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Biomedical subjects

R B Clark

Publications and source records attributed to R B Clark.

At least 199 records · Page 11Linked to original sources
PubMed1981

Alloreactive T cells from individual soft agar colonies specific for guinea pig Ia antigens. I. Production and initial characterization.

Allo-reactive T cells reactive with Ia antigens from strain 2 and 13 guinea pigs were cloned by plating T cells from primary mixed leukocyte cultures in soft agar and expanding the resulting colonies in liquid culture. Optimal expansion of the cultures from an individual colony was achieved by repeated stimulation with allogeneic cells in conditioned media. Mapping studies using stimulator cells from inbred and outbred guinea pigs and blocking studies with alloantisera and xenogeneic monoclonal antibodies to guinea pig Ia antigens indicated that these T cell colonies were specifically stimulated to proliferate by Ia molecules. In addition, these xenogeneic monoclonal anti-Ia antibodies had selective and differential inhibitory effects on the proliferative response of these alloreactive colonies. This result suggested that each T cell colony is specific for a distinct epitope expressed on Ia molecules. Furthermore, unlike heterogeneous cell populations in guinea pig primary and secondary mixed leukocyte cultures, most alloreactive T cell colonies expressed significant cytotoxic T lymphocyte activity that was also specific for Ia. Thus, the same cell proliferating to Ia is also a T killer cell. These T cell colonies will be useful for further analysis of the specificity and functional significance of the T cells responding in the mixed leukocyte reaction.

Animals↗
PubMed1981

Epinephrine desensitization of adenylate cyclase from cyc- and S49 cultured lymphoma cells.

The characteristics of the specific beta-adrenergic desensitization of adenylate cyclase from the adenylate cyclase deficient lymphoma cell line (cyc-) and the wild type S49 (WT) are presented in detail in this report. We have previously shown that the cyc- adenylate cyclase desensitized with 1-3 hr pretreatment of the cells with the beta-adrenergic agonist epinephrine. Adenylate cyclase of cyc- was measured after reconstitution with cholate extracts of the coupling proteins (G/F) from WT. We have now demonstrated that: (i) the initial epinephrine-induced desensitization of adenylate cyclase from either cyc- or WT was similar and occurred rapidly, with a half-life of approximately 2 min, although WT desensitized to a greater extent with prolonged hormone treatment (18 hr pretreatment of cyc- or WT with 0.1 mM terbutaline resulted in a 92% desensitization of the WT adenylate cyclase and only a 48% desensitization of cyc-); (ii) the 60 min epinephrine desensitization of cyc- was reversed after addition of propranolol and continued (40-60 min) incubation, while that of WT was only partially reversed; (iii) similar concentrations of epinephrine (0.2-0.4 muM) were required for half-maximal desensitization of both cell lines; and (iv) the Kact for epinephrine stimulation of reconstituted adenylate cyclase from cyc- or WT was increased after a 1 hr desensitization with 10 muM epinephrine. Kact was approximately 5-fold less than the half-maximal concentration required for desensitization. The data support the conclusion that the mechanisms of the rapid, reversible specific beta-adrenergic desensitization of adenylate cyclase in cyc- and WT cells are similar and occur independently of the G/F proteins of the adenylate cyclase complex, whereas the slower, apparently irreversible phase of desensitization occurs only in WT.

Adenylyl Cyclases↗
PubMed1980

Influence of glutamate and aspartate on time course of decay of excitatory synaptic currents at locust neuromuscular junctions.

The influence of glutamate and aspartate on the time course of decay of excitatory currents at neuromuscular junctions of locust skeletal muscle was examined. Both aspartate (up to 10 mM) and glutamate (up to 0.3 mM) had little influence on the decay time of the synaptic currents, whether measured intracellularly by voltage-clamp or extracellularly with a focal electrode. These results do not exclude a role for an active uptake system in sequestering neurally released transmitter at locust neuromuscular junctions, but they suggest that it has a negligible influence on the time course of the synaptic currents.

Animals↗
PubMed1980

Inhibition of cyclic AMP accumulation in hamster adipocytes with phosphatidic acid: differences and similarities with alpha adrenergic effects.

