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S V Jones

Publications and source records attributed to S V Jones.

31 records · Page 2Linked to original sources
PubMed1991

Electrophysiological responses to muscarinic receptor stimulation in cultured hippocampal neurons.

Electrophysiological recordings of responses to muscarinic receptor stimulation in cultured embryonic hippocampal neurons have been largely unsuccessful to date. In this study muscarinic receptor binding was demonstrated in 2-week-old embryonic rat hippocampal cultures. Using whole-cell patch-clamp recording we found that 1-5 microM carbachol produced multiple effects including depolarization, increased action potential firing rate, increased synaptic activity and a reduction in the amplitude of medium-duration afterhyperpolarizations. Voltage-clamp analysis revealed a time-dependent current relaxation with hyperpolarizing steps from a holding potential of about -40 mV which was inhibited by 10 microM muscarine or 50 microM carbachol and had characteristics similar to those of the m-current. Both atropine and pirenzepine inhibited all of these effects indicating that these cholinergic actions were mediated by muscarinic receptors. This study shows that muscarinic responses obtained classically in hippocampal brain slices can also be produced in cultured hippocampus.

Action Potentials↗
PubMed1991

Functional responses of cloned muscarinic receptors expressed in CHO-K1 cells.

The physiological responses to activation of the m5 muscarinic acetylcholine receptor were compared with those of m3 and m4 in transformed Chinese hamster ovary cells, using patch-clamp electrophysiological and biochemical techniques. Stimulation of the m5 receptor induced increases in both a calcium-dependent potassium conductance and phosphoinositide (PI) metabolism of similar magnitude to those activated by m3. Raising of intracellular calcium or injection of inositol-1,4,5-trisphosphate mimicked the activation of the calcium-dependent potassium conductance by both of these receptors. Although similar regarding these responses, the m3 and m5 receptors induced different cAMP responses. Stimulation of m5 receptors induced a 2-fold increase in cAMP levels, whereas m3 induced a 20-fold increase. These cAMP responses required greater than 100-fold more agonist than the PI responses, and both PI and cAMP responses were insensitive to pertussis toxin. Stimulation of m4 receptors caused little increase in PI metabolism and no electrophysiological effects. Stimulation of m4 receptors with low concentrations of agonist decreased cAMP levels, but at high agonist concentrations cAMP levels were elevated. After treatment with pertussis toxin, the decrease in cAMP levels induced by m4 was blocked and a marked increase in cAMP levels, comparable to those observed for m3 receptors, was uncovered at higher doses. The data indicate that each of the receptors has distinct functional properties.

Acetylcholine↗
PubMed1990

Inositol trisphosphate mediates cloned muscarinic receptor-activated conductances in transfected mouse fibroblast A9 L cells.

1. The mechanism by which cloned m1 and m3 muscarinic receptor subtypes activate Ca2+-dependent channels was investigated with whole-cell and cell-attached patch-clamp recording techniques and with Fura-2 Ca2+ indicator dye measurements in cultured A9 L cells transfected with rat m1 and m3 cDNAs. 2. The Ca2+-dependent K+ and Cl- currents induced by muscarinic receptor stimulation were dependent on GTP. Responses were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. Intracellular GTP-gamma-S activated spontaneous fluctuations and permitted only one acetylcholine-(ACh) induced current response. These results implicate GTP-binding proteins (G protein) in the signal transduction pathway. This G protein is probably not pertussis toxin-sensitive as the ACh-induced electrical response was not abolished by pertussis toxin treatment. 3. Cell-attached single-channel recordings revealed activation of ion channels within the patch during application of ACh outside the patch, implying that second messengers might be involved in the ACh-induced response. Two types of K+ channel were activated, a discrete channel of 36 pS and channel activity calculated to be about 5 pS. 4. Application of 8-bromo cyclic AMP or 1-oleoyl-1,2-acetylglycerol (OAG) produced no electrical response and did not affect the ACh-induced responses. Phorbol myristic acetate (PMA) evoked no electrical response, but reduced the ACh-induced responses. 5. Inclusion of inositol 1,4,5-trisphosphate (IP3) in the intracellular pipette solution activated outward currents at -50 mV associated with an increase in conductance. The IP3-induced current response reversed polarity at -65 mV and showed a dependence on K+. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) from 20 nM to 1 microM also induced an outward current response associated with an increase in conductance. Inclusion of inositol 1,3,4,5-tetrakisphosphate (IP4) in the intracellular solution had no effect on the A9 L cells. 6. Fura-2 measurements revealed ACh-induced increases in Cai2+. The Ca2+ responses were abolished by atropine showing that they were muscarinic in nature. Removal of extracellular Ca2+ did not affect the initial ACh-induced increase in Cai2+ but subsequent Cai2+ responses to ACh were depressed, suggesting depletion of Ca2+ intracellular stores. Residual though small responses continued to be elicited by ACh. Barium (5 mM) had little effect and cobalt slightly reduced the ACh-induced Ca2+ response. 7. The ACh-induced currents recorded at -50 mV were unaffected by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine↗
PubMed1988

Electrophysiological characterization of cloned m1 muscarinic receptors expressed in A9 L cells.

