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T G Smart

Publications and source records attributed to T G Smart.

At least 73 records · Page 4Linked to original sources
PubMed1990

Cloned GABA receptors are maintained in a stable cell line: allosteric and channel properties.

The cloned cDNAs encoding the alpha 1 and beta 1 subunits of the bovine brain GABA(A) receptor have been co-transfected, using a dexamethasone-inducible promoter, into cultured hamster ovary cells, with selection to form a stable cell line. The use, alternatively, of a much stronger constitutive promoter led to cell death consequent upon high receptor density. After induction, the cells contained the alpha 1 and beta 1 mRNAs. The expressed receptors showed the high-affinity binding of [3H]muscimol and of the GABA(A) receptor channel blocker, t-butylphosphorothionate (TBPS), and the characteristic enhancement of the former by a pregnanolone. Their GABA-activated current was potentiated by the barbiturate, pentobarbitone, was reversibly blocked by bicuculline and picrotoxin, but was not enhanced by benzodiazepines. In mouse spinal cord neurons GABA activates channel openings to at least four conductance states (45, 30, 19 and 12 pS) with the 30 pS state being the most frequently observed (main) state. However, the main state of the alpha 1/beta 1 GABA(A) receptor was the 19 pS state. The enhancement of GABA(A) receptor current by barbiturates wa due to prolongation of mean channel lifetime, whereas the reduction of GABA(A) receptor current by picrotoxin was due to reduction of channel opening frequency and mean channel lifetime. Stable cell lines containing subunit combinations of this receptor should provide a powerful tool for the elucidation of its channel features and control mechanisms.

Animals↗
PubMed1990

Differential effect of zinc on the vertebrate GABAA-receptor complex.

1. gamma-Aminobutyric acid (GABA) responses were recorded from rat superior cervical ganglia (SCG) in culture using the whole cell recording technique. 2. Zinc (50-300 microM) reversibly antagonized the GABA response in embryonic and young post-natal neurones, while neurones cultured from adult animals were far less sensitive and occasionally resistant to zinc blockade. Cadmium (100-300 microM) also antagonised the GABA response, while barium (100 microM-2 mM) was ineffective. 3. The differential blocking effect of zinc on cultured neurones of different ages also occurred in intact SCG tissue. 4. The GABA log dose-response curve constructed with foetal or adult cultured neurones was reduced in a non-competitive manner by zinc. This inhibition was minimally affected by the membrane potential. 5. The GABA response recorded intracellularly from guinea-pig pyriform cortical slices was enhanced by zinc (300-500 microM), which occurred concurrently with a decrease in the input conductance of the cell. The enhancement was unaffected by prior blockade of the GABA uptake carrier by 1 mM nipecotic acid. This phenomenon could be reproduced by barium (300 microM) and cadmium (300 microM). 6. We conclude that the vertebrate neuronal GABAA-receptor becomes less sensitive to zinc with neural (GABAA-receptor?) development, and the enhanced GABA response recorded in the CNS is a consequence of the reduction in the input conductance and not due to a direct effect on the receptor complex.

Aging↗
PubMed1989

Excitatory amino acids: the involvement of second messengers in the signal transduction process.

1. Excitatory amino acids (EAA) can activate second messenger systems in addition to a direct gating of ion channels. A discrete coupling between novel EAA receptor subtypes and second messenger systems has been previously proposed. 2. EAAs have been suggested to activate both adenylate and guanylate cyclases and also to induce phosphoinositide (PI) turnover. The increased PI turnover was observed in both central neurons and glia, and a "quisqualate-type" receptor has been most frequently involved, which may differ from the quisqualate receptor previously defined by electrophysiological studies. 3. The roles of EAA-induced calcium influx into neurons and raised intracellular calcium levels are discussed regarding the activation of phosphoinositide turnover. 4. This review examines the data supporting a link between EAA receptors and second messengers and considers whether there is any need for adopting new EAA receptor subtypes. Also, the use of the Xenopus laevis oocyte for expressing EAA receptors and studying any putative links to second messenger systems is discussed.

Adenylyl Cyclases↗
PubMed1989

Muscarinic suppression of the M-current in the rat sympathetic ganglion is mediated by receptors of the M1-subtype.

1. Under voltage-clamp dissociated adult and foetal rat superior cervical ganglion (s.c.g.) cells exhibited a non-inactivating voltage- and time-dependent component of K+ current termed the M-current (IM). IM was detected and measured from the current decay during hyperpolarizing voltage steps applied from potentials where IM was pre-activated. 2. Neither the resting membrane current nor the amplitude of these current decay relaxations were reduced by omitting Ca from the bathing fluid, showing that the M-current was not a 'Ca-activated' K-current dependent on a primary Ca-influx. Concentrations of (+)-tubocurarine sufficient to block the slow Ca-activated K-current IAHP did not inhibit IM or antagonize the effect of muscarinic agonists on IM, showing that IM was not contaminated by IAHP. Tetraethylammonium (1 mM), which blocks the fast Ca-activated K-current IC, produced a small inhibition of IM. This was not due to contamination of IM by IC since muscarinic agonists did not consistently block IC. 3. The muscarinic agonists muscarine, oxotremorine, McN-A-343 and methacholine reversibly suppressed IM, resulting in an inward (depolarizing) current. The rank order of potency was: oxotremorine greater than or equal to muscarine greater than McN-A-343 greater than methacholine. 4. The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5. IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 +/- 0.13 (n = 3) and 6.02 +/- 0.13 (n = 4) respectively. 6. The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 microM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nM. 7. Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 +/- 0.03 (n = 8). 8. These results suggest that the receptors mediating suppression of the M-current accord with those designated pharmacologically as M1 and that these receptors reach maturity at a very early stage in the development of the rat s.c.g.