Clark et al (Journal of Cyclic Nucleotide Research 6:37 (1980)) demonstrated phosphatidic acid inhibition of cyclic AMP formation in WI-38 fibroblasts and suggested the hypothesis that cholinergic inhibition of adenylate cyclase is mediated through accumulation of this phospholipid. In view of these data, we tested the hypothesis that phosphatidic acid is involved in alpha-adrenergic inhibition of cyclic AMP formation in hamster adipocytes. The effects of phosphatidic acid on hormone and methyl xanthine stimulated cyclic AMP accumulation and lipolysis were studied. Phosphatidic acid inhibited 3-isobutyl-1-methyl xanthine (IBMX) stimulated cyclic AMP formation. The maximum inhibition (85% to 100%) of IBMX stimulated cyclic AMP accumulation was detected at 1.0 microM phosphatidic acid. When lipolysis was measured, however, inhibitory effects of phosphatidic acid were not evident until the concentration of phospholipid was increased to 300 microM. The cyclic AMP lowering and antilipolytic effects were detected using egg yolk phosphatidic acid, dipalmitoyl phosphatidic acid, dimyristoyl phosphatidic acid, distearoyl phosphatidic acid and palmitoyl lysophosphatidic acid but not with phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine or 1,2- diglyceride. In contrast to phosphatidic acid, clonidine or N6-phenylisopropyl adenosine inhibited lipolysis in concert with inhibition of methyl xanthine stimulated cyclic AMP accumulation. Cyclic AMP accumulation increased by isoproterenol in combination with IBMX was partially blocked by clonidine but not by phosphatidic acid. These results show similarities as well as differences between clonidine and phosphatidic acid actions on hamster fat cells and do not, therefore, provide unequivocal support for the possibility that phosphatidic acid is intimately involved in alpha-adrenergic effects on hamster adipocytes. However, the high selectivity of phosphatidic acid suggests a physiological role of this agent in regulation of adenylate cyclase.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Phosphatidic acid inhibition of PGE1-stimulated cAMP accumulation in WI-38 fibroblasts: similarities with carbachol inhibition.

To test the hypothesis that phosphatidic acid (PhA) is involved in the carbachol inhibition of hormone stimulated accumulation of cAMP we observed the effects of PhA on PGE1-stimulation of cAMP in WI-38 fibroblasts. PhA inhibited PGE1-stimulated cAMP accumulation of WI-38 fibroblasts; maximum inhibition (approximately 50-80%) occurred at a PhA concentration of 1.0 microM and significant inhibition was observed with a concentration of 0.1 microM. The full effects of PhA were evident within 15 sec after the co-addition of PGE1 and PhA. Addition of PhA to cells which had been pre-stimulated with PGE1 resulted in the rapid decay of cAMP levels to a new steady state level with a t 1/2 of approximately 65 sec. The inhibition produced by PhA did not appear to be simply attributable to a depolarization or increased intracellular Ca2+, since addition of either KCl or the Ca2+ ionophore A23187 did not lower PGE1-stimulated cAMP accumulation. When intact cells were pretreated with PhA then lysed and adenylate cyclase immediately assayed, no detectable changes in broken cell adenylate cyclase activities were observed. Also, PhA added directly to adenylate cyclase assays at concentrations as high as 100 microM produced no detectable inhibition of the membrane fraction adenylate cyclase activities. Nonetheless, our results suggest that adenylate cyclase activity in intact cells may be directly affected by physiological levels of PhA . Further, the similarities of carbachol [Butcher, R. W., Journal of Cyclic Nucleotide Research, 4:411 (1978)] and PhA inhibition support the hypothesis that carbachol (acetylcholine) exerts its effect on adenylate cyclase through alterations of the plasma membrane phospholipid composition.

Adenylyl Cyclases↗
PubMed1979

Responses to DL-ibotenic acid at locust glutamatergic neuromuscular junctions.

