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When rats were made tolerant to the benzodiazepine tranquilizer chlordiazepoxide (CDPX) by its steady administration, a particular gamma-aminobutyric acid type A (GABAA) receptor in cerebral cortex was modified. Its rate of desensitization in the absence of CDPX was enhanced (3-fold with 10 microM GABA) below saturation with GABA, and the dependence of this rate on GABA concentration was changed from sigmoid to hyperbolic. This mimicked the effect of the presence of CDPX on desensitization of the naive receptor. This receptor has been characterized by its rapid desensitization (t1/2 = 30 msec at saturation). In contrast, a different, slower desensitizing GABAA receptor, on the same membrane, was unaffected, and the initial transmembrane halide exchange rate of the faster desensitizing receptor was unaltered. In the presence of CDPX, the initial halide exchange rate of the modified receptor was enhanced, but the already enhanced desensitization rate was not altered. During chronic presence of CDPX and the development of tolerance, the total signal due to this receptor remained constant at the value before exposure. After discontinuation, the total signal decreased but could be restored to the original value by the presence of CDPX. It was postulated that dependence and withdrawal syndromes result from a decreased ratio of initial chloride flux rate to desensitization rate, caused by an increase in desensitization. The contribution of this effect in vivo would depend on desensitization making a contribution to signal termination [or the fraction of receptors that are inactive (desensitized)]. In the quench flow experiments, the total signal due to this receptor from naive rat did not depend much on GABA concentration or the presence of CDPX because the result of increased channel opening was counterbalanced by increased desensitization. In contrast, the total signal of this receptor from tolerant rat was significantly increased by CDPX or increased GABA concentration. Differences between these experiments and measurements reported with other drugs could be explained if, in those experiments, the halide exchange rate, as well as its desensitization rate, retained an enhanced value in the absence of the drug.

Enantiomers of norbicuculline, (+)[1S,9R] and (-)[1R,9S]erythro-1-[1'-(4',5'-methylenedioxyphthalidyl)]-6,7-meth ylenedioxy-1,2,3,4-tetrahydroisoquinoline and of the N-methyl derivatives {(+)[1S,9R] and (-)[1R,9S]bicuculline} were found to inhibit the progress of the gamma-aminobutyric acid transporter-mediated uptake of 40 microM [14C]gamma-aminobutyric acid into native plasma membrane vesicles from the rat cerebral cortex at 30 degrees C. The values for the dissociation constants of the reversible inhibition, relative to (+)[1S,9R]bicuculline, in order of increasing inhibition, were: (-)[1R,9S]bicuculline, 3.3; (+)[1S,9R]-bicuculline, 1.0; (-)[1R,9S]norbicuculline, 0.4 approximately (+)[1S,9R]norbicuculline; guvacine, 0.02. The norbicucullines have higher potencies than (+)[1S,9R]bicuculline for the gamma-aminobutyric acid transporter, in contrast to the relative potencies of these inhibitors for the inhibition of function and gamma-aminobutyric acid binding of the gamma-aminobutyric acid type A receptor.

Retinoic acid is a signaling molecule involved in the regulation of growth and morphogenesis during development. There are three types of nuclear receptors for all-trans retinoic acid in mammals, RAR alpha, RAR beta, and RAR gamma, which transduce the retinoic acid signal by inducing or repressing the transcription of target genes (Leid, M., P. Kastner, and P. Chambon. 1992. Trends Biochem. Sci. 17:427-433). While RAR alpha, RAR beta, and RAR gamma are expressed in distinct but overlapping patterns in the developing mouse limb, their exact role in limb development remains unclear. To better understand the role of retinoic acid receptors in mammalian limb development, we have ectopically expressed a modified RAR alpha with constitutive activity (Balkan, W., G.K. Klintworth, C.B. Bock, and E. Linney. 1992. Dev. Biol. 151:622-625) in the limbs of transgenic mice. Overexpression of the transgene was associated with marked pre- and postaxial limb defects, particularly in the hind limb, where expression of the transgene was consistently seen across the whole anteroposterior axis. The defects displayed in these mice recapitulate, to a large degree, many of the congenital limb malformations observed in the fetuses of dams administered high doses of retinoic acid (Kochhar, D.M. 1973. Teratology. 7:289-295). Further analysis of these transgenic animals showed that the defect in skeletogenesis resided at the level of chondrogenesis. Comparison of the expression of the transgene relative to that of endogenous RAR alpha revealed that downregulation of RAR alpha is important in allowing the chondrogenic phenotype to be expressed. These results demonstrate a specific function for RARalpha in limb development and the regulation of chondroblast differentiation.