Animals↗
PubMed1989

On the transduction mechanism for muscarine-induced inhibition of M-current in cultured rat sympathetic neurones.

1. Dissociated adult or fetal rat superior cervical ganglion cells were voltage-clamped through a single patch pipette. The voltage-dependent K+ current, IM (M-current), was maintained by including MgATP in the pipette solution and by buffering the solution pH to 6.7. 2. Bath-applied muscarine (0.4 microM) produced a reversible inhibition of IM. 3. Addition of Gpp(NH)p (200 microM) or GTP-gamma-S (500 microM) to the pipette solution induced a slowly developing inhibition of IM and prevented recovery from subsequent muscarine-induced inhibition. 4. Addition of GDP-beta-S (500 microM) to the pipette solution reduced the amount of IM inhibition produced by 0.4 microM-muscarine by 42% and reduced the associated inward shift of the holding current by 56%. 5. Cells responded normally to muscarine after pre-treatment for 4-27 h with 500 ng ml-1 pertussis toxin (PTx). 6. IM was not diminished by extracellular addition of 1 mM-dibutyryl cyclic AMP, 8-bromo-cyclic AMP or dibutyryl cyclic GMP, or of 10 microM-forskolin. 7. IM was not reduced by inclusion of Li+ (2 mM) or inositol 1,4,5-trisphosphate (IP3, 100 microM) in the patch pipette, nor by ionophoretic injection of IP3 from an inserted micropipette. 8. Addition of 4-beta-phorbol 12,13-dibutyrate (PDBu, 0.5-2 microM) to the extracellular medium partly inhibited IM and reduced an additional component of resting membrane current. This effect was not replicated by 4-alpha-phorbol 12,13-didecanoate. 9. It is concluded that the inhibition of IM by muscarine is mediated through activation of a PTx-insensitive GTP-binding protein. The effect of muscarine appears not to be mediated by cyclic nucleotides or IP3 but may possibly involve the generation of diacylglycerols and activation of protein kinase C.

Animals↗
PubMed1987

Membrane currents in adult rat superior cervical ganglia in dissociated tissue culture.

Intracellular and whole-cell recordings were made from young adult superior cervical ganglion neurones maintained in dissociated tissue culture. The neurones possessed an M-current, A-current and calcium-activated potassium currents in addition to inward sodium and calcium currents. The properties of these currents closely resembled those observed in intact sympathetic ganglia. In contrast to neurones cultured from foetal tissue, these adult cultures exhibited consistent responses to nicotinic agonists.

Animals↗
PubMed1987

Functional expression in the Xenopus oocyte of messenger ribonucleic acids encoding brain neurotransmitter receptors: further characterisation of the implanted GABA receptor.

The Xenopus oocyte is the only system currently available for the full expression of mRNAS encoding membrane receptors and ion channels. Its application in the case of brain mRNAs for the GABAA receptor is illustrated, including the production of receptor subunits and its chloride channel with characteristic pharmacology.

Animals↗
PubMed1987

GABA receptors induced in Xenopus oocytes by chick brain mRNA: evaluation of TBPS as a use-dependent channel-blocker.

Sensitivity to GABA was induced in Xenopus laevis oocytes by injection of poly(A)+ mRNA extracted from 19-day chick embryo brain. The effect of the convulsant drug t-butylbicyclophosphorothionate (TBPS) was studied on the responses to bath-applied GABA using voltage-clamp techniques. TBPS reversibly inhibited the GABA-evoked current (IGABA) in a dose-dependent manner; however, the chloride currents evoked by carbachol or serotonin, or the spontaneous chloride fluctuations, were unaffected. The onset of the block by TBPS was faster in the presence of GABA. The recovery from the block after TBPS wash-out was also agonist-stimulated. At the steady state block, TBPS showed a mixed type of inhibition: increasing the GABA concentration decreased but failed to abolish completely the inhibition by TBPS. The TBPS block was independent of the direction of the chloride flow: both inward and outward IGABA were blocked. The time course of the decay of IGABA was markedly changed in the presence of TBPS: above 40 microM GABA, this time course showed essentially two exponentials and TBPS abolished only the fast component, whereas at a low concentration (less than or equal to 4 microM), IGABA was relatively constant and uniformly reduced by TBPS. It is suggested that TBPS may stabilize a closed form of the liganded receptor-channel complex.

