Dennis D. Johnson

Studies on the mechanism of the protective and antidotal actions of diazepam in organophosphate poisining

The effect of diazepam on soman-induced bradycardia and respiratory depression in rabbits has been investigated. Diazepam prevents the bradycardia produced by soman in conscious rabbits. Although atropine will reverse the respiratory depression produced by soman in anesthetized rabbits, diazepam enhances the depression and renders rabbits less susceptible to this action of atropine.

Reduction by diazepam of repetitive electrical activity and toxicity resulting from soman

Abstract The organophosphate cholinesterase inhibitor soman (methyl pinacolylphosphufluoridate) produces potentiation of twitch tension in rat anterior tibial and phrenic nerve-diaphragm preparations by initiating repetitive electrical activity in the vicinity of the motor nerve terminals. Diazepam abolished this repetitive electrical activity and hence the associated potentiation of muscle twitch tension. These effects are produced by concentrations of iazepam that are without other apparent effects on neuromuscular transmission, as indicated by the absence of effect on normal twitch responses. The combination of diazepam with atropine provides additional protection against soman toxicity in rabbits.

A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays

Two commonly used procedures for removing endogenous GABA from brain homogenates were evaluated by measuring residual GABA using high performance liquid chromatography (HPLC). The effect of these treatments on [3H]muscimol binding to the GABA receptor was also determined. Membranes subjected to osmotic lysing and eight washes with Tris-citrate buffer contained significant quantities of residual GABA whereas lysing and incubation with Triton X-100 followed by three buffer washes resulted in GABA levels below the limits of detection. The apparent affinity for [3H]muscimol was significantly higher in the Triton X-100 treated membranes and this was probably a result of the lower amount of GABA present in these membranes. The effect of Triton treatment or buffer washing on residual levels of glutamate, glutamine, aspartate, and taurine were also determined.

Experimental Febrile Convulsions in Epileptic Chickens: The Anticonvulsant Effect of Elevated γ-Aminobutyric Acid Concentrations

Summary: The high seizure susceptibility in epileptic chickens is due to an autosomal recessive mutation. In 3‐day‐old chicks homozygous for the epilepsy gene (epileptics), elevation of body temperature using microwave diathermy evoked an initial febrile seizure resembling the clonic seizures evoked in epileptic chicks by photic stimulation. After complete recovery, this was followed by a clonic‐tonic seizure. In nonepileptic heterozygote hatchmates (carriers) of the same age, only the latter seizure pattern was observed. In 16‐ to 17‐day‐old chicks of either phenotype, both seizure patterns were observed during hyperthermia. In all cases, the temperature at which seizures occurred was significantly lower in epileptic than in nonepileptic chicks, indicating a lower threshold for febrile seizures when there is an inherited predisposition to convulse. The occurrence of seizures was dependent on the body temperature and not on the rate of rise of temperature. Elevation of the brain γ‐aminobutyric acid (GABA) concentrations by administration of the GABA transaminase inhibitor γ‐vinyl GABA reduced the incidence of the initial febrile seizures and increased the latency in those birds that were not fully protected.

Phorbol ester inhibits myoblast fusion and activates β-adrenergic receptor coupled adenylate cyclase

Primary cultures of myoblasts, derived from embryonic chick pectoral muscle, were treated with phorbol ester (TPA) for 8–96 h. TPA treatment blocked the fusion of myoblasts along with the expression of the MM form of creatine kinase. Interestingly, TPA treatment markedly increased the activity of β‐adrenergic receptor coupled adenylate cyclase (AC) activity. The study suggests that TPA treatment augments the functional interaction between a coupling Ns protein and catalytic unit of AC. The likely significance of these results is briefly presented.

