Subbiah P. Sivam

Effect of Acute and Chronic Cholinesterase Inhibition with Diisopropylfluorophosphate on Muscarinic, Dopamine, and GABA Receptors of the Rat Striatum

Abstract: The effects of acute and chronic administration of diisopropylfluorophosphate (DFP) to rats on acetylcholinesterase (AChE) activity (in striatum, medulla, diencephalon, cortex, and medulla) and muscarinic, dopamine (DA), and γ‐aminobutyric acid (GABA) receptor characteristics (in striatum) were investigated. After a single injection of (acute exposure to) DFP, striatal region was found to have the highest degree of AChE inhibition. After daily DFP injections (chronic treatment), all brain regions had the same degree of AChE inhibition, which remained at a steady level despite the regression of the DFP‐induced cholinergic overactivity. Acute administration of DFP increased the number of DA and GABA receptors without affecting the muscarinic receptor characteristics. Whereas chronic administration of DFP for either 4 or 14 days reduced the number of muscarinic sites without affecting their affinity, the DFP treatment caused increase in the number of DA and GABA receptors only after 14 days of treatment; however, the increase was considerably lower than that observed after the acute treatment. The in vitro addition of DFP to striatal membranes did not affect DA, GABA, or muscarinic receptors. The results indicate an involvement of GABAergic and dopaminergic systems in the actions of DFP. It is suggested that the GABAergic and dopaminergic involvement may be a part of a compensatory inhibitory process to counteract the excessive cholinergic activity produced by DFP.

An assessment of comparative acute toxicity of diisopropyl-fluorophosphate, Tabun, Sarin, and Soman in relation to cholinergic and GABAergic enzyme activities in rats

The sc LD50s (mumol/kg) in rats for diisopropylfluorophosphate (DFP), Tabun, Sarin, and Soman were 14.5, 1.9, 1.4, and 0.88, respectively. The relative potency was as follows: DFP less than Tabun less than Sarin less than Soman (1:7.6:10.4:16.4). The relative potencies correlated with the in vitro acetylcholinesterase (AChE) inhibition (in terms of the IC50) by these compounds, in whole brain homogenates or the purified bovine erythrocyte AChE. There was a dose versus time for mortality relationship for all four compounds; the average time for death decreased with increase in dose. However, there was no correlation between time for death and the extent of AChE inhibition. The striatal as well as other regional (medulla, diencephalon, cortex, and cerebellum) AChE activity was inhibited over 90% of the control, by the lethal doses of these compounds. None of the lethal or sublethal doses had any apparent effect on choline acetyltransferase (CAT) or GABA-transaminase activities. Glutamic acid decarboxylase activity was increased by Soman, Sarin, and Tabun at certain lethal doses but was not affected by DFP even at the lethal dose. The results indicate that (a) the acute toxicity of organophosphate acetylcholinesterase inhibitors is directly related to the inhibition of AChE though there is a wide difference in their potency; (b) a substantial inhibition of AChE activity (over 90% of control) is necessary for lethality to ensue after an acute exposure and the margins between lethal and nonlethal doses are extremely small; and (c) qualitative differences seem to exist among the various organophosphates in affecting noncholinergic neurotransmitter enzymes.

Gabaergic drugs, morphine and morphine tolerance: A study in relation to nociception and gastrointestinal transit in mice

Agonists and antagonists of gamma-aminobutyric acid, i.e. GABAergic drugs, such as muscimol, baclofen or bicuculline, alone or in combination, exhibited analgesic effects per se and enhanced the analgesia induced by morphine. The analgesic effects of GABAergic drugs were unaffected by administration of naloxone in a dose which antagonized the analgesia induced by morphine. The ED50 for the antinociceptive effect of muscimol, bicuculline, picrotoxin, gabaculline or aminooxyacetic acid (AOAA) was not affected in the morphine-tolerant group as compared to the control group, in contrast to the increase in the ED50 for morphine under similar conditions; this indicated that there was no development of cross-tolerance between morphine and GABAergic drugs. Muscimol suppressed the abrupt withdrawal-jumping induced by morphine and enhanced the suppression of this phenomenon by morphine. The GABAergic drugs also shared with morphine the property of inhibiting gastrointestinal (GIT) motility. Naloxone reversed the inhibition of motility induced by morphine but failed to influence that induced by GABAergic drugs. In the morphine-tolerant state, the sensitivity of gastrointestinal motility to morphine decreased, whereas, the sensitivity to GABAergic drugs remained unaltered. The results indicate that GABAergic drugs share some of the classical properties of morphine, such as analgesia and inhibition of gastrointestinal motility, but they probably do so by different mechanisms.

An analysis of GABA receptor changes in the discrete regions of mouse brain after acute and chronic treatments with morphine

Abstract: The effects of morphine on the affinity and distribution of GABA receptors in the mouse regions (striatum, medulla, diencephalon, cortex, and cerebellum) were investigated in relation to: (a) acute administration, (b) chronic administration (tolerance), (c) precipitated withdrawal by naloxone, an opiate antagonist, and (d) abrupt withdrawal for 8 and 24 h. The alterations in the affinity as reflected by the dissociation constant (KD) and the number of receptors (Bmax) in the synaptic membranes obtained from controls and various treatments were determined by radioligand binding assay using [3H]muscimol as a ligand. Significant changes were observed in striatum, medulla, and diencephalon, whereas other regions including whole brain exhibited marginal changes. In general the number of GABA receptors increased after tolerance development, which upon abrupt withdrawal returned to control levels except in the case of naloxone‐induced precipitated withdrawal. The affinity changes in different regions were diverse in nature and were not evident in the whole brain membranes. These results indicate that: (a) the regional alterations in the affinity and distribution of GABA receptors may play a role in the induction, maintenance, and regression of morphine tolerance; (b) abrupt withdrawal and antagonist precipitated withdrawal affect the GABA system differently, (c) chronic morphine treatment appears to influence the GABA receptors in the cerebellum, a region generally known for its lack of opiate receptors.

