Dopamine uptake in five structures of the brain: comparison of rate, sodium dependence and sensitivity to cocaine.

The rate of uptake of dopamine (DA) per microgram protein and the sensitivity of uptake to sodium ([Na]o) and cocaine, have been measured in synaptosomes from five structures in the brain of the rat: striatum, nucleus accumbens, neocortex, limbic cortex and thalamus. Probably reflecting the number of DA terminals, there was a wide variation in the rate of uptake in the different structures: uptake was far greater in the striatum and nucleus accumbens (10-20-fold) than in the neocortex, limbic cortex or thalamus. Uptake in all structures was inhibited by cocaine. With high [Na]o, the IC50's varied from 1.10 microM for the thalamus to 3.32 microM for the nucleus accumbens. Maximum percentage inhibition varied from 76.0 for limbic cortex to 96.8 for nucleus accumbens and 97.7 for striatum; thus most uptake was carrier-mediated. With low [Na]o, IC50 for the nucleus accumbens was unchanged, while the IC50's for the striatum and limbic cortex were less. Maximum percentage inhibition was similar to that found for high [Na]o. Although the sensitivity to [Na] varied, synaptosomes from all areas showed Na-dependent uptake; lowering [Na] in the incubation medium from 133.2 to 9.5 mM reduced the uptake by a minimum of 36.9% in the neocortex to a maximum of 78.0% in striatum. Neither the rate of uptake nor the Na-dependence correlated precisely with the sensitivity to cocaine but the two structures which showed the greatest rate of uptake (nucleus accumbens and striatum) also showed the greatest sensitivity to [Na]o. Generally, the rate of uptake correlated with the density of DA terminals and number of cocaine binding sites as reported by others.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of cocaine on membrane potential, on membrane depolarization by veratridine or elevated [K]o and on sodium/potassium permeability ratios in synaptosomes from the limbic cortex of the rat.

Effects of cocaine on the synaptosomal membrane potential (Em), on membrane depolarization induced by veratridine or elevated [K]o and on sodium/potassium permeability ratios (pNa/pK), have been measured in buffer containing either low or high [Na]. Fluorescence of the dye rhodamine 6G was used to measure the membrane potential. Cocaine began to reduce the Em (depolarized) at concentrations between 10(-6) and 10(-5) M in low [Na] buffer and between 10(-5) and 10(-4) M in high [Na] buffer. Maximum depolarization (with 10(-3) M cocaine) was 21 mV in low [Na] buffer and 11 mV in high [Na] buffer. Cocaine also reduced the depolarization caused by veratridine or elevated [K]o; the effective concentration of cocaine in reducing the response to veratridine was one-tenth that necessary to reduce the response to elevated [K]o. The antagonism by cocaine of the response to veratridine was similar to that found by other investigators; however, this action would tend to oppose depolarization and thus cannot explain the depolarizing effect of cocaine alone. The antagonism by cocaine of the depolarization caused by elevated [K] was consistent with a reduction in pK; such a change in pK could explain the observed reduction in Em caused by cocaine alone. The effect of cocaine (10(-3) M) on the Em was also measured as a function of [K]o at low and high [Na]o. Cocaine caused membrane depolarization at all [K]o's (3.9-19.2 mV), an effect that was somewhat greater in the low [Na] medium. These measurements of Em were fitted to the Goldman equation and the ratio of pNa/pK estimated. The presence of cocaine increased the estimate of pNa/pK by 45.7%, presumably by reducing pK.

Aging of membrane transport mechanisms in the central nervous system--high affinity choline transport in rat cortical synaptosomes.

High affinity choline transport has been studied in cortical synaptosomes from rats ranging from 2 months to 30 months of age. Initial velocities were measured as a function of both choline and sodium concentration. These data give similar fits to a kinetic model which was previously found to give minimal best fit to data from 2-month-old animals. Thus, aging has apparently not altered the fundamental mechanism by which carrier, sodium, and choline interact in the process of uptake. Although some small differences in initial velocities were found between data from young and older animals, these differences showed no pattern and were not statistically significant. It is concluded that the choline transporter is unaffected by age, in contrast to the transporters for GABA and glutamic acid, whose transport capacities were found to decline with age.

Aging of membrane transport mechanisms in the central nervous system. GABA transport in rat cortical synaptosomes.