Synthesis of erythro-(±)-[1SR,9RS]-norbicuculline and threo-(±)-[1SR,9SR]-noradlumidine from piperonal was performed using Bischler-Napieralski cyclization as a key step. Resolution gave rise to (+)-[1S,9R]-norbicuculline ([1S,9R] norBIC) and (-)-[1R,9S]-norbicuculline ([1R,9S] norBIC) in >99.5% enantiomeric purity. Bicuculline enantiomers were readily obtained by methylation of the latter products. [1S,9R]BIC was about 70 times more potent than [1R,9S] BIC as an inhbitor of GABA(A) receptor binding and was about 100 and 900 times more potent than [1S,9R] norBIC at pH 7.1 and 5.0 respectively. Similarly, [1S,9R] norBIC was much less potent than [1S,9R] BIC as an inhibitor of GABA-specific (36)Cl(-) ion flux. The observed increase of about two orders of magnitude in the in vitro biological activity caused by N2-CH(3) substitution in [1S,9R] norBIC was attributed to different conformations for erythro- and nor-erythro-bicucullines indicated by (1)H nuclear Overhauser enhancements of [1S,9R] BIC and [1S,9R] norBIC.

We used the short-lived radionuclide, 82Br- to follow gamma-aminobutyrate (GABA) receptor-mediated halide exchange into membrane vesicles from rat cerebral cortex in millisecond and second time regions using quench-flow technique. The radioisotope was prepared by neutron capture [81Br-(n,gamma)82Br-] on irradiation of a natural isotope of bromine, 81Br- in a neutron flux. 82Br- decays by beta-emission with secondary gamma-emission. Possible advantages of 82Br- over 36Cl- in anion tracer measurements include, (a) a short lifetime (t1/2 = 35.3 hr), which alleviates contamination and disposal problems, (b) high counting efficiency (1.54) due to the secondary radiation, (c) measurement with a gamma-counter as well as a beta-counter, (d) a simple preparation not requiring subsequent purification steps giving a specific activity depending on the irradiation time. With 6 hr irradiation time the specific activity was sufficient to make measurements with < 1 mM Br-, which is less than the bromide concentration known to affect the properties of GABAA receptor. The radiotracers, 82Br- and 36Cl- could be compared with the same solution composition. In conditions where a direct effect of binding of halide to receptor does not contribute to a difference in measured ion-flux, 82Br- was translocated only marginally faster than 36Cl-. The effect of chlordiazepoxide (CDPX) (2-250 microM) on the progress of GABA (10 microM)-mediated 82Br- uptake was measured in a time range of 200 msec to 20 sec using quench-flow technique. The two phases of anion exchange previously reported in this experimental model with GABA alone were observed. The rate of 82Br- exchange was increased 2.3-fold at 30-60 microM CDPX and was not further increased with increasing [CDPX]. The rate of halide exchange is a measure of open channel concentration. The isotope exchange rate constant, J, in a membrane vesicle preparation, is a measure of the membrane permeability per internal volume/surface area, J = PmA/V. Receptor desensitization rate was also increased by CDPX, but unlike the isotope exchange rate, it continued to increase up to at least 250 microM CDPX.

L-Glutamate-mediated transmembrane influx of 22Na+ into a native membrane vesicle preparation from rat hippocampus was resolved into components due to glutamate receptor and Na(+)-glutamate symport, by kinetic analysis and pharmacological specificity. Measurements made with a quench-flow technique showed that receptor-mediated cation exchange proceeded in two phases, the faster phase progressively attenuated by a desensitization process with a half time of approximately 90 ms. The influx of 22Na+ continued in the second phase with a half time of 6 s. Receptor-mediated 22Na+ influx was replicated with the glutamate mimetics, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate and kainate but not with N-methyl-D-aspartate and was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione but not by 2-amino-5-phosphonovaleric acid. Receptor-mediated 22Na+ influx did not require glycine and was pH dependent; influx was inhibited at pH values less than pH 5.0. Thus, the receptor-mediated activity was of the gamma-amino-3-hydroxy-5-methyl-4-isoxazole propionate-kainate subtype. The N-methyl-D-aspartate receptor inhibitor 5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine or MK801), increased receptor-mediated transmembrane 22Na+ flux in both phases, but did not accelerate receptor desensitization. Receptor-mediated radiotracer exchange was the major contribution to 22Na+ influx in times less than 5 s and the receptor-mediated responses could be measured with a small correction for sodium-glutamate symport or in the presence of a glutamate-uptake inhibitor. A third phase of 22Na+ influx accessed a different internal volume and proceeded with the same first-order rate constant as [3H]glutamate uptake when measured simultaneously. This was shown to be mediated by the glutamate uptake mechanism, with a half time of 20-40 s varying with the preparation. The Na(+)-glutamate symport became the largest cause of 22Na+ influx after at least 5 s, the final magnitude being, on average, twofold larger than the receptor-mediated 22Na+ influx.