Animals↗
PubMed1987

Single calcium-activated potassium channels recorded from cultured rat sympathetic neurones.

1. The properties of single Ca2+-activated K+ channels in cultured rat superior cervical ganglionic neurones were studied in cell-attached and excised patches using the patch-clamp technique. 2. In cell-attached patches using an external K+ concentration ([K+]o) of 150 mM, approximately equal to the internal [K+], the channel slope conductance was approximately 200 pS and independent of membrane voltage between -50 and +50 mV. Using [K+]o of 4.7 mM (providing a near physiological K+ gradient), the I-V relationship was non-linear with a slope conductance of approximately 120 pS at 0 mV. 3. The channel was selective for K+ over Cs+ and Na+ which were impermeant from either side of the membrane. Both Na+ and Cs+ also blocked the movement of K+ through the channel. Cs+ was active on either side of the membrane, whereas Na+ apparently blocked the channel only when applied to the cytoplasmic side. 4. The channel was activated by increasing the Ca2+ concentration on the inside of the membrane ([Ca2+]i). The channel was virtually inactive when [Ca2+]i = 0.01 microM. Depolarizing the patch at a constant [Ca2+]i usually further increased the opening probability. 5. The gating properties of the channel were studied using cell-attached patches. At potentials more negative than the resting membrane potential, the open-time distribution was described by a single exponential. On depolarization, two exponentials were required. The closed-time distribution was fitted by three exponentials. 6. Depolarization of the patch caused the long mean open lifetime to increase whilst the short mean open and closed lifetimes were unaffected. Both the intermediate and long mean closed lifetimes decreased with depolarization from -60 to +60 mV. 7. In cell-attached patches, the long mean open lifetimes were usually smaller than those observed in excised patches at depolarized potentials (greater than 0 mV). 8. A fourth closed state, possibly representing an inactivated form of the channel, was infrequently observed. A 50% substate of the full single-channel current was also observed occasionally. This substate was always associated with openings to the full current state. 9. The channel was blockable by external tetraethylammonium (25 microM-1 mM), Ba2+ (1-10 mM), and quinine (10-200 microM). External d-tubocurarine (25-100 microM) also blocked this IC channel. However it was insensitive to apamin (100-300 nM), muscarine (10 microM) and 4-aminopyridine (1-3 mM). The channel was also blocked by internal tetraethylammonium (5-10 mM) or Ba2+ (0.3-1 mM).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1984

A system for the translation of receptor messenger-RNA and the study of the assembly of functional receptors.

The Xenopus oocyte translation system has been developed for the recognition of receptor messenger-RNA molecules. Observations with the nicotinic acetylcholine, GABA, glycine, glutamate and serotonin receptors show that the mRNAs coding for their subunits can be faithfully translated in the oocyte and the products processed, glycosylated, assembled to a receptor structure, inserted correctly in the cell membrane and organised to form the functional ion channel. The specific regulatory interactions between different sites on one receptor, as exemplified in the GABAA receptor, are established in this process. This system is of value for the detection of mRNAs for receptor gene cloning and for the study of receptor assembly and of the expression of multiple receptor genes. The receptor ion channels formed can also be studied in their own right in a highly accessible and controlled situation.

Acetylcholine↗
PubMed1983

Pre- and postsynaptic effects of zinc on in vitro prepyriform neurones.

Intracellular recordings from guinea-pig olfactory cortex neurones revealed a dual effect of zinc: firstly (at 100-500 microM), the responses to bath-applied GABA, muscimol and 3-aminopropanesulphonate were reversibly enhanced, and secondly (at 25-500 microM), the excitatory postsynaptic potential was dramatically prolonged. At Zn2+ doses higher than 50 microM, transmission was eventually blocked irreversibly. These effects of zinc were not produced by 4-aminopyridine, or other divalent cations. The GABA-enhancement is suggested to occur via an interaction of Zn2+ with the post-synaptic GABA receptor, and the prolonged transmitter release probably via blockade of an outward current in nerve terminals. The latter effect may be a contributory factor in the epileptogenic activity of zinc.

Animals↗
PubMed1983

Synthesis of functional chick brain GABA-benzodiazepine-barbiturate/receptor complexes in mRNA-injected Xenopus oocytes.

Membrane conductance changes evoked by bath-applied GABA (2-640 microM) and some related agonists were recorded intracellularly in Xenopus oocytes previously injected with chick brain mRNA. Muscimol and 3-aminopropanesulphonate were approximately 4 and 0.25 times as potent as GABA in producing a conductance increase. GABA responses were antagonized by bicuculline (10 microM) or picrotoxinin (10-100 microM) but were clearly enhanced by the benzodiazepine (BZ) receptor ligand chlorazepate or by pentobarbitone. We conclude that an appropriate fraction of brain mRNA was capable of directing the synthesis and correct insertion of functional GABA-BZ-barbiturate/receptor complexes in the oocyte membrane.

Animals↗