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K Subbarao

Publications and source records attributed to K Subbarao.

At least 19 recordsLinked to original sources
PubMed1992

New CNS-specific calcium antagonists.

Ischemic insults to the brain in stroke or traumatic brain injury produce excessive release of glutamate from depolarized nerve terminals. This excessive glutamate release in turn stimulates massive calcium entry into nerve cells, activating a biochemical cascade that results in cell death. A major pathway of calcium entry into depolarized nerve cells is through voltage-sensitive, high threshold calcium channels. A large fraction of this calcium entry is mediated through "R-type" calcium channels, channels resistant to blockage by dihydropyridine calcium antagonists such as nimodipine. A newly discovered compound derived from spider venom, CNS 2103, antagonizes both R-type channels and dihydropyridine-sensitive ("L-type") calcium channels. This broad spectrum of action, coupled with selectivity for calcium channels over other classes of voltage-sensitive and ligand-gated ion channels, makes CNS 2103 an interesting lead for development of drugs to treat ischemic brain injury. Activation of presynaptic ("N-type") calcium channels in nerve terminals is a primary cause of excessive neurotransmitter release in brain ischemia. Prevention of glutamate release by blockade of N-type channels in glutamatergic nerve terminals may, at an early stage in the pathophysiological cascade, abort the process leading to nerve cell death. Cambridge NeuroScience has developed a novel rapid kinetic approach for monitoring glutamate release from brain nerve terminals in vitro, and this has led to CNS 1145, a substituted guanidine that selectively blocks a kinetic component of calcium-dependent glutamate release mediated by persistent depolarization. Additional evidence suggests that CNS 1145 antagonizes presynaptic N-type calcium channels, and this may account at least in part for its ability to block glutamate release.

Animals↗
PubMed1991

Filter assay technique and quench-flow experiments: examples of receptor-mediated transmembrane ion-exchange measured with membrane vesicles.

Modifications to a quench-flow apparatus are described which allow a rapid, in-line filter assay with immediate washing, in conditions to give minimum background. A design for an effluent spout is presented, which decelerates the liquid by a large factor, prevents splashes, limits the area of the filter exposed to the sample and allows an immediate wash over a larger area. A design for a filter assay funnel for general use is also presented. These devices feature minimal contact of the funnels with the filter disc. Examples are given in which in-line filtration was used to follow transmembrane ion flux in membrane vesicle preparations. In measurements of transmembrane flux with membrane vesicles and radioisotope the filter assay background can be resolved into three components. These are, (1) the uptake of radioactivity by the filter, (2) the radioactivity inside the vesicles not taking part in the specific measurement and (3) the occlusion of radioactivity in aggregated membrane particles on the filter. These different components depend on the conditions in different ways. Techniques for minimizing the background in filter assays are discussed. The importance of rapid filtration and immediate washing is demonstrated. The examples given illustrate that the function of the acetylcholine receptor from E. electricus is not affected by diisopropylfluorophosphate in the conditions used, and that added GABA is not removed from solution in a brain membrane preparation by the GABA uptake mechanisms in the short times of the experiments.

Animals↗
PubMed1989

Responses of gamma-aminobutyrate receptor from rat brain: similarity of different preparation methods; muscimol induced desensitization and chloride exchange.

Chloride-36 exchange into three different membrane vesicle preparations from rat brain homogenate was followed. The different preparations all contained the same sealed vesicular components characterized by their rates of chloride exchange. The GABA-mediated 36Cl- exchange in all the preparations occurred in two phases shown to be mediated by two distinguishable receptors present in the activity ratio of 5:1 as previously described (Cash, D.J., Subbarao, K. 1987. Biochemistry 26:7556, 7562). Reported differences do not result from differences in the membrane preparations used or from the use of a GABA-mimetic instead of GABA, but from experimental differences. The preparations compared were made with mild or vigorous homogenization and with different extents of purification from solutes or membrane components: (i) a synaptoneurosome preparation, (ii) a Ficoll gradient preparation, and (iii) a washed P2 preparation. In each preparation the same four populations of membrane vesicles were characterized by their 36Cl- influx rates: (i) a major population (40-50%) (tau 1/2 = 1.4 min), (ii) a slower exchanging major population (40-55%) (tau 1/2 = 24 min), (iii) a minor population (5-12%) containing active GABA receptor and having the GABA-independent permeability of the slower exchanging population, and (iv) a very small exchange (approximately 2%) (tau 1/2 approximately 0.2 sec). The GABA-independent 36Cl- exchange processes were kinetically first order. The relative quantities of the different vesicle populations varied slightly with the preparation and purification technique. The identity of these components, observed in the different preparations, was attributed to the vesicle formation being dependent on the morphology and properties of the membrane rather than the preparation method. The soluble brain extract was GABA-mimetic with the two observed receptors, causing channel opening and desensitization. But little washing of the membrane was required to observe the function of both receptors. Muscimol was GABA-mimetic with both receptors. With muscimol, channel opening occurred at 2.6-fold lower concentrations while desensitization was unaltered relative to GABA. This is additional evidence that these responses are mediated by different pairs of binding sites. The dependence of desensitization rate on muscimol concentration indicated that there are two binding sites mediating desensitization, as described with GABA.