1 The responses of excitatory junctions on locust skeletal muscle fibres to iontophoretically applied L-glutamic acid and DL-ibotenic acid, a rigidly extended analogue of glutamate, were recorded by means of intracellular microelectrodes.2 Iontophoresis of L-glutamate to junctional sites produced transient depolarizations. Ibotenate applied iontophoretically to these sites usually evoked small hyperpolarizations which probably resulted from the activation of glutamate H-receptors on the extrajunctional membrane surrounding the junctions. However, at a minority ( approximately 20%) of junctions, ibotenate iontophoresis evoked transient depolarizations.3 Iontophoretically applied glutamate desensitized the ibotenate receptors, and vice versa. In experiments performed at junctional sites at which ibotenate depolarizations were absent, ibotenate had no effect on the responses to glutamate.4 Glutamate and ibotenate junctional currents had similar reversal potentials, measured under voltage-clamp, suggesting that the ionic bases for these currents are identical.5 It is proposed that the excitation caused by ibotenate results from the activation of receptors for extended L-glutamate and that these receptors co-exist on the post-junctional membranes of locust excitatory nerve-muscle synapses with ibotenate-insensitive glutamate receptors activated by glutamate in partially folded conformation.

Animals↗
PubMed1979

Desensitization of glutamate receptors on innervated and denervated locust muscle fibres.

Depolarizations to L-glutamate, applied locally by microinophoresis to the extrajunctional membrane of locust extensor tibiae muscle fibres and measured either in current clamp or voltage clamp, increased in amplitude for equivalent doses of glutamate following chronic denervation of the muscle. 2. A two-pulse method was used to examine recovery from desentization of junctional and extrajunctional receptors. A 'response ratio', i.e. the amplitude of response to the second (test) of a pair of glutamate pulses over to the pulses. The 'response ratio' for extrajunctional depolarizations of innervated fibres increased exponentially with pulse interval, with a time constant of 15.6 +/- 4.7 sec (n = 11). Recovery of extrajunctional receptor populations from desensitization was accelerated after denervation. The recovery kinetics for responses from fibres 6-22 days after denervation were generally described by two exponential terms, with time constants in the range 0.5-10 sec which were inversely related to the glutamate sensitivity of the extrajunctional membrane. For junctional receptors on both innervated and denervated fibres the recovery kinetics were described by a single exponential with a time constant of 0.2-1 sec. 3. The results suggest that the increased extrajunctional glutamate sensitivity which occurs after denervation results from the 'appearance' of glutamate receptors with properties similar to those found at the post-junctional membrane on locust muscle fibres.

Animals↗
PubMed1978

Two components of extracellularly-recorded photoreceptor potentials in the cephalopod retina: differential effects of Na+, K+ and Ca2+.

The ERG of the isolated, superfused half-eye of the cephalopod Sepiola atlantica, evoked by a brief (10 micro second) light flash, has been studied by recording intraretinal potentials with glass microelectrodes. The intensity-response characteristics of the potentials recorded at an electrode fixed at the surface (Vs) can be fitted by a simple equation derived from an equivalent circuit model based on a sodium conductance increase mechanism. Raising the external potassium level reduces the maximal response (deltaVm), but does not alter the half-saturation intensity value (I0). Reducing external sodium does not affect deltaVm, but increase I0. Reducing external calcium also does not affect delta Vm, but decrease I0. These effects are adequately described by the model if it is also assumed (a) that changing the external sodium does not significantly alter the transmembrane sodium gradient, and (b) that sodium and calcium ions compete for the sensitivity control mechanism. Differential-depth recording between the fixed electrode at the surface and another electrode that could be moved into the retina revealed that the two component appearance of the transretinal ERG arose from the superposition of two vitreal-negative waveforms. An initial "fast" component was mainly recorded in the photoreceptive distal segments while a "slow" component was prominent in the more proximal regions of the retina. Perfusion with high K+ salines resulted in a decrease in the amplitudes of both fast and slow components of the response whereas reducing external Na+ reduced the amplitude of the fast component at all light intensities but reduced the amplitude of the slow component only at low intensities. The amplitudes of both the fast and slow components increased on reducing external calcium, but the rate of rise and fall of the fast component was independent of external calcium. The rate of rise of the slow component was also independent of the external Ca2+ level but a minimum in the recovery time (tF) was shifted to a lower intensity value at lower calcium concentrations. The shift of the minimum was to a higher intensity value with lowered sodium perfusing solutions. On the basis of the differential sensitivity of the two components to ion changes, as well as stimulus intensity and intraretinal distribution of the components, it is suggested that they reflect two distinct processes in the light-evoked potential of the photoreceptor cells.

Animals↗