John H. Skerritt

Adenosine agonists reduce voltage‐dependent calcium conductance of mouse sensory neurones in cell culture.

Adenosine and several of its analogues produced a concentration‐dependent shortening of calcium‐dependent action potential (c.a.p.) duration of mouse dorsal root ganglion (d.r.g.) neurones in dissociated cell culture. The following rank order of potency was obtained: N6‐(L‐phenylisopropyl)adenosine greater than N6‐(D‐phenylisopropyl)adenosine greater than N6‐cyclohexyladenosine greater than 2‐chloroadenosine much greater than 1‐methylisoguanosine greater than adenosine. Effects of adenosine agonists on c.a.p. duration were blocked by methylxanthine adenosine antagonists. Adenosine monophosphate (AMP) and cyclic AMP shortened c.a.p.s in d.r.g. neurones, while ATP also depolarized cells. Voltage‐clamp analysis revealed that the effect arose from reduction of a voltage‐dependent calcium conductance. Adenosine agonists reduced depolarization‐evoked inward currents but did not alter membrane conductance following blockade of calcium channels by cadmium. Additionally, adenosine reduced the instantaneous current‐voltage slope (chord conductance) during step commands that produced maximal activation of voltage‐dependent calcium conductance. If effects of adenosine on neuronal somata and synaptic terminals are similar, adenosine agonists may inhibit neurotransmitter release in the central nervous system by inhibiting a voltage‐dependent calcium conductance. Since effects of adenosine agonists did not correspond with their relative potencies as modulators of adenylate cyclase activity or inhibitors of neurotransmitter release in peripheral tissues, a novel adenosine receptor may be involved in regulation of this conductance.

Diazepam and its anomalous p-chloro-derivative Ro 5-4864 : comparative effects on mouse neurons in cell culture

The actions of diazepam and its p-chloro-derivative Ro 5-4864 were compared on mouse spinal cord and dorsal root ganglion neurons in cell culture. Diazepam enhanced but Ro 5-4864 reduced iontophoretic GABA responses in a concentration-dependent manner. Both diazepam and Ro 5-4864 limited sustained, high frequency repetitive firing of spinal cord neurons but diazepam was more potent. Ro 5-4864 was, however, more potent than diazepam in inhibiting spontaneous neuronal activity of spinal cord neurons and reducing the duration of calcium-dependent action potentials of dorsal root ganglion neurons. The differing actions of diazepam and Ro 5-4864 may account for the contrasting pharmacological spectra of the two benzodiazepines.

Benzodiazepine Ro 15-1788: Electrophysiological evidence for partial agonist activity

The effects of diazepam and Ro 15-1788 were assessed upon responses of mouse spinal cord (SC) neurons in cell culture to the amino acid neurotransmitters 4-aminobutyric acid (GABA) and S-glutamic acid. Diazepam (100 nM) enhanced GABA responses by 65 +/- 3% (113 cells), while Ro 15-1788 (100 nM) failed to alter GABA responses but reduced their enhancement by diazepam. Higher Ro 15-1788 concentrations (1 microM or 10 microM) enhanced GABA responses to a moderate extent, while blocking further enhancement of GABA by diazepam. Neither diazepam nor Ro 15-1788 affected glutamate responses or resting membrane potential or conductance of spinal cord neurons. These results provide electrophysiological support for partial agonist, rather than pure antagonist, activity of Ro 15-1788.

BENZODIAZEPINE RECEPTOR LIGAND ACTIONS ON GABA RESPONSES, BENZODIAZEPINES, CL 218872, ZOPICLONE

The effects on GABA (4-aminobutyric acid) responses of several benzodiazepine and nonbenzodiazepine benzodiazepine receptor ligands were examined using mouse spinal cord neurons in dissociated cell culture. Diazepam, clonazepam and nitrazepam enhanced GABA responses potently at low nanomolar concentrations. Diazepam and clonazepam were most potent with significant enhancement at 1 nM and peak enhancement of 80.7 and 50.2% at 10 nM respectively. Nitrazepam was least potent with no significant enhancement at 1 nM and enhancement of only 20.7% at 10 nM. The benzodiazepine antagonist, Ro 15-1788, blocked enhancement by diazepam but also weakly enhanced GABA responses at low micromolar concentrations, suggesting partial agonist activity. The convulsant benzodiazepine, Ro 5-4864, did not enhance GABA responses at any concentration tested but antagonized GABA responses at 1 microM and above. Diazepam shifted GABA dose-response curves to the left by decreasing the apparent KD but without altering the apparent Vmax (Lineweaver-Burk analysis). Two nonbenzodiazepine anxiolytic/anticonvulsants, CL 218872 and zopiclone, were weak enhancers of GABA responses at high nanomolar concentrations. These results with benzodiazepines, CL 218872 and zopiclone are consistent with their anxiolytic and anticonvulsant profile in vivo and with studies of their effects upon low affinity GABA binding in vitro.

