picrotoxinin and alphaxalone

When the vertebrate retina is stimulated by light, a class of amacrine or interplexiform cells release dopamine, a modulator responsible for neural adaptation to light. In the intact retina, dopamine release can be pharmacologically manipulated with agonists and antagonists at GABA(A) receptors, and dopaminergic (DA) cells receive input from GABAergic amacrines. Because there are only 450 DA cells in each mouse retina and they cannot be distinguished in the living state from other cells on the basis of their morphology, we used transgenic technology to label DA cells with human placental alkaline phosphatase, an enzyme that resides on the outer surface of the cell membrane. We could therefore identify DA cells in vitro after dissociation of the retina and investigate their activity with whole cell voltage clamp. We describe here the pharmacological properties of the GABA(A) receptors of solitary DA cells. GABA application induces a large inward current carried by chloride ions. The receptors are of the GABA(A) type because the GABA-evoked current is blocked by bicuculline. Their affinity for GABA is very high with an EC(50) value of 7.4 microM. Co-application of benzodiazepine receptor ligands causes a strong increase in the peak current induced by GABA (maximal enhancement: CL-218872 220%; flunitrazepam 214%; zolpidem 348%) proving that DA cells express a type I benzodiazepine-receptor (BZ1). GABA-evoked currents are inhibited by Zn(2+) with an IC(50) of 58.9 +/- 8.9 microM. Furthermore, these receptors are strongly potentiated by the modulator alphaxalone with an EC(50) of 340 +/- 4 nM. The allosteric modulator loreclezole increases GABA receptor currents by 43% (1 microM) and by 107% (10 microM). Using outside-out patches, we measured in single-channel recordings a main conductance (29 pS) and two subconductance (20 and 9 pS) states. We have previously shown by single-cell RT-PCR and immunocytochemistry that DA cells express seven different GABA(A) receptor subunits (alpha1, alpha3, alpha4, beta1, beta3, gamma1, gamma2(S), and gamma2(L)) and by immunocytochemistry that all subunits are expressed in the intact retina. We show here that at least alpha1, beta3 and gamma2 subunits are assembled into functional receptors.

Topics: Alkaline Phosphatase; Animals; Benzodiazepines; Bicuculline; Binding Sites; Dopamine; Dose-Response Relationship, Drug; GABA Antagonists; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid; Humans; Mice; Mice, Transgenic; Neurons; Osmolar Concentration; Patch-Clamp Techniques; Picrotoxin; Pregnanediones; Protein Isoforms; Receptors, GABA-A; Retina; Sesterterpenes; Triazoles; Zinc

gamma-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the mammalian nervous system, is known to operate bicuculline-sensitive Cl- channels through GABAA receptors and bicuculline-insensitive cation channels through GABAB receptors. Recent observations indicate that the retina may contain GABA receptors with unusual pharmacological properties. Here we report that GABA gates bicuculline-insensitive Cl- channels in rod bipolar cells of the rat retina, which were not modulated by flunitrazepam, pentobarbital and alphaxalone and were only slightly blocked by picrotoxinin. Moreover, the GABAB receptor agonist baclofen, and the antagonist 2-hydroxysaclofen had no effect. The underlying single-channel conductance was 7 pS and the open time 150 ms. These values are clearly different from those obtained for GABAA receptor channels recorded in other neurons of the same preparation, and in other parts of the brain. The bicuculline- and baclofen-insensitive GABA receptors were activated selectively by the GABA analogue cis-4-aminocrotonic acid (CACA). Hence they may be similar to those receptors termed GABAC receptors.

Topics: Animals; Animals, Newborn; Bicuculline; Cells, Cultured; Chloride Channels; Flunitrazepam; gamma-Aminobutyric Acid; Ion Channels; Membrane Potentials; Membrane Proteins; Neurons; Pentobarbital; Photoreceptor Cells; Picrotoxin; Pregnanediones; Rats; Receptors, GABA-A; Sesterterpenes; Strychnine

1. Alfaxalone (1-100 nM) potentiated gamma-aminobutyric acidA (GABAA)-receptor-mediated contractile responses in the guinea-pig isolated ileum, with a leftward shift of the GABA concentration-response curve, and a significant potentiation of the GABA-induced contractions over the lower concentration-range for GABA (3-30 microM). Alfadalone on the other hand, did not affect contractile responses to GABA. 2. Picrotoxinin (10 microM) induced a non-parallel rightward shift of the GABA concentration-response curve, with a 50% depression of the maximum response to GABA. Alfaxalone (100 nM) potentiated the responses to GABA in the presence of picrotoxinin (10 microM) over the GABA concentration-range of 10-100 microM, causing a leftward shift of the concentration-response curve, but without affecting the depression of the maximum response by picrotoxinin. 3. Bicuculline methochloride (10 microM) caused a parallel rightward shift of the GABA concentration-response-curve; the ratio of this shift was unchanged in the presence of alfaxalone (100 microM), although the latter itself displaced the curve leftwards. 4. Alfaxalone (1-100 mM) also induced a similar potentiation of contractile responses to 3-amino-1-propanesulphonic acid (3-APS), a GABA agonist not subject to uptake. Such concentrations of alfaxalone were ineffective against contractile responses to exogenous acetylcholine. 5. Higher concentrations of alfaxalone (1 microM and above), however, elicited a GABA-like ileal contraction, sensitive to both picrotoxinin (10 microM) and bicuculline (10 microM). 6. In conclusion, alfaxalone potentiated GABAA-receptor-mediated contractile responses in the guinea-pig isolated ileum by acting at a modulatory site on GABAA-receptor-chloride-ionophore complexes of GABA-sensitive myenteric neurones, whilst high concentrations of alfaxalone exhibited a GABA-mimetic action at GABAA-receptors in the ileum. It is suggested that more than one site may exist where steroids interact with the GABAA-receptor-ionophore complexes.

Topics: Anesthetics; Animals; Bicuculline; Female; gamma-Aminobutyric Acid; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Picrotoxin; Pregnanediones; Sesterterpenes