Effects of cocaine on sodium dependent dopamine uptake in rat striatal synaptosomes.

Initial velocity of uptake of dopamine (DA) has been measured in the presence of 1 microM cocaine as a function of both [DA] and [Na]. Although DA uptake is overwhelmingly dependent on sodium, it appears that a small amount of DA uptake takes place in the absence of sodium. This contrasts with a previous study of the sodium dependence of uptake without cocaine (referred to below as control), in which uptake was found to be 100% sodium dependent. The data were fitted to several rapid equilibrium models and the minimal best fit model identified. The interaction of transporter (C), DA (S), and Na+ (Na) in this present model is identical to the reaction scheme found previously to fit control data (no cocaine). Whereas the control model required translocation only as CNa2S, in the presence of cocaine (I), two additional translocated species are required to fit the data (CS and CNaS). Another previous study of the interaction of carrier and cocaine at a constant [Na]0 predicted that cocaine interacts with a transporter site other than the DA binding site and that uptake takes place as CS and CSI. The present results are consistent with the assumption that the CS and CNaS forms of the present model are actually CSI and CNaSI, since they are required to fit a model of the sodium dependence in the presence of cocaine, but are not required in the absence of cocaine.

Effects of veratridine and cocaine on the kinetics of synaptosomal dopamine release.

Following preloading of striatal synaptosomes with 3H-dopamine (DA), the kinetics of release have been followed for a 60-min incubation period. DA appears to be totally releasable under both depolarizing (veratridine) and nondepolarizing conditions. Cocaine has no significant effect on release under either condition. Release is consistent with a model consisting of two parallel, linear compartments (when plotted as a log function). It is proposed that the slower compartment might represent the operation of the DA transporter, while the faster compartment might represent vesicular release.

A model of the sodium dependence of dopamine uptake in rat striatal synaptosomes.

Initial velocity of uptake of dopamine (DA) has been measured in rat striatal synaptosomes as a function of both [DA] and [Na]. Carrier mediated uptake is totally dependent on external sodium. The data were fitted to a rapid equilibrium model which has been found in previous studies to fit, with appropriate simplification, uptake data for glutamate, GABA, and choline in several brain regions under varying conditions. This model also gives a good fit to the dopamine data. The minimal best fit simplification of this model allows for DA uptake along with two sodium ions and predicts that apparent maximal velocity of uptake should increase with [Na], while the Michaelis-Menten constant should decrease. The minimal best fit model for DA, and a number of kinetic parameters which quantitate the model, are compared to those for the GABA, glutamate, and choline transporters. The results are consistent with a symmetrical, rapid equilibrium model, which has been presented previously for other neurotransmitters and precursors (18). This model offers a unifying basis for understanding the sodium and membrane potential dependence of neurotransmitter transport and the possible participation of transporters in depolarization induced release throughout the CNS.

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.

Central actions of adenosine on pituitary secretion of prolactin, luteinizing hormone and thyrotropin.

Pituitary hormones were measured in plasma in unanesthetized male and female rats prepared with venous and ventricular cannulae following ventricular infusions of adenosine and two adenosine receptor agonists. Adenosine (10, 100, and 200 nmol) injected intraventricularly caused dose-related rises in plasma prolactin (PRL) in males; in females, only the 100- and 200-nmol doses increased PRL. Similar doses of adenosine had little effect on plasma levels of luteinizing hormone (LH) and thyrotropin (TSH) in the same animals. The adenosine receptor agonists L-N6-phenylisopropyladenosine (L-PIA) and 5'-N-ethyl-carboxamideadenosine (NECA) potently stimulated prolactin secretion at doses of 10 and 50 nmol when administered into the lateral ventricle, and at a dose of 2.5 nmol when administered into the third ventricle. The secretion of PRL was antagonized by the adenosine receptor antagonist theophylline (25 nmol), when theophylline was coadministered with NECA and L-PIA. TSH levels were reduced slightly but significantly following the 10- and 50-nmol infusions of L-PIA into the lateral ventricle. The less potent D-isomer of PIA (D-PIA) did not significantly stimulate PRL release. Coupled with studies indicating the presence of adenosine in the basal hypothalamus, our observations indicate a potential neuroendocrine role for this purine in prolactin secretion.

Effect of central administration of phencyclidine on plasma concentrations of luteinizing hormone.

Previous research has demonstrated that excitatory amino acids acting at N-methyl-D-aspartate (NMDA) receptors stimulate the release of luteinizing hormone (LH). Castration also elevates LH, an effect that may also involve NMDA receptors since the specific NMDA antagonist, DL-2-amino-5-phosphonopentanoic acid (AP5), antagonizes this action. Since PCP antagonizes a variety of actions of NMDA agonists, we hypothesized that it would diminish the ability of glutamate, homocysteic acid and castration to elevate LH. Our data support this hypothesis.

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.

The arcuate nucleus: a site for gamma-aminobutyric acid regulation of prolactin secretion.