The electrophysiological properties of A9 L cells stably transfected with m1 muscarinic receptor cDNA were examined by using the whole-cell patch-clamp technique. In current-clamp recordings, acetylcholine (AcCho) elicited a hyperpolarization of all transfected cells studied but had no effect on nontransfected A9 L cells. In voltage-clamp recordings, AcCho elicited an outward current at -50 mV accompanied by an increase in conductance. The onset of the current response was consistently delayed by several seconds with respect to the onset of the application of AcCho and could not be accounted for by diffusion. The AcCho-induced currents were reversibly inhibited by the muscarinic receptor antagonist atropine (1 microM) but were unaffected by the nicotinic receptor antagonist tubocurarine (50 microM). Ion-substitution experiments replacing K+ with N-methyl-D-glucamine and Cl- with methanesulfonate indicated that the current was carried mainly by K+, although a minor part appeared to be carried by Cl-. The AcCho-induced current could be blocked by the K+ channel blocking agents tetraethylammonium ion, 4-aminopyridine, apamin, and Ba2+ but not by Cs+. The AcCho-induced current was inhibited when 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was included in the patch pipette or when extracellular Cd2+ or Co2+ was applied, indicating a role for intracellular Ca2+ in the generation of the response. Thus, these results show that cloned m1 muscarinic receptors expressed in A9 L cells can activate a Ca2+-dependent K+ conductance, possibly via a second-messenger system.

Animals↗
PubMed1988

Cloned muscarinic receptor subtypes expressed in A9 L cells differ in their coupling to electrical responses.

The electrophysiological responses to cholinergic stimulation of four cloned muscarinic receptor subtypes (m1-m4) were studied in A9 L cells transfected with the expression plasmids of each of the different subtypes, using the tight-seal whole-cell recording technique. Cells transfected with m1 and m3 muscarinic receptor subtypes were hyperpolarized by acetylcholine (ACh), whereas m2- and m4-transfected cells did not respond to ACh concentrations of up to 1 mM. Stimulation of both m1 and m3 muscarinic receptor subtypes evoked outward currents in cells voltage-clamped at -50 mV, associated with an increase in membrane conductance. These outward currents were blocked by atropine but not by tubocurarine. The ACh-induced currents of m1- and m3-transfected cells primarily involved potassium ions, although chloride ions also contributed to a minor extent. The potassium and chloride conductances were blocked by barium or cobalt and by buffering the intracellular calcium to low levels with 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, showing a dependence of these conductances on calcium. Thus, m1- and m3-transfected cells respond to ACh in a manner that is qualitatively similar, evoking calcium-dependent potassium and chloride conductances, whereas m2- and m4-transfected cells are not coupled to electrically detectable responses in A9 L cells.

Acetylcholine↗
PubMed1987

Non-competitive effects of disopyramide at the neuromuscular junction: evidence for endplate ion channel block.

The effects of disopyramide were studied at the neuromuscular junction in an attempt to elucidate the mechanism of its blocking action at this site. Disopyramide 5 X 10(-5) - 10(-3) mol litre-1 produced a concentration-dependent reduction of twitch amplitude in the indirectly stimulated chick biventer cervicis preparation, but greater concentrations were required to reduce twitches elicited directly in the presence of erabutoxin-b 1 microgram ml-1. Equieffective twitch blocking doses of either disopyramide or tubocurarine greatly reduced agonist responses to acetylcholine and carbachol, but the reduction was less for magnesium-blocked twitches. Neostigmine antagonized tubocurarine-induced, but not disopyramide-induced, blockade of twitches. Concentration-response profiles to acetylcholine and carbachol were shifted to the right in a non-parallel fashion and the maximal response was depressed by disopyramide 5 X 10(-5) - 10(-4) mol litre-1. Intracellular recording studies carried out in the cut, voltage-clamped costo-cutaneous muscle of the garter snake showed that disopyramide 5 X 10(-5) - 5 X 10(-4) mol litre-1 produced a concentration- and voltage-dependent reduction of the amplitude of neurally evoked endplate-currents (EPC) and of the time constant of decay (tau) of EPC. We conclude that disopyramide possesses a non-competitive blocking action at the neuromuscular junction, which is not reversible by anticholinesterase agents. The voltage-dependent nature of the block suggests that it is mediated via blockade of the open form of the acetylcholine-activated receptor-ion channel complex.