Animals↗
PubMed1988

Different effects of pentobarbital on two gamma-aminobutyrate receptors from rat brain: channel opening, desensitization, and an additional conformational change.

The effect of pentobarbital on the responses of the gamma-aminobutyric acid (GABA) receptor from rat brain was studied in quantitative measurements of GABA-mediated chloride-exchange rates (reflecting channel-opening equilibrium) and receptor desensitization rates by using 36Cl- tracer ion with native membrane vesicles. Pentobarbital effected the two phases of 36Cl- influx in different ways, supporting previous evidence that these are mediated by two different receptors [Cash, D. J., & Subbarao, K. (1987) Biochemistry 26, 7556; Cash, D. J., & Subbarao, K. (1987) Biochemistry 26, 7562]. Both the chloride-exchange rate and the desensitization rate of the faster desensitizing receptor were increased by pentobarbital at concentrations above 20 microM by an allosteric effect shifting the response curve to lower GABA concentrations. A similar enhancement of the responses of the slower desensitizing receptor occurred up to 200 microM pentobarbital. Two pentobarbital effector sites were involved in the allosteric mechanism. Above 500 microM pentobarbital, both the initial chloride-exchange rate and the desensitization rate of the slower desensitizing receptor were decreased. This inhibition, which was immediate, occurred with saturating as well as low GABA concentrations and therefore was not attributed to decreased GABA binding but to inhibitory sites for pentobarbital, different from the allosteric activating sites and the GABA binding sites. The chloride ion exchange activity was seen to recover with time, at concentrations above 1000 microM pentobarbital, in a process with a very steep dependence on pentobarbital concentration. This reactivation was attributed to the conversion of an initial form of the receptor to a final form that was less inhibited by pentobarbital. The similarity of the effects of pentobarbital on the chloride ion exchange with its effects on electrophysiological measurements supports the fact that these different techniques study the same phenomena. Comparisons of the effects of pentobarbital on desensitization and on high-affinity ligand binding measurements suggest that increased GABA binding at equilibrium reflects an increased conversion to the desensitized state.

Animals↗
PubMed1988

Transmembrane flux and receptor desensitization measured with membrane vesicles. Homogeneity of vesicles investigated by computer simulation.

The use of membrane vesicles to make quantitative studies of transmembrane transport and exchange processes involves an assumption of homogeneity of the membrane vesicles. In studies of 86Rb+ exchange mediated by acetylcholine receptor from the electric organ of Electrophorus electricus and of 36Cl- exchange mediated by GABA receptor from rat brain, measurements of ion exchange and receptor desensitization precisely followed first order kinetics in support of this assumption. In other measurements a biphasic decay of receptor activity was seen. To elucidate the molecular properties of receptors from such measurements it is important to appreciate what the requirements of vesicle monodispersity are for meaningful results and what the effect of vesicle heterogeneity would be. The experiments were simulated with single vesicle populations with variable defined size distributions as well as with mixtures of different populations of vesicles. The properties of the receptors and their density in the membrane could be varied. Different receptors could be present on the same or different membrane vesicles. The simulated measurements were not very sensitive to size dispersity. A very broad size distribution of a single vesicle population was necessary to give rise to detectable deviations from first order kinetics or errors in the determined kinetic constants. Errors could become significant with mixtures of different vesicle populations, where the dispersity in initial ion exchange rate constant, proportional to the receptor concentration per internal volume, became large. In this case the apparent rate of receptor desensitization would diverge in opposite directions from the input value when measured by two different methods, suggesting an experimental test for such kinetic heterogeneity. A biphasic decrease of receptor activity could not be attributed to vesicle heterogeneity and must be due to desensitization processes with different rates. Significant errors would not arise from the size dispersity apparent in subpopulations of vesicles seen by imaging techniques in membrane preparations.

Animals↗
PubMed1987

Desensitization of gamma-aminobutyric acid receptor from rat brain: two distinguishable receptors on the same membrane.

Transmembrane chloride flux mediated by gamma-aminobutyric acid (GABA) receptor can be measured with a mammalian brain homogenate preparation containing sealed membrane vesicles. The preparation can be mixed rapidly with solutions of defined composition. Influx of 36Cl- tracer initiated by mixing with GABA was rapidly terminated by mixing with bicuculline methiodide. The decrease in the isotope influx measurement due to prior incubation of the vesicle preparation with GABA, which increased with preincubation time and GABA concentration, was attributed to desensitization of the GABA receptor. By varying the time of preincubation with GABA between 10 ms and 50 s with quench-flow technique, the desensitization rates could be measured over their whole time course independently of the chloride ion flux rate. Most of the receptor activity decreased in a fast phase of desensitization complete in 200 ms (t 1/2 = 32 ms) at saturation with GABA. Remaining activity was desensitized in a few seconds (t 1/2 = 533 ms). These two phases of desensitization were each kinetically first order and were shown to correspond with two distinguishable GABA receptors on the same membrane. The receptor activities could be estimated, and the faster desensitizing receptor was the predominant one, giving on average ca. 80% of the total activity. The half-response concentrations were similar, 150 and 114 microM for the major and minor receptors, respectively. The dependence on GABA concentration indicated that desensitization is mediated by two GABA binding sites. The fast desensitization rate was approximately 20-fold faster than previously reported rates while the slower desensitization rate was slightly faster than previously reported rates.

