Gaetano Riggio

Inhibition of morphine-induced analgesia and locomotor activity in strains of mice: A comparison of long-acting opiate antagonists

The long-acting opiate antagonistic potency of naloxazone (NXZ), beta-chlornaltrexamine (beta-CNA) and beta-funaltrexamine (beta-FNA) was compared using three inbred strains of mice, in which morphine induces either analgesia (DBA/2), locomotion (C57BL/6), or both responses (C3H/He). The antagonists were applied SC 24-120 hr before morphine (10 or 20 mg/kg, IP), followed by the tests after 30 min. The minimal dose which completely antagonized morphine-induced analgesia in DBA and locomotion in C57 mice during 24 hr were: for NXZ 50 and 100 mg/kg, for beta-CNA 0.8 and 6.2 mg/kg, for beta-FNA 1.6 and 12.5 mg/kg, respectively. beta-FNA and beta-CNA more potently blocked morphine-induced analgesia in DBA mice than the activity response in the C57 strain. In contrast, beta-FNA prevented morphine-induced locomotion at a lower dose (6.2 mg/kg) than analgesia (greater than 50 mg/kg) in C3H mice, while beta-CNA was equipotent (1.6 mg/kg). In general, beta-CNA turned out to be the most reactive compound, antagonizing morphine effects in low doses up to 120 hr. beta-FNA selectively antagonized either morphine-induced analgesia or locomotion, depending on the strain used. This suggests that a given morphine response might be caused by a genetically determined multiplicity of opiate receptor types and their mutual interactions.

Long-term analgesic reaction in attacked mice.

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.

Evaluation of different detection modes for the analysis of procyanidins in leaves and flowers of Crataegus spp. Part II. Liquid chromatography–mass spectrometry

Liquid chromatography–mass spectrometry, using electrospray (ESP) as well as atmospheric pressure chemical ionization (APCI), has been evaluated for the qualitative analysis of (−)-epicatechin and oligomeric procyanidins in reference solutions and in extracts of leaves and flowers of Crataegus spp. Applying the APCI interface, considerable fragmentation took place owing to cleavage of the inter-flavanoid bonds and to retro-Diels–Alder fission of the heterocyclic rings. Fragmentations occurred more extensively in the positive than in the negative ionization mode. Lowering the corona current or the vaporizer temperature did not increase the abundance of the molecular ions, but produced lower sensitivities and higher noise levels. The use of the ESP interface in the negative ionization mode yielded molecular ions as principal signals with all tested compounds. In addition, ions resulting from the cleavage of the inter-flavanoid bonds as well as cluster ions, generally composed of two analyte molecules, could be detected. The pyran ring systems were stable under these mild ionization conditions. The chromatographic analysis of extracts from leaves and flowers of Crataegus spp. revealed the presence of procyanidins up to the tetrameric level. Evidence for the occurrence also of pentamers and hexamers could be found in the chromatograms. The difficulties in interpreting mass spectra of higher molecular mass compounds as well as different approaches to the quantitative analysis of procyanidins using liquid chromatography–mass spectrometry are discussed. Copyright

Preexposure to a nonaggressive opponent prevents low-intensity, social conflict analgesia in mice.

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.

Blockade of acetylcholine synthesis in organophosphate poisoning

In spite of worldwide research efforts in the search for the treatment of organophosphate poisoning, the substances with practical antidotal capabilities remain to be discovered. This problem has generally been approached by attempting to reactivate the inhibited acetylcholinesterase. Our approach consisted of reducing the amount of the lethal agent acetylcholine by blocking its synthesizing enzyme cholineacetylase with methyl methane thiol sulfonate (MMTS). We have taken into consideration that we are dealing with acute toxicological problems. This applies for poisoning as well as for treatment, and therefore in the present stage we can only present minimal results. The time from sarin (2 mg/kg) injection to death in rats (controls) was 2:59 min. With a MMTS dosage of 133.5 mg/kg prior to sarin, it was prolonged to 20:55 min (p less than 0.01). With the same dosage of MMTS under identical conditions, the time from soman (2 mg/kg) injection to death was prolonged from 6:08 to 14:48 min (p less than 0.01). Although MMTS cannot be used as a therapeutic agent, our attempt has demonstrated a utility in treating organophosphate poisoning in mice and rats and points in a direction where further work might be fruitful.

Synthesis of specific cholinergic inhibitors for affinity chromatography

Our first aim was to simplify the spacer-synthesis for affinity-chromatography of cholinergic proteins. Further-more we synthesized 2 new inhibitors which proved to be useful for purification of acetylcholine-receptor protein.

Cholinergic Receptor Isolation

The isolation of specific cholinergic bindin protein (acetylcholine receptor = AChR) has been achieved successfully by numerous research groups (1-4, 7-9, 11, 12). The ideal source for such preparations was the electric organ of several species of Torpedo. All successful preparations notwithstanding, there are still some serious problems waiting to be solved. The first problem is the stability of AChR. Compared to acetylcholinesterase (AChE) which can be kept at room temperature for several days without losing enzymatic activity, the AChR is less stable. Regarding stability as shown by immunological properties only, the AChR is remarkably stable. Even methods such as freeze drying of the electric organ or protein fractions during preparation, binding to animal toxins (mostly snake venoms) and application of drastic methods to release it from affinity columns do not interfere with the immunological properties. Regarding stability in terms of pharmacological properties, however, the AChR is very unstable and continuously loses its binding properties during isolation and over the course of time. It is still an open question whether a free isolated receptor in solution will ever behave like a receptor in its natural environment embedded in membranes. Some factors which might interact with this very sensitive protein are 1) ambient oxygen, 2) proteolytic enzymes (a number of proteolytic enzymes are liberated in the course of isolation), 3) microbes, 4) acetylcholine (ACh) (reasonable amounts of ACh are liberated during preparation), and 5) other agonist type molecules (small molecules with depolarizing ability have been used for affinity chromatography and/or elution from the affinity column.