Pharmacology of methyl- and propyl-β-carbolines in a hereditary model of epilepsy

Abstract Intravenous administration of β-carboline-3-carboxylate methyl ester (β-CCM) produced convulsions at small doses (0.03 mg kg ) in adult chickens, homozygous for the epileptic gene. Non-epileptic heterozygote hatchmates (carriers) did not undergo seizures at doses of 1 mg kg , and doses of 3–5 mg kg produced only brief myoclonic responses. The convulsant effect of β-CCM could be prevented by pretreatment with large doses of β-carboline-3-carboxylate propyl ester (β-CCP). β-Carboline-3-carboxylate methyl ester displayed a higher affinity than diazepam in displacement studies on synaptosomal membrane preparations from brains of epileptic and carrier chickens.

The Relationship Between Anticonvulsant Activity and Receptor Affinity of N-methyl-D-aspartate Antagonists in Epileptic Fowl

The N-methyl-D-aspartate (NMDA) receptor antagonists [(3-(+/-)2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP), +/- 2-amino-7-phosphonoheptanoic acid (2AP7), +/- 2-amino-5-phosphonovaleric acid (2AP5), D-alpha-aminoadipic acid (alpha AA), and +/- alpha, epsilon-diaminopimelic acid (DAP)] were tested for anticonvulsant activity in epileptic chickens. There was a high correlation between anticonvulsant potencies (ED50) and the affinity for the NMDA receptor measured by displacement of L-[3H]glutamate from synaptosomal membranes. The high seizure susceptibility is not due to abnormalities in the NMDA receptor as comparison of KD, Bmax and Ki values in synaptosomal preparations from epileptic and non-epileptic chickens indicated no differences in NMDA receptor binding receptor characteristics.

Protection by GAB A Agonists, γ‐Hydroxybutyric Acid, and Valproic Acid Against Seizures Evoked in Epileptic Chicks by Hyperthermia

Summary: With microwave diathermy, febrile seizures were produced in epileptic chicks aged 2–5 days. Drugs that enhance GABAergic activity (i.e., GABA, muscimol, and progabide), as well as valproic acid and γ‐hydroxybutyric acid, produced dose‐dependent increases in latency to onset of seizures.

Quisqualate receptors in epileptic fowl: the absence of coupling between quisqualate and N-methyl-D-aspartate receptors

The ability of excitatory amino acid receptor agonists, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) and quisqualate to produce seizures was determined in 1-2 day old epileptic and non-epileptic (carrier) chicks. Both compounds produced prolonged clonic seizures in epileptic chicks at doses which were not convulsant in carrier chicks. Seizures produced in epileptics by AMPA were suppressed by the quisqualate antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione), but were not prevented by pretreatment with competitive (2-amino-7-phosphonoheptanoic acid, APH) or non-competitive (MK-801) NMDA (N-methyl-D-aspartate) receptor antagonists. These data do not support the hypothesis that NMDA receptors work in concert with quisqualate receptors. Binding sites for [3H]AMPA were characterized in cerebral hemispheres of both epileptic and carrier chicks. Analysis of the data revealed no significant alterations in the binding affinity (KD) or the number of binding sites (Bmax) of AMPA to tissue preparations from epileptic chickens when compared to carriers. The latter data does not explain the increased susceptibility of epileptic fowl to the convulsant effects of quisqualate and AMPA.

Benzodiazepine antagonist Ro 15-1788 (flumazepil) attenuates the anticonvulsant activity of diazepam in epileptic fowl

The ability of the imidazobenzodiazepine Ro 15-1788 to displace diazepam from brain membranes in vitro and to antagonize the anticonvulsant activity of diazepam in vivo was determined in epileptic fowl. At doses of 1.0 mg/kg and higher, Ro 15-1788 significantly attenuated the anticonvulsant action of diazepam (1.0 mg/kg) in epileptic chickens. Ro 15-1788 alone exerted no anticonvulsant activity even in doses as high as 10 mg/kg. Specific binding of 10 nM [3H]diazepam to whole homogenate fractions prepared from cerebral hemispheres of epileptic fowl was inhibited by Ro 15-1788 with an IC50 of 8.5 nM and the Ki was determined to be 4.25 nM. These results suggest that Ro 15-1788 competes directly with diazepam for a binding site involved in producing anticonvulsant activity.