Uffe Kristiansen

Activation of single heteromeric GABAA receptor ion channels by full and partial agonists

The linkage between agonist binding and the activation of a GABAA receptor ion channel is yet to be resolved. This aspect was examined on human recombinant α1β2γ2S GABAA receptors expressed in human embryonic kidney cells using the following series of receptor agonists: GABA, isoguvacine, 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP), isonipecotic acid, piperidine‐4‐sulphonic acid (P4S), imidazole‐4‐acetic acid (IAA), 5‐(4‐piperidyl)‐3‐isothiazolol (thio‐4‐PIOL) and 5‐(4‐piperidyl)‐3‐isoxazolol (4‐PIOL). Whole‐cell concentration–response curves enabled the agonists to be categorized into four classes based upon their maximum responses. Single channel analyses revealed that the channel conductance of 25–27 pS was unaffected by the agonists. However, two open states were resolved from the open period distributions with mean open times reduced 5‐fold by the weakest partial agonists. Using saturating agonist concentrations, estimates of the channel shutting rate, α, ranged from 200 to 600 s−1. The shut period distributions were described by three or four components and for the weakest partial agonists, the interburst shut periods increased whilst the mean burst durations and longest burst lengths were reduced relative to the full agonists. From the burst analyses, the opening rates for channel activation, β, and the total dissociation rates, k−1, for the agonists leaving the receptor were estimated. The agonist efficacies were larger for the full agonists (E∼7−9) compared to the weak partial agonists (∼0.4–0.6). Overall, changes in agonist efficacy largely determined the different agonist profiles with contributions from the agonist affinities and the degree of receptor desensitization. From this we conclude that GABAA receptor activation does not occur in a switch‐like manner since the agonist recognition sites are flexible, accommodating diverse agonist structures which differentially influence the opening and shutting rates of the ion channel.

GABA-A Receptor Ligands and their Therapeutic Potentials

The GABA(A) receptor system is implicated in a number of neurological diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP or isoguvacine. Using recombinant GABA(A) receptors, functional selectivity has been shown for a number of compounds such as the GABA(A)agonists imidazole-4-acetic acid and THIP, showing highly subunit-dependent potency and maximal response. In the light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogues of 4-PIOL, a low-efficacy partial GABA(A) agonist, have been performed. In this connection, a series of GABA(A) ligands has been developed showing pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effect. Only little information about direct acting GABA(A) receptor agonists in clinical studies is available. Results from clinical studies on the effect of the GABA(A) agonist THIP on human sleep pattern shows that the functional consequences of a direct acting agonist are different from those seen after administration of GABA(A) receptor modulators.

GABAA and GABAB receptor agonists, partial agonists, antagonists and modulators: design and therapeutic prospects

Abstract A large number of highly selective GABAA and, more recently, GABAB receptor ligands have been developed and used for receptor characterization. Whereas full agonists and antagonists at GABAA receptors, for different reasons, may be difficult to use therapeutically, partial GABAA agonists may have therapeutic interest. The efficacious partial GABAA agonist, THIP, shows analgesic and anxiolytic effects in man, but THIP is ineffective as an antiepileptic agent, and PET studies have disclosed that THIP increases glucose metabolism in epileptic patients and human volunteers. In principle, GABAA antagonists may be used therapeutically in Alzheimers disease and schizophrenia, but low-efficacy partial GABAA agonists may have particular interest in these disorders. Using the nonannulated THIP analogue, 4-PIOL, as a lead, a series of partial GABAA agonists showing a broad spectrum of relative efficacies have been developed.

Electrophysiological studies of the GABAA receptor ligand, 4-PIOL, on cultured hippocampal neurones.

1 Whole‐cell, patch‐clamp recordings from cultured hippocampal neurones have been used to characterize the action of the GABAA ligand, 5‐(4‐piperidyl)isoxazol‐3‐ol (4‐PIOL). The action of 4‐PIOL was compared with that of the established GABAA agonist, isoguvacine. 2 With a symmetrical Cl−gradient across the membrane and a holding potential of −60 mV, both isoguvacine and 4‐PIOL evoked an inward current. The reversal potentials of the responses to both agents were identical (+8.8 mV, n = 4) and the current/voltage relationships showed outward‐going rectification. 3 The response to 300 μm 4‐PIOL was completely blocked by the GABAA antagonist, bicuculline methobromide (BMB, 10 μm). The pA2 of BMB was >6.46. With 2 mm 4‐PIOL about 15% of the response remained in the presence of 100 μm BMB. This may represent a non‐specific component of the response to large concentrations of 4‐PIOL. 4 4‐PIOL was about 200 times less potent as an agonist than isoguvacine. Because of the rapid fade (desensitization) of isoguvacine‐induced currents, the maximum response to this agonist was not determined. However, the response to 2 mm 4‐PIOL was only a small fraction of that evoked by submaximal concentrations of isoguvacine. 5 Setting the response to 1 mm 4‐PIOL as maximum, the EC50 for 4‐PIOL was 91 μm (95% confidence limits: 73–114 μm). 6 4‐PIOL antagonized the response to isoguvacine with a parallel shift to the right of the dose‐response curve. The antagonist action of 4‐PIOL was about 30 times weaker than that of BMB. When allowance was made for the intrinsic agonist action of 4‐PIOL, the Ki was 116 μm (95% confidence limits: 102–130 μm). This was not significantly different from EC50 (P = 0.86; non‐parametric Mann‐Whitney test). 7 It is concluded that 4‐PIOL is a partial agonist at the GABAA receptor on cultured hippocampal neurones.