Development of dispositional tolerance to phencyclidine by osmotic minipump in the mouse

Development of tolerance to phencyclidine (PCP) was assessed in male ICR mice, using motor incoordination as a parameter. The implantation of a PCP (1-3 mg/day/mouse for 1-5 days)-containing osmotic minipump, induced tolerance, as evidenced by a gradual reduction of the duration of motor incoordination. The degree of tolerance exhibited dose and time dependency. Even after the removal of the PCP pump (1 mg/day/mouse for 5 days), the tolerance remained to the same degree for at least 4 days. The hepatic microsomal cytochrome P-450, cytochrome b5 and nicotinamide adenine dinucleotide phosphatase (NADPH)-cytochrome c reductase activities were found to be elevated in tolerant mice (2 mg/day/mouse for 5 days). The half-life of PCP in the brains of tolerant mice was likewise decreased. These data indicate a dispositional tolerance for PCP. It appears that the administration of PCP by the osmotic minipump offers a convenient method for inducing PCP tolerance.

Alterations of synaptic high and low affinity opiate binding sites after acute and chronic morphine administration in mice

1. The opiate receptor binding to whole brain synaptic membranes obtained from mice subjected to acute and chronic morphine administration and precipitated withdrawal was investigated. 2. The number of high and low affinity binding sites was significantly higher in morphine tolerant group than in control; acute administration of morphine also induced an increase in the number of binding sites, though this increase was significantly lower as compared to tolerant group. 3. The affinity of both high and low affinity sites, however remained unchanged after acute or chronic treatment. In contrast, both the affinity and the number of binding sites were significantly reduced after precipitated withdrawal, compared to the tolerant group. 4. Sodium chloride enhanced the antagonist binding and inhibited the agonist binding in both tolerant and non-tolerant groups. 5. It is concluded that (a) the increase in the number of receptors during tolerance development is an extension of the acute effect of morphine, (b) the character of tolerant membrane is qualitatively same as that of non-tolerant membrane, (c) upon withdrawal, the receptor population is brought back to normal; the altered higher, affinity after withdrawal may be a compensatory effect as a corollary to the withdrawal-induced decrease in receptor population.

Analgesic cross-tolerance between morphine and opioid peptides.

The analgesic effect, of intracerebroventricular administration of morphine, ketocyclazocine, [D-ala2]-methionine enkephalinamide (DAM), [D-ala2-D-leu5]-enkephalin. (DADLE), leuenkephalin, metenkephalin, and β-endorphin on acetic acid-induced abdominal writhing (AAW) was investigated in naive and morphine-tolerant mice. It was found that the relative potencies of a series of opioids are different in naive and morphine-tolerant groups. In naive animals, the order of potency (ED50, nmol) was β-endorphin > morphine=DAM > DADLE > ketocyclazocine=leuenkephalin=metenkephalin. The morphine-tolerant animals were cross-tolerant to ketocyclazocine and to all the peptides studied; DAM and β-endorphin exhibited the highest degree of tolerance. In morphine-tolerant animals, the order of potency was morphine=DADLE=β-endorphin > DAM=ketocyclazocine =metenkephalin > leuenkephalin. The results indicate that endogenous opioid systems may be affected by tolerance development to morphine.

Antinociceptive and gastrointestinal effects of opiates: an analysis of the nature of the involvement of mu and delta receptors of the central nervous system in morphine-tolerant and non-tolerant mice

This study attempted to distinguish between mu (morphine) and delta [(D-Ala2-D-Leu5)-enkephalin; DADLE] receptors, with regard to both in vivo effects (analgesia and gastrointestinal motility) and the location of binding activity in the brain. Analgesia and motility are distinguishable both by dose (intracerebroventricular) and by ligand selectivity with mu ligands more potent for the former and delta for the latter. Tolerance and cross-tolerance are exhibited for both effects, with the relationships between mu and delta ligand potencies preserved. In vitro receptor binding revealed an affinity decrease for delta in medulla and an increase in medulla and diencephalon for mu receptors after tolerance development to morphine. The results indicate that the mu receptors in medulla and diencephalon mediate analgesia, while medullary delta receptors control motility.

INTERACTION BETWEEN MORPHINE AND PHENCYCLIDINE: INTOXICATION AND CROSS-TOLERANCE

The LD50s of PCP were estimated to be 74.0 mg/kg, 8.5 mg/kg and 70.0 mg/kg in control, acute morphine (30 mg/kg, s.c.) and tolerant groups, respectively. Acute morphine administration potentiated the PCP induced motor incoordination in a dose dependent fashion; the degree of suppression of ambulation and rearing also was enhanced. In contrast, PCP induced motor incoordination was attenuated in morphine tolerant animals, as evidenced by shortening of the recovery time and a decrease in duration of motor incoordination. A similar attenuation of the effect of PCP on ambulation and rearing also was observed. On the other hand, in PCP tolerant mice, the ED50 of morphine sulfate in tail flick test or acetic acid-induced writhing test was higher than that of the controls. Morphine-induced ambulation was attenuated in PCP tolerant group. These results suggest that acute morphine administration enhances PCP effects and toxicity and there is cross-tolerance development between PCP and morphine.