A kinetic study of sodium dependent GABA transport has been made in synaptosomes from 30-month old Long-Evans rats and compared to results from 2-month old animals. Initial velocity of uptake was measured as a function of both sodium and GABA concentration, and these data were than fitted to the model which was found to give best fit for the 2-month data. An excellent fit was also obtained for the 30-month data. Thus there has been no change with age in the fundamental mechanism by which carrier, sodium, and GABA interact in the process of transport. However, quantitative changes were found to occur with age both in initial velocity of uptake and in those constants which quantitate the model. The rate equation for the model was utilized along with the best fit constants to define and calculate certain parameters which were then used to quantitatively compare the transport mechanism in young and aged animals.

Aging of the high affinity GABA transporter in synaptosomes from the hypothalamus of the rat.

Initial velocity of GABA uptake in hypothalamic synaptosomes has been measured in aged animals as a function of both sodium and GABA concentration and compared to previous studies in young animals. The data give good fit to the model found previously to give minimal best fit to control data. Thus it appears that aging has not resulted in a change in fundamental mechanism by which carrier, sodium, and GABA interact in the process of transport. However, changes were found in the constants which quantitate the model. As a consequence, there is a reduction in transport capability in the aged animals which is particularly apparent at the higher sodium concentrations. The best fit constants were used along with the rate equation from the model to calculate several kinetic parameters which are useful in comparing transport mechanisms. In the physiological range of sodium concentration, those kinetic parameters related to maximal transport capabilities declined with age, while apparent carrier affinities increased with age. Similar results were found in previous studies of GABA (Wheeler, 1982) and glutamic acid (Wheeler, 1980a) transport in cortical synaptosomes.

Endogenous GABA concentration in cortical synaptosomes from young and aged rats.

The objectives of this study are threefold: to compare the gamma-aminobutyric acid (GABA) concentration of a preparation of cortical synaptosomes from young male rats to that of aged rats; to compare the GABA concentration in synaptosomes retained by Millipore filtration for young and aged rats; and to compare the GABA concentration in the synaptosomal preparation to that in synaptosomes retained by Millipore filtration for young and aged rats. The GABA concentration is measured in cortical synaptosomes from Long-Evans rats at 2 months and 30 months of age. Concentration of the synaptosomal preparation declined significantly from 2.97 mM in 2-month animals to 2.68 mM in 30-month animals. In order to compare the results with previous studies of GABA transport, in which Millipore filters are used to separate synaptosomes from incubation medium, the GABA concentration is also measured following placement of synaptosomes on 0.65 mu pore size Millipore filters. For both young and aged animals, this population of synaptosomes is found to contain GABA at a concentration more than triple that in the overall population. This is the expected result if the filtration process reduces the non-synaptosomal component of the total preparation, since GABA uptake is known to be a function of the nerve-ending component. Comparison of 2-month and 30-month synaptosomes reveals that the GABA concentration of those synaptosomes retained by the filtration process declined to a greater extent in the aged group than did the content of the overall population (from 9.33 mM to 8.37 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of the aging of the high affinity L-glutamate transporter in rat cortical synaptosomes.

Initial velocities of cortical synaptosomal glutamate uptake are measured as a function of both glutamate and sodium concentration in 10- and 18-month old animals and compared to previous results from 2- and 30-month old animals. As a percentage of 2-month values, mean velocity of uptake falls to 87% in the 10-month group and to 79% in the 18-month group, with no further change between 18 and 30 months. The data give minimal best fit to the same model for interaction of carrier with glutamate and sodium as found previously for 2- and 30-month old animals. Thus the basic mechanisms involved in transport do not change with age. However, the kinetic parameters which describe the transporter do change with age, the primary changes being in those parameters reflective of the transport capacity of the carrier. Va, apparent maximal velocity of uptake, declines most rapidly between 2 and 30 months, continues to decline rapidly between 10 and 18 months, and then declines slowly between 18 and 30 months. Jm, rate of uptake at infinite sodium concentration, declines most rapidly between 2 and 10 months, with a much slower decline between 10 and 30 months. Kt, apparent Michaelis-Menten constant, changes but little with age; at 30 months, Kt is only 6-11% less than at 2 months. Although the total change is only 9-10%, there appears to be a slow, steady decline in KNa, the sodium concentration giving an uptake equal to Jm/2, between 2 and 30 months of age.

