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Four experiments were designed to characterize long-term analgesic (LTA) reaction in attacked mice. In Experiment 1 we showed that analgesic reaction in DBA mice, induced by the stress of being attacked (30 or 50 bites), is reinstated upon reexposure to seven bites 24 hr later. The magnitude of the LTA response depended on the level of analgesia on Day 1 and was smaller than the original response. In Experiment 2 we showed that LTA was prevented by naloxone or beta-chlornaltrexamine given before exposure (50 bites) on Day 1. Results of Experiment 3 revealed that naloxone or beta-chlornaltrexamine injected before reexposure to seven bites on Day 2 antagonized LTA measured 10 min, but not 1 min, after reexposure. In Experiment 4 we showed that morphine substituted for being attacked on Day 1 failed to produce LTA. We concluded that pain inhibitory mechanisms remain in a state of increased readiness for at least 24 hr after attack stress and that activation of opioid systems is necessary but not sufficient to produce LTA, a response that is only partly sensitive to opioid antagonists.

In a first experiment, exposure of DBA/2 mice to a small number of attack bites by a C57BL/6 mouse resulted in low-intensity analgesia as assessed by the tail-flick test. The analgesia dissipated within 10 min and was insensitive to naloxone (10 mg/kg, sc) but was antagonized by the irreversible opioid antagonist beta-chlornaltrexamine (5 mg/kg, sc). In a second experiment, preexposure to a nonaggressive C57BL/6 opponent prevented low-intensity analgesia induced by a small number of attack bites 24 hr later. The preexposure effect was abolished by naloxone (10 mg/kg, sc) given before the nonaggressive confrontation. This suggests that the release of endogenous opioids during preexposure interferes with the subsequent activation of endogenous opioid-mediated pain control mechanisms.

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SummaryAffinity chromatography of proteins requires a ligand covalently bound to a solid support separated by a spacer of sufficient length. In the specific case of acetylcholinesterase we have reduced the conventional spacer synthesis from five to three steps.For affinity chromatography of cholinergic proteins the ideal ligand would be acetylcholine which, however, could not be used because it is easily hydrolyzed. We synthesized hydrolysis-resistent ligands. Different specific ligands were synthesized for the affinity chromatography of serum esterase.Affinity chromatography has achieved great importance in the last decade for the purification of biologically active proteins [l-31. In this method a ligand bound covalently to a solid support creates an affinity for the desired protein. As proteins generally have large molecular volumes, the ligand must not be too closely bound onto the support. Cuatrecasas [ 11 first mentioned the importance of a 'spacer'. In the case of acetylcholinesterase, from both theoretical and practical experience, the ligand must be bound to a spacer of 45-58 A of length to achieve optimum interaction with the active centre of the enzyme. The definition 'affinity chromatography' has been used differently by several authors. Unspecific elution of the desired protein with increasing ionic strength of buffer solutions after more or less specific adsorption on a support has also been defined as 'affinity chromatography' [4] [5]. We use the term affinity chromatography to mean specific adsorption and subsequent specific desorption chromatography.An adequate support for affinity chromatography is agarose'). Cuatrecasas [ 11 has already described in detail the spacer synthesis on agarose. Generally the following sequence leads to good results. Bromocyano-activation of agarose, coupling with a diamine, elongation with succinic anhydride, another treatment with diamine catalyzed by water-soluble carbodiimide, again elongation with succinic anhydride, and finally, carbodiimide-catalyzed coupling of the ligand.