Rates of desensitization of a rapidly desensitizing 4-aminobutyrate (4-Abu) receptor (t1/2 = 33 ms at saturation) were measured in a previously characterized experimental system from rat cerebral cortex. Desensitization was measured at times between 100 ms and several s. Measurements with 300-ms or 1-s 82Br- influx assays after prior incubation of the membrane with 4-Abu covered the whole 4-Abu concentration range of response. Desensitization was not affected by the low Br- concentrations (< or = 1 mM) present. The presence of the tranquilizing drug chlordiazepoxide (Librium) in the prior incubation increased the desensitization rate at 4-Abu concentrations below saturation. This drug changed the dependence of desensitization rate on 4-Abu concentration from the sigmoid response, previously reported with 4-Abu alone, to a hyperbolic dependence. In the presence of chlordiazepoxide the measurements approximated linear Eadie-Hofstee and Lineweaver-Burk plots. This extended the range of response to lower 4-Abu concentrations, so that the relative increase of desensitization rate became larger with decreasing 4-Abu concentration and was about sixfold at 1 microM 4-Abu. The maximum rate, at saturation with 4-Abu was unchanged by chlordiazepoxide. This observation parallels the change in the mechanism of channel opening due to chlordiazepoxide previously reported. All the measurements could be fitted to a simple minimal kinetic model, in which the effect of chlordiazepoxide could be attributed to a change in the receptor with one bound 4-Abu molecule. This could be an increase in desensitization rate or a decrease in dissociation constant of the receptor with one bound 4-Abu molecule and no other change. The effects of the tranquilizer, chlordiazepoxide with this receptor are to extend the 4-Abu concentration range of response 10-fold, to lower 4-Abu concentrations, so that the major part of the response occurs between 0.3 microM and 1000 microM 4-Abu. In addition to the effect on channel opening, the receptor desensitization rate is affected in the same way.

Retinoid receptors are ligand activated transcription factors that regulate gene transcription through a complex network of interactions with members of the nuclear hormone receptor superfamily. Although ligand is required for trans-activation, addition of ligand to mammalian cells in vitro complicates the study of individual activated retinoid receptors. In order to circumvent this problem we have constructed a series of retinoid receptors which do not require ligand for trans-activation. This was accomplished by fusing the acidic activation domain of the herpes simplex viral protein VP16 to the carboxyl terminus of individual retinoid receptors. All of the chimeric receptors were found to exhibit constitutive trans-activation activity in CV-1 and P19 cells when cotransfected with a reporter that contained a trimerized retinoic acid receptor-beta 2 (RAR beta 2) retinoic acid response element. Further analysis conducted on reporters containing either the RAR beta 2 promoter or the rat cellular retinol binding protein II (rCRBPII) promoter showed that promoter specificity was well conserved between the chimeric receptors in the absence of exogenous retinoid and their ligand-induced native counterparts. Moreover, on the RAR beta 2 promoter reporter construct, the chimeric retinoid receptors displayed both cell type and inter- and intrafamily differences in trans-activation, whereas, trans-activation of the rCRBPII in the absence of exogenous ligand in CV-1 and P19 cells was found to be stimulated only by chimeric retinoid X receptor-alpha (RXR alpha). In P19 cells trans-activation of the rCRBPII promoter by RXR alpha v in the absence of exogenous ligand was inhibited by RAR alpha and the constitutive forms of RAR alpha, RAR beta, RAR gamma, RXR beta, and to a lesser extent RXR gamma.

Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA-mediated influx of 36Cl-. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 microM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10-fold at 0.3 microM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 microM GABA, was unaltered. The half-response concentration was reduced from 80 microM to 50 microM. CDPX alone caused no measurable 36Cl- exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel opening mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.