Hypothalamic site of action for N-methyl-D-aspartate (NMDA) on LH secretion.

Excitatory amino acids have been shown to increase luteinizing hormone (LH) secretion following ventricular or systemic administration. In the present study we attempted to determine possible hypothalamic sites of action for the potent excitatory amino acid agonist, N-methyl-D-aspartate (NMDA). The ability of NMDA to enhance LH release was tested in male rats following infusion into the medial preoptic nucleus (MPO), anterior hypothalamic nucleus (AHY), ventromedial hypothalamic nucleus (VMH), and arcuate nucleus (ARC). In the MPO, infusion of 50 or 500 pmole NMDA increased pituitary LH secretion, resulting in a 2-7 fold increase in plasma LH. The 50 pmole dose was selected to test more caudal hypothalamic sites. Plasma LH levels were not affected following microinfusion of NMDA (50 pmole) into the AHY, VMH, and ARC. The present results indicate a regional specificity for NMDA in the enhancement of LH secretion. This regional specificity may reflect either a greater density of LHRH perikarya in the MPO or the presence of specific amino acid receptors on neuronal elements in the MPO, but not on neuronal elements in the other areas tested.

Locus of interference on the Stroop test.

On the Stroop test subjects are presented with a random sequence of color names printed in random colors of ink. They are asked to go through the list twice, once reading the words aloud and once naming the ink colors. In this experiment 36 college students were also asked to go through the lists using their fingers to make push button responses. With verbal responses naming the ink colors took nearly twice as long as reading the words, thus replicating the usual Stroop test results. With either the left hand or the right there was no difference between the time required by the subjects to respond to the words and to the ink colors. The disappearance of the Stroop effect with finger responses suggests strongly that the interference normally shown on the Stroop test occurs primarily in the processing of the verbal response.

Are there both low- and high-affinity glutamate transporters in rat cortical synaptosomes?

Kinetics of sodium dependent glutamic acid transport have been studied in rat cortical synaptosomes at sufficiently high glutamic acid concentrations ([G]) to delineate the "low affinity" transporter. Computer optimization techniques were used to fit the data to models which account for the sodium and substrate dependence of uptake. The data fit about equally well models consisting of two carriers (Model 1) or one carrier plus a linear component (Model 2). However, the results of further studies were inconsistent with Model 1, but totally consistent with Model 2. Thus the results are incompatible with the presence of both high- and low-affinity carriers. The carrier model found in previous studies of high affinity glutamate transport predicts the effects of high [G] and [Na] observed in the present study. The biphasic effect of [Na] on velocity of uptake is the logical consequence of the operation of this model. The rate equation for this model has been utilized to define and compute kinetic parameters which characterize the transporter. These kinetic functions are remarkably similar in shape and magnitude to previous estimates from the studies of the high affinity transport (low [G]). The results of other studies by the author which corroborate and expand the predictions of the kinetic model are discussed. These have been combined with the present results to formulate a rather comprehensive model of glutamate function. This model can be used to describe function in terms of mathematical equations and to make predictions from these equations. These equations relate velocity of uptake and the kinetic parameters to sodium and substrate concentration, velocity to membrane potential, distribution ratio to the electrochemical potential, and release to time, compartment sizes, and exchange constants. Such processes as concentration in the presynaptic terminal, depolarization induced release, re-uptake following stimulus induced release, and postsynaptic depolarization are all possible consequences of the operation of this model. The wide applicability of the model to the transport of other substrates in addition to glutamate is discussed.

Sodium dependence of GABA transport in rat hypothalamic synaptosomes.

The sodium dependence of gamma-aminobutyric acid (GABA) uptake has been studied in rat hypothalamic synaptosomes, and the results compared to previous studies in cortical synaptosomes. Initial velocity of GABA uptake was measured as a function of both GABA and sodium concentration, and these data were fitted to the rate equation for each of several plausible models. The minimal best-fit model was found to be identical to that for cortical synaptosomes. However, the constants which quantitate the model were found to differ for hypothalamus and cortex. As a result, uptake at any combination of [Na] and [G] in hypothalamic synaptosomes is approximately double that in cortical synaptosomes. The rate equation for the minimal best-fit model was utilized to define and compute certain parameters which are useful in comparing transport mechanisms (Vmax, Va, Kt, Jm, and k Na). In all cases, differences were found between hypothalamus and cortex.