The effects on prolactin (Prl) secretion following microinfusion of muscimol or N-methyl-aspartate (NMA) into the medial basal hypothalamus (MBH) of male rats was determined. A highly significant increase in Prl occurred following microinfusion of muscimol (500 pmol) into the arcuate nucleus. Microinfusion of muscimol into nearby sites, such as the ventromedial nucleus and dorsal medial nucleus, was not effective in altering Prl secretion. Similarly, microinfusions of artificial cerebrospinal fluid were not effective. NMA (50 pmol), a dose found to affect hormone secretion in other CNS areas, did not alter Prl secretion when infused into the MBH. The present study was able to discriminate between CNS areas by using small volumes (250 nl) and small quantities of the amino acid agonists. This data indicates that GABAergic systems in the arcuate nucleus control Prl release from the pituitary, and that one possible mechanism is via the tuberoinfundibular dopamine system.

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.

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)

The antagonistic action of 6-aminomethyl-3-methyl-4H,1,2, 4-benzothiadiazine-1,1-dioxide (TAG) on the prolactin-releasing action of taurine.

The action of the putative taurine antagonist 6-aminomethyl-3-methyl-4H-1, 2,4-benzothiadiazine-1,1 dioxide (TAG) was examined on the prolactin-releasing action of taurine. The study utilized unanesthetized male rats with indwelling jugular and cerebroventricular cannulas. The intracerebroventricular (I.C.V.) infusion of taurine (0.5 mumole) elicited an increase in prolactin (PRL) secretion elevating plasma levels significantly by 10 min. The I.C.V. infusion of TAG (0.01 mumole) did not alter baseline plasma PRL levels. When a combined I.C.V. infusion of taurine and TAG was given the PRL levels normally induced by taurine were significantly reduced. Neither taurine nor the antagonist TAG altered pituitary secretion of luteinizing hormone.

The stimulation of prolactin secretion by taurine: studies on the site of action.

Taurine's site of action within the central nervous system was localized by studying its effect on prolactin secretion following the microinfusion of 125 nmoles of this amino acid into discrete regions of the brain. Taurine was found to stimulate prolactin secretion only when microinfused into the arcuate nucleus of the hypothalamus. Taurine was not effective in other areas of the hypothalamus or in several extra-hypothalamic sites. Mannitol infusions (125 nmoles) into the arcuate nucleus, used as a control, were without effect on prolactin secretion. These studies extend previous observations on the prolactin-releasing action of centrally administered taurine and suggest further that the arcuate nucleus is one target site for this action.

The central actions of glycine and strychnine on prolactin and LH secretion.

Unanesthetized diestrous female rats were tested for the prolactin response following an intraventricular injection of several doses of the neuroinhibitory amino acid glycine, and the antagonist strychnine. Glycine in doses of 1.0 or 0.1 mumoles increased pituitary prolactin release, significantly elevating plasma hormone levels. Direct pituitary effects for glycine were not observed. Strychnine, a glycine antagonist, was effective in blocking the prolactin release caused by glycine in doses as low as 5 nmoles. Intraventricular glycine administration did not alter pituitary LH release significantly. These studies suggest a central stimulatory role for the neuroinhibitory amino acid glycine in provoking prolactin secretion, and that this effect is strychnine sensitive.

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.

Effects of the neuroexcitatory amino acids aspartate and glutamate on LH secretion.

The effects of infusing aspartate or glutamate into the third ventricle of unanesthetized female animals was examined. LH levels were significantly enhanced within 5-15 min following aspartate (0.5 and 0.2 micromoles), separated by one hour and twenty-four hours, promoted consecutive increases in plasma LH. Plasma FSH levels, however, were not altered in any of the animals examined. This study further supports the findings that neuroexcitatory amino acids promote LH secretion.

Stimulation of LH and FSH secretion following intraventicular injection of cysteic acid but not taurine.

Taurine and related neurally active metabolites, were tested for their ability to influence basal secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH). The animal model used was the unanesthetized, unrestrained adult rat with an indwelling catheter in the jugular vein and a cannula implanted in the right lateral cerebroventricle or anterior pituitary. The proposed inhibitory neurotransmitter traurine, and its metabolic precursor, hypotaurine, did not affect the secretion of LH or FSH following infusion of 0.2 or 2.0 mumol intraventricularly or into the pituitary. In contrast, intraventricular injection of cysteic acid, a neurally excitant amino acid, in doses of 2.0 and 0.2 mumol promoted pituitary secretion of LH in both male and female rats. FSH secretion was also increased slightly by cysteic acid (2.0 mumol). These studies provide additional evidence that excitatory amino acids have a stimulatory role in the release of pituitary gonadotropins.

Stimulation of prolactin secretion by taurine, a neurally depressant amino acid.

The neurally depressant amino acid taurine and related metabolites were tested for their ability to alter prolactin (PRL) secretion in conscious, unrestrained male rats. The intraventricular infusion of taurine (0.2 and 2.0 mumol) elicited a significant increase in PRL secretion. Hypotaurine (0.2 and 2.0 mumol), cysteic acid (2.0 mumol), mannitol (2.0 mumol) and 0.9% saline were ineffective in altering PRL secretion. When infused directly into the pituitary of conscious, unrestrained male rats none of the substances tested stimulated PRL secretion. Taurine (50.0 mumol) was similarly ineffective in stimulating PRL secretion when an in vitro pituitary preparation was used. These studies indicate that taurine is capable of stimulating PRL secretion in the male rat. This effect appears to be mediated centrally, presumably at the hypothalamus.