Animals↗
PubMed1987

A kinetic analysis of the endplate ion channel blocking action of disopyramide and its optical isomers.

The effects of the antiarrhythmic agent disopyramide was studied on responses from voltage-clamped endplates at the neuromuscular junction of the garter snake. Disopyramide reduced endplate current amplitude and decay time constant in a concentration- and voltage-dependent manner. Endplate current decays remained monophasic in the presence of the drug. These results were interpreted in terms of the drug blocking the open form of the acetylcholine receptor-ion channel complex. Disopyramide produced a greater reduction of the amplitude of endplate currents than of miniature endplate currents. The reduction in miniature endplate current amplitude was not voltage-dependent. Analysis of endplate current driving functions showed that this was due to the rapid occurrence of channel block during the rising phase of the endplate current. The residual reduction, apart from that produced by channel block, is most probably due to receptor block. Disopyramide had a voltage-dependent blocking rate constant of about 10(7) M-1 S-1 at -90 mV. The unblocking rate constant was estimated from the results of experiments using paired ionophoretically applied pulses of acetylcholine. This value was again voltage-dependent and approximately 1 s-1. The actions of the (+)- and (-)-stereoisomers of disopyramide on endplate current decay were identical, indicating that the channel binding site at the neuromuscular junction is not stereoselective.

Acetylcholine↗
PubMed1987

Expression of a cloned muscarinic receptor in A9 L cells.

Using an oligonucleotide based on the sequence of a porcine brain muscarinic receptor cDNA, we recently cloned four distinct muscarinic receptors from the rat and human genomes. In the present study we transfected the rat homolog of the porcine brain muscarinic receptor cDNA into A9 L cells using a mammalian expression vector and a calcium phosphate precipitation procedure. Before transfection, A9 L cells do not bind muscarinic ligands and do not express muscarinic receptor mRNA. After transfection, A9 L cells expressed muscarinic receptor mRNA and saturable, high affinity binding sites for the muscarinic antagonists 3H-quinuclidinyl benzilate and 3H-pirenzepine. The muscarinic receptor antagonists AF DX-116 and pirenzepine displaced bound 3H-quinuclidinyl benzilate with inhibition curves suggestive of a single high affinity binding site. Competition of 3H-quinuclidinyl benzilate-labeled sites with the agonists acetylcholine and carbachol yielded broad inhibition curves, consistent with a heterogeneity of binding sites. In the presence of guanine nucleotide, the agonist inhibition curves were steeper, suggesting the presence of a single low affinity site. The effects of guanine nucleotides on agonist binding are consistent with coupling of these receptors to a guanine nucleotide-binding protein (G-protein) endogenous to A9 L cells. The electrical properties of the transfected A9 L cells were examined using the whole cell patch-clamp technique. Fifty microM acetylcholine induced a conductance which reversed in polarity at -60 mV. This conductance could be reversibly blocked by atropine. These data illustrate the utility of stable transfection of A9 L cells for the characterization of individual cloned muscarinic receptors, their G-protein coupling mechanisms, and resultant physiological responses.

Animals↗
PubMed1975

T and B cells in myelomatosis.

The majority of treated patients with myelomatosis had a significantly decreased number of T cells as measured by both lymphocyte stimulation to phytohaemagglutinin (PHA) and by their ability to form spontaneous rosettes with sheep red blood cells (SRBC). Seven untreated patients also had markedly depressed T cells. On the other hand, B-cell numbers' were significantly increased in the majority of the patients as judged by increased lymphocyte stimulation to pokeweed mitogen (PWM) and the markedly increased numbers of rosettes that were formed with antibody-coated complement activated SRBC, as well as by immunofluorescence cell markers to monospecific immunoglobulin antiserum.

B-Lymphocytes↗
PubMedDate not supplied

Assisted reproduction techniques.

Many couples with infertility will require a form of assisted reproduction to achieve a pregnancy. Assisted reproduction techniques, including intrauterine insemination, gamete intrafallopian transfer and in-vitro fertilisation, are considered in this review.

Female↗