Sodium dependence of high affinity glutamic acid transport in cortical synaptosomes--a comparison of Long-Evans and Sprague-Dawley rats.

The sodium dependence of high affinity glutamic acid transport has been studied in cortical synaptosomes from Sprague-Dawley rats and compared to previous studies with Long-Evans rats. Initial velocity of uptake was measured as a function of both glutamate and sodium concentration, and the data fitted to the rate equations for the same set of models that was utilized in the Long-Evans studies. The minimal best-fit model was found to be identical to that found previously for Long-Evans animals. Thus there are no fundamental differences in the basis of the sodium dependence of high affinity glutamate transport between the two groups. However, since the constants describing the best fit model are different for the two groups of animals, there are quantitative differences in the transport mechanism between the two groups. The rate equation for the minimal best-fit model permits certain functions to be defined in terms of dissociation and translocation constants, substrate, sodium, and total carrier concentrations. These functions were calculated and then utilized to demonstrate the quantitative differences in the transport mechanism in the two groups.

Effects of polarizing currents and repetitive stimulation on the uptake of amino acids by peripheral nerve.

The effects of polarizing currents and repetitive stimulation on the uptake of representative amino acids have been studied in peripheral nerve. Glutamic acid, lysine, pheylalanine, and GABA were chosen for study as representatives of different carrier-mediated transport mechanisms. Depolarizing currents inhibited uptakes of glutamic acid, lysine, and GABA; maximum inhibitions were 56.1%, 20.0%, and 16.7%, respectively. Pheylalanine uptake was not inhibited by polarizing currents under any experimental conditions. Only glutamic acid uptake was inhibited by hyperpolarizing current, with a maximum inhibition of 43.9%. Stimulation had no significant effect on uptake of lysine, phenylalanine, or GABA but was found to potentiate uptake of glutamic acid by 12%.

The effect of membrane potential on initial velocity of GABA uptake and steady state distribution ratio in rat cortical synaptosomes.

Initial velocity of synaptosomal GABA uptake and steady state distribution ratio (DR) have been measured as a function of medium potassium concentration [( K]o) and in the presence of veratridine. Previous studies of the effect of [K]o and veratridine on membrane potential have been utilized to relate GABA uptake to membrane potential. Both initial velocity of uptake and steady state DR are functions of membrane potential; initial velocity and in DR are linearly related to membrane potential. Final DR is determined by the electrochemical potential gradient for the transported ions. Previous studies of the relation of [Na]o to uptake have shown that either one or two sodium ions may be translocated with each GABA molecule. Present results are consistent with this assumption if it is also assumed that an anion is co-transported with each GABA molecule.

Kinetics of D-aspartic acid release from rat cortical synaptosomes.

A kinetic study has been made of the release of D-aspartate from rat cortical synaptosomes following pre-loading with labelled D-aspartate, and the results compared to a previous study of the release of the acidic amino acids glutamate plus aspartate following pre-loading with labeled L-glutamate. Qualitatively, the results of the two studies are similar. The D-aspartate taken up during the pre-load period appears to be totally releasable. However, release is greatly increased by depolarizing media. The increased rate of release induced by increasing [K]o is independent of the [Ca]o, while veratrine-induced release is inhibited by [Ca]o. Release is from more than a single compartment, since plots of the log10 of the synaptosomal D-aspartate content (calculated from the label content) as a function of the incubation time are non-linear for all incubation solutions. In the previous study which utilized L-glutamate pre-loading, the results were consistent with either a model consisting of two passive compartments (that is, synaptosomal content T as a function of time is given by Ae-Kat + Be-Kbt, in which A and B are compartment sizes, Ka and Kb are exchange constants, and t is incubation time) or a model consisting of one passive compartment (Ae-Kat) and one saturated carrier compartment (T-Kbt, in which T = total content at zero time and Kb = maximal velocity). The present results with D-aspartate also give excellent fits to these models. However, there are some quantitative differences in the estimates of the compartment sizes and exchange constants, which are obtained by optimizing the fit of the data to the equation for each model. Although most of these quantitative differences appear to be minor, one difference between the two studies is of potential significance in interpretation of the results. In the glutamate study, all depolarizing media were found to reduce the exchange constant for the carrier mechanism, while in the present study, depolarizing media were found to increase the exchange constant, with the exception of veratrine-containing medium without calcium.