Cholinergic degeneration is associated with increased plaque deposition and cognitive impairment in APPswe/PS1dE9 mice.

Cholinergic dysfunction and deposition of plaques containing amyloid β-peptides (Aβ) are two of the characteristics of Alzheimers disease. Here, we combine APPswe/PS1dE9 (APP/PS1) mice with the cholinergic immunotoxin mu p75-saporin (SAP) to integrate partial basal forebrain cholinergic degeneration and the neuropathology of APP/PS1 mice. By 6 months of age, APP/PS1 mice and wild type littermates (Wt) received intracerebroventricular injection of 0.6 μg SAP (lesion) or PBS (sham). Two months following surgery, APP/PS1 mice treated with SAP were significantly impaired compared to sham treated APP/PS1 mice in a behavioural paradigm addressing working memory. Conversely, the performance of Wt mice was unaffected by SAP treatment. Choline acetyltransferase activity was reduced in the hippocampus and frontal cortex following SAP treatment. The selective effect of a mild SAP lesion in APP/PS1 mice was not due to a more extensive cholinergic degeneration since the reduction in choline acetyltransferase activity was similar following SAP treatment in APP/PS1 mice and Wt. Interestingly, plaque load was significantly increased in SAP treated APP/PS1 mice relative to sham lesioned APP/PS1 mice. Additionally, APP/PS1 mice treated with SAP showed a tendency towards an increased level of soluble and insoluble Aβ1-40 and Aβ1-42 measured in brain tissue homogenate. Our results suggest that the combination of cholinergic degeneration and Aβ overexpression in the APP/PS1 mouse model results in cognitive decline and accelerated plaque burden. SAP treated APP/PS1 mice might thus constitute an improved model of Alzheimers disease-like neuropathology and cognitive deficits compared to the conventional APP/PS1 model without selective removal of basal forebrain cholinergic neurons.

Ketogenic diet is antiepileptogenic in pentylenetetrazole kindled mice and decrease levels of N-acylethanolamines in hippocampus

The ketogenic diet (KD) is used for the treatment of refractory epilepsy in children, however, the mechanism(s) remains largely unknown. Also, the antiepileptogenic potential in animal models of epilepsy has been poorly addressed. Activation of cannabinoid type-1 receptor (CB(1)-R) upon seizure activity may mediate neuroprotection as may several N-acylethanolamines. It is unknown how the KD interfere with the endocannabinoid system. We investigated the antiepileptogenic potential of the KD in the pentylenetetrazole kindling model in young mice and measured the hippocampal levels of CB(1)-R by Western blot and of endocannabinoids and N-acylethanolamines by mass spectrometry. The KD significantly decreased incidence and severity of seizures, and significantly increased the latency to clonic convulsions. There were no changes in levels of endocannabinoids or CB(1)-R expression by either seizure activity or type of diet. The level of oleoylethanolamide as well as the sum of N-acylethanolamines were significantly decreased by the KD, but were unaffected by seizure activity. The study shows that the KD had clear antiepileptogenic properties in the pentylenetetrazole kindling model and does not support a role of endocannabinoids in this model. The significance of the decreased hippocampal level of oleoylethanolamide awaits further studies.

Uterine contraction induced by Tanzanian plants used to induce abortion.

ETHNOPHARMACOLOGICAL RELEVANCE Women in Tanzania use plants to induce abortion. It is not known whether the plants have an effect. AIMS OF STUDY Collect data on plant use in relation to induced abortion and test the effect of plant extracts on uterine contraction. MATERIALS AND METHODS During interviews with traditional birth attendants and nurses, plants were identified. Cumulative doses of plant extracts were added to rat uterine tissue in an organ bath, and the force and frequency of contractions recorded. Acetylcholine was used as positive control. RESULTS 21 plant species were tested for effect on uterine contraction. 11 species increased the force of contraction, and 12 species the frequency of contractions. The strongest contractions comparable to the maximum response obtained with acetylcholine were obtained with extracts of Bidens pilosa, Commelina africana, Desmodium barbatum, Manihot esculenta, Ocimum suave, Oldenlandia corymbosa and Sphaerogyne latifolia. 7 species increased both the force and frequency of contractions. CONCLUSION Several of the plant species induced strong and frequent contractions of the uterus, and can be used to induce an abortion.