Animals↗
PubMed1987

Channel opening of gamma-aminobutyric acid receptor from rat brain: molecular mechanisms of the receptor responses.

The function of gamma-aminobutyric acid (GABA) receptors, which mediate transmembrane chloride flux, can be studied by use of 36Cl- isotope tracer with membrane from mammalian brain by quench-flow technique, with reaction times that allow resolution of the receptor desensitization rates from the ion flux rates. The rates of chloride exchange into the vesicles in the absence and presence of GABA were characterized with membrane from rat cerebral cortex. Unspecific 36Cl- influx was completed in three phases of ca. 3% (t 1/2 = 0.6 s), 56% (t 1/2 = 82 s), and 41% (t 1/2 = 23 min). GABA-mediated, specific chloride exchange occurred with 6.5% of the total vesicular internal volume. The GABA-dependent 36Cl- influx proceeded in two phases, each progressively slowed by desensitization. The measurements supported the presence of two distinguishable active GABA receptors on the same membrane mediating chloride exchange into the vesicles with initial first-order rate constants of 9.5 s-1 and 2.3 s-1 and desensitizing with first-order rate constants of 21 s-1 and 1.4 s-1, respectively, at saturation. The half-response concentrations were similar for both receptors, 150 microM and 114 microM GABA for desensitization and 105 microM and 82 microM for chloride exchange, for the faster and slower desensitizing receptors, respectively. The two receptors were present in the activity ratio of ca. 4/1, similar to the ratio of "low-affinity" to "high-affinity" GABA sites found in ligand binding experiments. The desensitization rates have a different dependence on GABA concentration than the channel-opening equilibria.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

gamma-Aminobutyric acid (GABA) mediated transmembrane chloride flux with membrane vesicles from rat brain measured by quench flow technique: kinetic homogeneity of ion flux and receptor desensitization.

Transmembrane chloride flux mediated by the GABAA receptor and the desensitization of the receptor were followed using quench flow technique with 36Cl- and a membrane preparation from rat cerebral cortex. Measurements in short times allowed these two processes to be resolved. In general the ion-flux activity was desensitized in two phases. A fast phase took place in circa 200 ms (100 microM GABA) followed by a slower phase in several seconds. A minority (10%) of the membrane preparations did not display the fast phase. It is desirable to be able to separate these two phases of desensitization to facilitate analysis of the responses of the receptor. A short preincubation with GABA removed the fast phase from a subsequent measurement. In the absence of the fast phase the whole ion-flux equilibration was seen as a single phase. The measurements presented covering a time range of 0.01 seconds to 10 seconds show a single phase of ion flux which can be described by a first order ion influx process and a single first order desensitization process with a half time of circa 1 s (100 microM GABA). The results imply a single kinetically homogeneous population of vesicles containing a single population of GABA receptor (remaining active) with a single phase of desensitization. An understanding of this homogeneity, and how to ensure it, gives a basis for quantitatively testing the effects of drugs on these responses. Ion flux measurements with quench flow technique are a suitable tool for investigation of the mechanism of action of neurotransmitter receptors from brain.

Animals↗
PubMed1987

Two desensitization processes of GABA receptor from rat brain. Rapid measurements of chloride ion flux using quench-flow techniques.

Two rapid phases of GABA receptor desensitization, which proceeded with a 10-fold difference in rates, were detected in two types of experiment with membrane vesicle preparations from rat cerebral cortex. The time course of GABA-mediated 36Cl- influx progressed in two phases. The 36Cl- influx was decreased, by preincubation with GABA, in two phases. Measurements were made in the time range 10-1000 ms. The major loss of channel opening activity occurred in the faster phase, which was complete in 100 ms with saturating GABA concentrations. The remaining activity decreased in a slower phase in a few seconds with a 10-fold slower rate. The faster phase of desensitization was more than 10-fold faster than previously observed and the slower phase was slightly faster than previously reported measurements with GABA receptor. Both desensitization processes had a similar dependence on GABA concentration with a half response at approximately 100 microM GABA.

Animals↗
PubMed1984

Systemic disorders affecting the thoracic cage.

Conventional chest roentgenograms provide an opportunity to study changes in the thoracic cage produced by systemic or localized disorders. Careful evaluation of abnormalities often gives a clue to the diagnosis of clinically unsuspected disorders. In this article, the authors present a review of developmental, granulomatous, collagen vascular, hematopoietic, metabolic, endocrine, and neoplastic disorders affecting the thoracic cage.

Acromegaly↗
PubMed1983

Deformed skull.

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Humans↗