A model of GABA transport by cortical synaptosomes from the Long-Evans rat.

The initial velocity of uptake of GABA by cortical synaptosomes from the Long-Evans rat has been measured as a function of both sodium and GABA concentration. These data were then fitted to the rate equation for each of several possible models, and the models giving least error were identified. Although one cannot unequivocally distinguish between two of the models on the basis of the goodness of fit, deductions based on the fits of subsets of the models point to one of the two models, The major predictions from this model are as follows: 1) Uptake of GABA is totally sodium-dependent. 2) Although plots of 1/v versus 1/[Na]2 are nonlinear, the coupling ratio for transport (Na/GABA) is 2. 3) For transport to take place, the order of combination with carrier must be Na, Na, GABA. 4) Maximal velocity will occur only at infinite Na and GABA concentrations. 5) There is a sigmoidal relationship between apparent maximal velocity (Va) and [Na]. 6) Kt, the [GABA] that gives a velocity equal to Va/2, rises and then falls as [Na] is increased from zero, passing through a maximum at 33.52 mM [Na]. 7) The relationship between initial velocity and [Na] is sigmoidal. 8) Jm, rate of uptake with infinite [Na], is hyperbolically related to [GABA]; Jm approaches Vmax as [GABA] becomes very large. 9) kappa Na, the [Na] giving a velocity equal to Jm/2, declines rapidly from 10(-7) M to 10(-5)M GABA, but is essentially constant at 10(-4)M above. 10) One GABA molecule is translocated per carrier molecule. The model is consistent with observations in the literature concerning the effects of sodium on the kinetics of transport.

Increase in K+ and alpha-AIB active transport in CHO cells after low [K+] treatment.

We have studied the effects of prolonged incubation in low [K+] medium (approximately 0.3 mM) on both K+ and amino acid transport in Chinese hamster ovary (CHO) cells. When incubated in low [K+] medium, CHO cells redressed partially the loss of intracellular K+ after 12 h. After 24 h of incubation, both the activity of Na+-K+-ATPase in crude homogenates, and the transport capacity (Vmax) for ouabain-sensitive (i.e., active) K+ influx approximately doubled. The magnitude of the ouabain-insensitive (i.e., passive) K+ influx decreased by 50%. Thus the regulatory response involves an apparent increase in Na+-K+ pump and a decrease in K+ leak. The transport capacity for the nonmetabolized amino acid, alpha-aminoisobutyric acid (alpha-AIB), also increased after 24 h in low [K+] medium. The Vmax for the Na+-dependent (i.e., active) alpha-AIB influx increased by about 150%, and the magnitude of the Na+-independent influx increased by 20-40%. These changes in alpha-AIB transport result in a twofold greater capacity to accumulate this amino acid. Thus the regulation of K+ and alpha-AIB transport systems appears to be linked and possible mechanisms of this linkage are discussed.

Effect of castration on the high affinity glutamate transporter in rat hypothalamic and cortical synaptosomes.

High affinity transport of glutamic acid has been studied in cortical and hypothalamic synaptosomes from castrated male rats and compared to normal controls. For hypothalamic synaptosomes, both initial velocity of uptake of Va (apparent maximal velocity) were found to be about one-third lower in the castrated animals. Kt (glutamate concentration giving Va/2), however, was reduced by only 5%. Initial velocity of uptake in cortical synaptosomes was measured as a function of both sodium and glutamate concentration. Reductions in uptake subsequent to castration were found to be much less for cortical synaptosomes (2-15%) than for hypothalamic synaptosomes. Fit of these data to various models for the sodium dependence of transport resulted in the same minimal best fit model as that found for control animals. Thus castration does not alter the fundamental nature of the mechanism by which carrier, sodium and glutamate interact in the process of transport. However, quantitative changes were found to occur, as reflected in the best fit constants. These constants were used along with the rate equation for the minimal best fit model to calculate certain parameters which were then used to delineate the quantitative changes in the transporter following castration. A neuroregulatory role for glutamate in gonadotropin secretion has been recently proposed; the present study now provides additional information on the relationship between reproductive function and one aspect of glutamatergic synaptic function, namely, the high affinity transport system.