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.

Some data on the concentration range of response and the concentration for half-response (EC50) of gamma-aminobutyric acid (GABA) for the GABAA receptor are reviewed and compared. An analysis of the 36Cl- flux assay demonstrates that both the EC50 and the slope of a Hill plot depend on the ion influx or efflux assay time. The effects of depletion of the 36Cl- concentration gradient during the assay and of receptor desensitization on the result for a range of assay times are considered. The EC50 can be decreased by orders of magnitude by increasing the assay time. The EC50 measured in a finite time is less than the half-response concentration for the response(s) of the receptor. The extent of this difference depends on the receptor concentration per internal volume. The maximal decrease of EC50 depends on the rate of receptor desensitization. The computer simulations showed that a GABAA receptor with a half-response concentration of 100 microM GABA can give 36Cl- flux measurements with an EC50 value 100-fold lower.

Synopsis Few systematic studies have examined individual differences in women's use of facial make-up or the possible psychosocial effects of such use. In the present investigation, the researchers developed several measures of the amount and the situational and temporal patterning of cosmetics use. Among forty-two female college students, differential use was associated with a number of selected personality variables-public self-consciousness, public body-consciousness, social anxiety, and various body-image factors. Through the imagery-induction of cosmetics use and non-use conditions, cosmetics users' self-evaluative responses were studied. In a variety of imagined situations, subjects reported being more self-confident and sociable when wearing as opposed to not wearing their customary cosmetics. Findings were discussed in the context of the role of cosmetics in self-image enhancement and social impression management.

The development of a spectrophotometric stopped-flow method to measure ion flux in membrane vesicles in the millisecond to minute time region is described in detail. The technique is based on fluorescence quenching of an entrapped fluorophore (anthracene-1,5-disulfonic acid) by Cs+. The method has been applied to the measurement of acetylcholine-receptor-mediated ion flux in membrane vesicles prepared from the electric organs of both Electrophorus electricus and Torpedo californica. The method is applicable to any vesicle system in which Cs+ can substitute for either Na+ or K+. Loading of vesicles with the fluorescent dye is accomplished using the routine procedure for making the vesicles. The dye-loaded vesicles can be stored in liquid nitrogen before use. Neither the dye-loading procedure nor the presence of Cs+ changes the permeability of the membrane to ions, allowing ion-translocation measurements to be made in the millisecond to minute time region. The stopped-flow design presented allows two sequential mixings of solutions. The relationship between fluorescence quenching and ion flux as well as the interpretation of the ion flux data is described. It is shown that the data obtained with stopped-flow and Cs+ is identical to data obtained previously using a quench-flow technique and 86Rb+. The advantages of the present method over the quench-flow technique and a similar stopped-flow technique developed previously based on T1+ are described in detail.

The dependence of acetylcholine receptor-controlled transmembrane ion flux on acetylcholine concentration was measured in the msec time region using membrane vesicles and a quench flow technique. Four measurements were made: (1) transmembrane ion flux, (2) rate of inactivation of the receptor, (3) rate of recovery, and (4) ion flux mediated by "inactivated" receptor. A minimum model which relates the ligand binding and ion translocation processes, which has been previously proposed to account for carbamylcholine-induced ion flux, can also account for acetylcholine-induced flux. The integrated rate equation, based on the model, predicts the time dependence of the ion flux over the 160-fold concentration range of acetylcholine investigated. The receptor-controlled ion flux exhibits simple kinetics, and this has permitted the use of simple analytical expressions for the dependence on acetylcholine concentration of the various constants of the minimum mechanism. The evaluation of the constants, and methods for the separation of vesicles which contain functional receptors from those which do not, have led to the determination of the specific reaction rate, J , of the acetylcholine receptor-controlled translocation of inorganic ions. J = 3 x 10 (7) M(?1) sec(?1). The value for J allows one to calculate the number of ions translocated per receptor per msec, ?6 x 10(3). A value of 1 x 10(7) ions translocated per receptor site per unit time has also been determined by analysis of acetylcholine-induced noise in cells. Therefore it becomes possible to integrate the results obtained in two types of measurement of receptor function: chemical kinetics, which establishes the relationship between the ligand binding and ion translocation processes, and noise analysis, which measures elementary steps in the formation of receptor-formed ion channels.