Effects of GABAA receptor partial agonists in primary cultures of cerebellar granule neurons and cerebral cortical neurons reflect different receptor subunit compositions

Based on an unexpected high maximum response to piperidine‐4‐sulphonic acid (P4S) at human α1α6β2γ2 GABAA receptors expressed in Xenopus oocytes attempts to correlate this finding with the pharmacological profile of P4S and other GABAA receptor ligands in neuronal cultures from rat cerebellar granule cells and rat cerebral cortex were carried out. GABA and isoguvacine acted as full and piperidine‐4‐sulphonic acid (P4S) as partial agonists, respectively, at α1β2γ2, α6β2γ2 and α1α6β2γ2 GABA receptors expressed in Xenopus oocytes with differences in potency. Whole‐cell patch‐clamp recordings were used to investigate the pharmacological profile of the partial GABAA receptor agonists 4,5,6,7‐tetrahydroisoxazolo‐(5,4‐c)pyridin‐3‐ol (THIP), P4S, 5‐(4‐piperidyl)isoxazol‐3‐ol (4‐PIOL), and 3‐(4‐piperidyl)isoxazol‐5‐ol (iso‐4‐PIOL), and the competitive GABAA receptor antagonists Bicuculline Methbromide (BMB) and 2‐(3‐carboxypropyl)‐3‐amino‐6‐methoxyphenyl‐pyridazinium bromide (SR95531) on cerebral cortical and cerebellar granule neurons. In agreement with findings in oocytes, GABA, isoguvacine and P4S showed similar pharmacological profiles in cultured cortical and cerebellar neurones, which are known to express mainly α1, α2, α3, and α5 containing receptors and α1, α6 and α1α6 containing receptors, respectively. 4‐PIOL and iso‐4‐PIOL, which at GABAA receptors expressed in oocytes were weak antagonists, showed cell type dependent potency as inhibitors of GABA mediated responses. Thus, 4‐PIOL was slightly more potent at cortical neurones than at granule neurones and iso‐4‐PIOL was more potent in inhibiting isoguvacine‐evoked currents at cortical than at granule neurons. Furthermore the maximum response to 4‐PIOL corresponded to that of a partial agonist, whereas that of iso‐4‐PIOL gave a maximum response close to zero. It is concluded that the pharmacological profile of partial agonists is highly dependent on the receptor composition, and that small structural changes of a ligand can alter the selectivity towards different subunit compositions. Moreover, this study shows that pharmacological actions determined in oocytes are generally in agreement with data obtained from cultured neurons.

Impaired GABAergic Inhibition in the Prefrontal Cortex of Early Postnatal Phencyclidine (PCP)-Treated Rats

A compromised γ-aminobutyric acid (GABA)ergic system is hypothesized to be part of the underlying pathophysiology of schizophrenia. N-methyl-D-aspartate (NMDA) receptor hypofunction during neurodevelopment is proposed to disrupt maturation of interneurons causing an impaired GABAergic transmission in adulthood. The present study examines prefrontal GABAergic transmission in adult rats administered with the NMDA receptor channel blocker, phencyclidine (PCP), for 3 days during the second postnatal week. Whole-cell patch-clamp recordings from pyramidal cells in PCP-treated rats showed a 22% reduction in the frequency of miniature inhibitory postsynaptic currents in layer II/III, but not in layer V pyramidal neurons of the prefrontal cortex. Furthermore, early postnatal PCP treatment caused insensitivity toward effects of the GABA transporter 1 (GAT-1) inhibitor, 1,2,5,6-tetrahydro-1-[2-[[(diphenyl-methylene)amino]oxy]ethyl]-3-pyridinecarboxylic acid, and also diminished currents passed by δ-subunit-containing GABAA receptors in layer II/III pyramidal neurons. The observed impairments in GABAergic function are compatible with the alteration of GABAergic markers as well as cognitive dysfunction observed in early postnatal PCP-treated rats and support the hypothesis that PCP administration during neurodevelopment affects the functionality of interneurons in later life.

GABAA receptor subunit interactions important for benzodiazepine and zinc modulation: a patch‐clamp and single cell RT‐PCR study

The expression of mRNAs for the GABAA receptor subunits α1, α6, β2, β3, γ2 and δ in single mouse cerebellar granule cells and cortical interneurons were analysed by RT‐PCR and correlated to their midazolam and zinc modulation of agonist‐induced receptor currents. The registration of molecular and electrophysiological data from each cell allowed us to estimate the significance of individual subunits and their two‐factor interaction for modulation. The presence of α6 decreased midazolam modulation, but statistical analysis also suggested interactions of α6 with β3 and γ2 with respect to midazolam modulation. Zinc modulation was decreased by the presence of γ2, and analysis points to an β3 effect as well as an interaction between γ2 and δ in zinc modulation. Thus, our model confirmed, in single native cells, the known effects of α6 in midazolam and γ2 in zinc modulation, and additionally pointed to significant subunit interactions that need to be further tested in recombinant receptors. The present study offers a method to identify subunit interactions in heteromeric receptor complexes.