Benzodiazepine receptor ligand actions on GABA responses. β-carbolines, purines

Abstract The effects of several β-carboline and purine ligands for benzodiazepine receptors were studied upon GABA (4-aminobutyric acid) responses and upon diazepan enhancement of GABA responses, using mouse spinal cord neurons in dissociated cell culture. While the potent convulsant β-carboline DMCM (methyl-6,7-dimethyoxy-4-ethyl-carboline-3-carboxylate), reduced GABA responses, methyl-carboline-3-carboxylate (βCCMe) and the corresponding ethyl ester (βCCEt) did not alter GABA responses. The propyl ester (βCCPr) enhanced GABA responses in a concentration-dependent fashion, while both βCCMe and βCCPr blocked diazepam enhancement of GABA responses. βCCPr may thus have partial agonist activity. Two purines with moderate benzodiazepine receptor affinity, 1-methylisoguanosine (MeIG) and 6-dimethylaminopurine (DMAP), weakly enhanced GABA responses. MeIG also significantly antagonized diazepam enhancement of GABA responses. Inosine and hypoxanthine had no apparent actions upon GABA responses or upon diazepam enhancement of such responses. The results with β-carbolines are consistent with their behavioural profile in vivo and with neurochemical studies of their effects upon GABA-benzodiazepine receptor complexes. Furthermore, certain purines are also able to interact with these complexes.

Diazepam enhances the action but not the binding of the GABA analog, THIP

GABA (4-aminobutyric acid) and its bicyclic analog THIP (4,5,6,7-tetrahydroisoxazolo-[4,5-c]-pyridin-3-ol) produced membrane hyperpolarization and increased chloride ion conductance of mouse spinal cord neurons in cell culture. Above 1 nM diazepam enhanced the actions of both GABA and THIP with similar potency and efficacy. Diazepam has been shown to enhance the binding of [3H]GABA to rat brain membranes over similar concentration ranges, with the EC50 values for enhancement of [3H]GABA binding and increase in membrane conductance being similar. In contrast, binding of [3H]THIP has been shown to be unaltered by diazepam under a variety of conditions. The possible reasons for such a discrepancy between these electrophysiological and neurochemical results with THIP are discussed.

Anticonvulsant drug actions on GABA responses and sustained repetitive firing of neurons in cell culture

Abstract We have studied barbiturate, benzodiazepine (BDZ), β-carboline, triazolopyridazine, piperazine carboxylate, hydantoin, iminostilbene, valproate, and succinmide actions on GABA responses and sustained, high frequency repititive firing of mouse neurons in cell culture. Barbiturates, clinically used BDZ, zopiclone and C1 218,872 enhanced GABA responses. Ro 15-1788 and propyl-β-carboline were partial agonists at BDZ receptors while DMCM was an inverse agonist. Phenytoin, carbamazepine, phenobarbital, diazepam and valproic acid limited sustained repetitive firing. We suggest that enhancement of GABAergic inhibition and limitation of sustained high frequency repetitive firing may be anticonvulsant mechanisms of action.

Differential modulation of γ-aminobutyric acid receptors by caprolactam derivatives with central nervous system depressant on convulsant activity

The effects of a series of caprolactam derivatives with central depressant, convulsant or muscle relaxant activity were investigated upon gamma-aminobutyric acid (GABA) receptor-ionophore binding to rat brain membranes using [3H]GABA, [3H]GABA, [3H]muscimol and [35S]-tert.-butylbicyclophophorothionate ([35S]TBPS) as ligands, and GABA responses in mouse spinal cord neurones in dissociated cell culture. Some caprolactams produced a picrotoxin-like chloride-dependent partial inhibition of muscimol binding and were potent inhibitors of TBPS binding. One compound that was further investigated (4,4,6,6-tetramethylhexahydro-2H-azepin-2-one), inhibited GABA responses and increased the frequency of paroxysmal depolarizations in cultured neurones. Other caprolactams enhanced muscimol binding and were relatively weak inhibitors of TBPS binding, and one (3,3-diallyl-6,6-dimethylhexahydro-2H-azepin-2,4-dione) was shown to enhance GABA responses and produced quiescence of activity in cultured neurones. There was a direct correlation between caprolactam effects on muscimol binding in the presence of chloride ions and their effects on TBPS binding suggesting a similar site of action for the caprolactams influencing the binding of these two ligands. For the two classes of caprolactams, with respect to inhibition or enhancement of muscimol binding, there appeared to be a relationship between in vitro effects and their convulsant or depressant activity in mice. Caprolactams may be useful low molecular weight probes for the study of GABA receptor-ionophore complexes.