Two molecular processes, the binding of acetylcholine to the membrane-bound acetylcholine receptor protein and the receptor-controlled flux rates of specific inorganic ions, are essential in determining the electrical membrane potential of nerve and muscle cells. The measurements reported establish the relationship between the two processes: the acetylcholine receptor-controlled transmembrane ion flux of (86)Rb(+) and the concentration of carbamoylcholine, a stable analog of acetylcholine. A 200-fold concentration range of carbamoylcholine was used. The flux was measured in the millisecond-to-minute time region by using a quench flow technique with membrane vesicles prepared from the electric organ of Electrophorus electricus in eel Ringer's solution at pH 7.0 and 1 degrees C. The technique makes possible the study of the transmembrane transport of specific ions, with variable known internal and external ion concentrations, in a system in which a determinable number of receptors is exposed to a known concentration of ligand. The response curve of ion flux to ligand was sigmoidal with an average maximum rate of 84 sec(-1). Carbamoylcholine induced inactivation of the receptor with a maximum rate of 2.7 sec(-1) and a different ligand dependence so that it was fast relative to ion flux at low ligand concentration but slow relative to ion flux at high ligand concentration. The simplest model that fits the data consists of receptor in the active and inactive states in ligand-controlled equilibria. Receptor inactivation occurs with one or two ligand molecules bound. For channel opening, two ligand molecules bound to the active state are required, and cooperativity results from the channel opening process itself. With carbamoylcholine, apparently, the equilibrium position for the channel opening step is only one-fourth open. The integrated rate equation, based on the model, predicts the time dependence of receptor-controlled ion flux over the concentration range of carbamoylcholine investigated. The values of the constants in the rate equation form the basis for predicting receptor-controlled changes in the transmembrane potential of cells and the conditions leading to transmission of signals between cells.

Exposure of the acetylcholine receptor to acetylcholine, or its stable analogue carbamylcholine, inactivates (desensitizes) the receptor. Inactivation of receptor-controlled ion (86Rb+) flux in the presence of different concentrations of carbamylcholine (12.5 microM to 28 mM) was measured in the millisecond to minute time region, using a quench flow technique and membrane vesicles prepared from the electric organ of Electrophorus electricus. Three different kinetic measurements were made to establish the relationship between carbamylcholine concentration and the ion translocation process: (i) the rate of inactivation of the ion translocation process; (ii) the rate of recovery of the inactivated receptor upon removal of carbamylcholine; and (iii) the rate of the ion flux mediated by equilibrium mixtures of active and inactive receptor forms. The kinetics of these three processes follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence could be used. Therefore, it was possible to determine the value of the rate constants in a scheme relating the ligand binding steps to ion translocation, and to predict the dependence of these rate constants on carbamylcholine concentration over the 200-fold range investigated.

The specific reaction rate (J) of the acetylcholine receptor-controlled ion translocation has been determined. In eel Ringer's solution (pH 7.0) at 1 degrees C, J = 3 X 10(7) M-1 sec-1. J is an intrinsic constant that is characteristic of the receptor and independent of other properties of a receptor-containing cell that also determine the rates of ion translocation. Membrane vesicles (prepared from the electric organ of Electrophorus electricus) and a flow-quench technique that has a millisecond time resolution were used to measure the receptor-controlled ion translocation. Using the value of J and the molar concentrations of receptor sites and inorganic ions, we calculated that 6 X 10(3) ions are translocated per msec per receptor. Analysis of electrical noise in frog muscle cells at temperatures above 8 degrees C [Nether, E. & Stevens, C. F. (1977) Annu. Rev. Biophys. Bioeng. 6, 345-381] gave a value of about 1 X 10(4) ions msec-1 per channel. Thus, each technique gives essentially the same result. It is now possible, therefore, to correlate the results obtained when receptor function is measured in two different ways in membrane vesicles and in muscle cells: (i) chemical kinetic measurements, using membrane vesicles, which relate the ligand binding and ion translocation processes and (ii) analysis of acetylcholine noise in muscle cells [Katz, B. & Miledi, R. (1972) J. Physiol. (London) 224, 665-699], which allows one to measure elementary steps in the formation of ion channels through the cell membrane.

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.

