and 1 minute after the addition of the aggregating substance minus the pre-addition percentage of large particles) for each experiment was calculated.

Juy 1978
Enzyme Release At 2 and 15 minutes after adding an aggregating substance to the PMN suspension, ½ml samples were taken, chilled in ice, and centrifuged at 4 C; their supernatant fluids were analyzed for lysozyme, ,B-glucuronidase, and lactate dehydrogenase (LDH), as previously described.cz  steadily increasing, monophasic dose-response curve, A23187-induced aggregation progressively increased over 15 minutes and had a biphasic dose-response curve: 10-5 M A23187 induced much less aggregation than did 10-6 M (Text- figure 2). The cause for this biphasic dose-response curve is unknown. However, cells exposed to 10-5 M A23187 showed an 18.5% decrease in mean cell volume, rapidly degranulated (Table 1), and did not aggregate in response to FMLP (not shown). Therefore, high Mean ± SD for four experiments, as percentage of total cellular enzyme t Mean of two experiments, as percentage of total cellular enzyme concentrations of A23187 may interfere with cell-cell adhesiveness either directly or by contracting or degranulating the cells.

Influence of P h on PMN Ago
FMLP-induced aggregation of PMNs suspended in the medium free of phosphates was significantly less than that found for PMNs suspended in the medium containing phosphates (aggregation index, 4.8 ± 0.4 and 9.9 ± 2.0 SEM, respectively, P < 0.005). Adding increasing amounts of phosphate (as pH 7.4 phosphate buffer) to PMN suspensions resulted in increasing enhancement of FMLP-induced aggregation (Text- figure 5).

TIME (MINUTES) AFTER ADDING FMLP
TEXT-FIGURE -Influence of 1 X 106 NI lanthanum chloride on the large particle percentage of PMIN suspension exposed or not exposed to the chemotactic factor FNMLP. The buffer contained 1.4 mM1 Ca2+, 0.7 m1 \Mg2+. and no PO43-.

Influence of Ca2+ and Mg2+ on PMN Aggregation
Both Ca2+ and Mg2+ were required for A23187-and FMLP-induced aggregation (Text-figures 6 and 7); neither bivalent cation was required for La3+-induced aggregation (Text- figure 7). Increases in Ca2+ with (Text- figure 6, upper panel) or without (Text- figure 6, center panel) equivalent increases in Mg2+ led to progressive increases in the magnitude of FMLP-induced aggregation. Increases in Mg2+ above 0.7 mM were not associated with these changes when Ca2+ was held constant at 1.4 mM (Text- figure 6, lower panel).

Influence of Aggregating Substances on Enzyme Rele
The cvtosolic enzvme LDH was not released from the PMN under any of the conditions studied (Table 1). Two aggregating substances, A283187 and FMLP (with cvtochalasin-B-treated cells), induced prominent release of the granule-bound enzymes, ,B-glucuronidase and Iysozvme, whereas two other aggregating substances, La3+ and FMLP (with PMNs not exposed to cvtochalasin B), did not (Table 1). These results suggest that PMNs remain viable during the aggregation experiments. Apparently, aggregation can occur in the absence of prominent degranulation.

Discussion
Chemotactic factors and A23187 induce an influx of extracellular Ca 2 and an increase in the exchangeable pool of Ca2+ in the PMN. 13ff6171925 '26 High levels of extracellular Ca2+,aa,ae cytochalasin B," and possibly extra-w w .
Changes in Ca2+ fluxes and intracellular Ca2+ may also modulate PMN de nulation 915,18.n,u,n and chemotaxis.''202532 If Ca2+ changes do modulate all three cellular responses, then factors influencing the accumulation and fluxes of Ca2+ should have similar influences on each. This is not the case. For instance, concentrations of chemotactic factors above an optimal level inhibit maximal Boyden chamber chemotaxis 6,15 but do not inhibit degranulation6 or aggregation. '231 Second, high concentrations of extracellular Ca2+ inhibit chemotaxis 17,21 and,B-glucuronidase release 14 but enhance Iysozyme release 14 and aggregation (Text- figure 6). Third, cytochalasin B in low concentrations enhances chemotaxis but in high concentrations inhibits chemotaxis33; this agent can only enhance, not inhibit, degranulation -"" and aggregation (Text- figure 1). Fourth, A23187 inhibits chemotaxis 1 but stimulates degranulation 9,13A5,18,22 (Table 1) and aggregation (Text- figure 2). Fifth, at concentrations above 10-4 M, La3+ inhibits chemotaxis,16''9 does not influence degranulation 15 (Table 1), and induces prominent cellular aggregation (Text- figure 3); from 10-6 M to 10-5 M, La'+ does not influence chemotaxis 16,19 but inhibits degranulation 15 and aggregation (Text- figure 4). In these examples, an agent frequently inhibits chemotaxis while stimulating or enhancing degranulation and aggregation. It may be that degranulation or aggregation interfers with chemotaxis. For instance, chemotactic factors, Ca2+, cvtochalasin B, A23187, and La3+, in concentrations which inhibit chemotaxis, induce or promote sustained cellular aggregation. Aggregated cells may migrate poorly. If aggregation is responsible for the inhibition of chemotaxis, then events such as Ca2+ fluxes and accumulations may similarly influence all three PMN responses: aggregation could limit the detection of this stimulation in chemotactic assays.  (Text-figure 7). Having a higher valency but similar ionic radius to Ca2+, La3+ may bind to Ca2+ sites on surface membranes." Our data (Text- figure 3) suggest that, in concentrations of 10-4 M to 10-3 M, La3+ binding may overcome the repulsive forces between cells and allow the formation of intercellular adhesions. At lower concentrations, ie, 10 M to 1O6 M, La3+ binding may block Ca2+ influxes and, thereby, aggregation (Text- figure 4) and degranulation.1' Why chemotaxis is uninhibited by these levels of La3+ 16,19 is unknown.
Mg2+ appears essential for chemotactic-factor-induced and A23187induced aggregation (Text-figures 6 and 7). Since Mg2+ is required for PMN adherence to glass surfaces,'7 it also may be necessary for cell-cell adherence. La3, which does not require Mg2+ to aggregate cells (Text- figure 7), may substitute for Mg2+ in this role.
Although we suggest that intracellular Ca2+ modulates PMN aggregation, as others suggest it modulates chemotaxis and degranulation, we are aware that the available evidence does not prove this. Thus, chemotactic factors and A23187 stimulate Ca2+ efflux, K+ efflux, Na+ influx and efflux,25 "' and, perhaps, the transmembrane fluxes of other unmeasured and unidentified species. Any of these events may be important in modulating PMN function. Moreover, the aggregative response occurs before the changes in ionic fluxes and accumulations. Hence, ionic fluxes and accumulations may be epiphenomena reflecting surface membrane changes or other events which are more closely related to cell function. This area requires further study.