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)

Acetylcholine-induced flux of inorganic ions across membranes and inactivation of the acetylcholine receptor were measured at pH 7.0, 1 degrees C, over a 5000-fold concentration range of acetylcholine. Receptor-containing electroplax membrane vesicles prepared from Electrophorus electricus and a quench-flow technique were used, allowing flux to be measured in the 2-msec to 1-min time region. Five different measurements were made: (i) rate of ion translocation with the active state of the receptor, (ii) rate of the slower ion translocation after equilibration of active and inactive receptor states, (iii) rate of inactivation, (iv) equilibrium between active and inactive forms of the receptor, and (v) reactivation of inactivated receptor. The kinetics of the steps in the receptor-controlled ion flux follow single-exponential rate laws, and simple analytical expressions for their ligand concentration dependence can be used. Thus, the rate and equilibrium constants in a scheme that relates the ligand binding steps to ion translocation could be evaluated. It was found that the dependence of the receptor-controlled ion translocation over the concentration range investigated obeys the integrated rate equation based on the proposed mechanism. The flux rate before inactivation was approximately 10(7) ions sec-1 per receptor, which is comparable with that measured electrophysiologically in muscle cells. The half-time of inactivation is approximately 100 msec when the receptor is saturated with acetylcholine. The specific reaction rate of the ion translocation (J) is 3 X 10(7) M-1 sec-1. The results support a minimum reaction mechanism previously proposed on the basis of experiments in which carbamylcholine was used.

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.

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.

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.

Suberyldicholine-induced ion translocation in the millisecond time region in acetylcholine receptor rich membrane vesicles prepared from the electric organ of Electrophorus electricus was investigated in eel Ringer's solution, pH 7.0, 1 degree C. A quench-flow technique with a time resolution of 5 ms was used to measure the transmembrane flux of a radioactive tracer ion (86Rb+). JA, the rate coefficient for ion flux mediated by the active form of the receptor, and alpha, the rate coefficient for the inactivation of the ion flux, increase with increasing suberyldicholine concentrations and reach a plateau value at about 15 microM. At higher suberyldicholine concentrations (greater than 50 microM), a concentration-dependent decrease in the ion flux rate was observed without a corresponding decrease in the rate of receptor inactivation. This regulatory effect was not observed with acetylcholine or carbamoylcholine. The minimal kinetic scheme previously presented for acetylcholine and carbamoylcholine, modified by the inclusion of an additional regulatory ligand-binding site for suberyldicholine and characterized by a single dissociation constant, KR, is consistent with the results obtained over a 10 000-fold concentration range of this ligand. Rate and equilibrium constants pertaining to this scheme were elucidated. Suberyldicholine binds to the regulatory site (KR = 500 microM) approximately 100-fold less well than to its activating sites, and the binding to the regulatory site has no effect on the inactivation (desensitization) rate coefficient alpha [alpha(max) = 5.7 s-1], which is comparable to that observed with acetylcholine. The maximum influx rate coefficient [JA(max) = 18.5 s-1] is approximately twice that obtained when carbamoylcholine is the activating ligand and somewhat higher than when acetylcholine is used.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The marine mollusc Aplysia californica has proved to be a useful preparation for analyzing the development of learning and memory on both behavioral and cellular levels. An important issue in this analysis concerns the anatomical substrate upon which learning is superimposed during development. As a first step in examining this question, in the present study we have determined the number of neurons in all the major central ganglia at each stage during juvenile development, a time when several forms of learning first emerge in Aplysia. We found that a large and highly nonlinear proliferation of neurons occurs during juvenile development, with the greatest increase in cell number occurring during a specific juvenile stage: Stage 12. The neuronal proliferation is system-wide, occurring in each of the central ganglia simultaneously, suggesting the action of a general developmental signal or trigger (perhaps a hormone). Accompanying the increase in neuron number in Stage 12 there is a large increase in neuropilar volume (150-fold), which significantly increases the opportunity for synaptic interactions late in juvenile development.

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.

A simple technique utilizing a bifurcated fiber light guide for obtaining the reflectivity spectra of specular and diffusely reflecting surfaces is described. The technique can be used in certain situations where conventional reflectometers are not practical.

The thicknesses of UO(2) films from 100 A to 1800 A on uranium substrates were determined from reflectance measurements in the visible region. The reflectance measurements on the U-UO(2) system were analyzed by two different methods to determine film thicknesses. In the first method, film thicknesses were determined by comparing theoretical reflectance calculations with the experimental reflectance measurements. In the second method, film thicknesses were determined by obtaining the best match of the clorimetric properties (wavelength, excitation purity, and luminous reflectance) of the sample with the colorimetric properties of a predetermined film thickness calibration curve.