Interstitial 3-Methoxytyramine Reflects Striatal Dopamine Release: An In Vivo Microdialysis Study

Previous ex vivo studies have provided indirect evidence that the dopamine (DA) metabolite 3-methoxytyramine (3-MT) may be a useful index of DA release in vivo. In the present study, in vivo microdialysis was utilized to assess directly the relationship between extracellular DA and 3-MT in the striatum of rats following a variety of pharmacological manipulations. Apomorphine, a DA receptor agonist, produced a rapid, transient decrease in both DA and 3-MT. Conversely, the DA receptor antagonist haloperidol produced a concomitant increase in extracellular DA and 3-MT. Increases in DA and 3-MT were also noted following the administration of the DA uptake inhibitor, bupropion. Local application of tetrodotoxin resulted in the complete elimination of measurable amounts of DA and 3-MT in the dialysate, gamma-Butyrolactone also greatly decreased DA and 3-MT. Finally, d-amphetamine produced a large increase in DA and 3-MT in animals that had been treated previously with gamma-butyrolactone. The Pearson correlation coefficients for DA and 3-MT following these manipulations ranged from 0.87 to 0.97. These data indicate that interstitial 3-MT is an accurate index of DA release. However, when compared with previous ex vivo findings, the present results also suggest that changes in tissue concentrations of 3-MT may not reliably reflect DA release following certain pharmacological manipulations.     

Automated selective dissociation of cells from different regions of multicellular spheroids

Summary In this report we describe a new apparatus which has been developed for the automated selective dissociation of multicellular spheroids into fractions of viable cells from different locations in the spheroid. This device is based on the exposure of spheroids to a 0.25% solution of trypsin under carefully controlled conditions, such that the cells are released from the outer spheroid surface in successive layers. Study of the spheroid size, number of cells per spheroid, and sections through the spheroid with increasing exposure to trypsin demonstrate the effectiveness of this technique. The technique has been successfully used on spheroids from five different cell lines over a wide range of spheroid diameters. We also present data detailing the effect of varying the dissociation temperature, the mixing speed, the trypsin concentration, and the number of spheroids being dissociated. The new apparatus has several advantages over previous selective dissociation methods and other techniques for isolating cells from different regions in spheroids, including: a) precise control over dissociation conditions, improving reproducibility; b) short time to recover cell fractions; c) ability to isolate large numbers of cells from many different spheroid locations; d) use of common, inexpensive laboratory equipment; and e) easy adaptability to new cell lines or various spheroid sizes. Applications of this method are demonstrated, including the measurement of nutrient consumption rates, regrowth kinetics, and radiation survivals of cells from different spheroid regions. This work was supported by grants CA-36535, CA-22585, and RR-02845 from the National Institutes of Health, Bethesda, MD, the National Flow Cytometry Resource (NIH grant RR-01315), and by the Department of Energy, Washington, DC.     

Pharmacologic features and effects of neuroleptics.

Neuroleptic drugs reduce the severity and prevent the recurrence of symptoms of schizophrenia. Recent studies indicate that these drugs probably produce their antipsychotic effects by blocking dopamine receptors in the brain, although they also block acetylcholine and norepinephrine receptors. The potency of commercially available neuroleptics in blocking dopamine receptors varies widely, being related to the compound''s lipid solubility. Neuroleptics predispose the patient to short-term and long-term medical hazards that must be weighed against the benefits of reduced symptom intensity, shortened psychotic episodes and lessened likelihood of recurrence of acute schizophrenic epidoses. The side effects associated with short-term therapy are either extremely rare or are treatable by dose change, medication change or the use of additional drugs. In long-term therapy the risks are more problematic in that they are sometimes irreversible. These include tardive dyskinesia, skin discoloration and corneal deposits. The clinician must consider the pattern aand severity of each patient''s present and past psychotic episodes before deciding whether maintenance therapy with neuroleptics is justified. If it is, doses should be re-evaluated frequently and kept as low as possible. Concomitant administration of anticholinergic agents should be avoided if possible. Most important, the long-term administration of neuroleptics should be prescribed only for patients with schizophrenia and not for those with conditions that respond to other treatments.     

Cooperative cyanide dissociation from ferrous hemoglobin.

Rapid reduction of cyano-met hemoglobin (Hb+CN-) leads to the formation of an intermediate species, the cyanide derivative of ferrous hemoglobin, which dissociates to unliganded hemoglobin because of the extremely low affinity of the ligand for the ferrous heme iron. The properties of the intermediate were studied by transient spectroscopy in human hemoglobin and its isolated alpha and beta chains, in the presence and absence of CO. When mixing with dithionite, the time courses of reduction of the heme iron and dissociation of cyanide overlap considerably; addition to the reaction mixture of the redox indicator methyl viologen considerably increases the rate of reduction and allows unequivocal determination of the spectroscopic and kinetic properties of the intermediate. The results show that (i) the dissociation of cyanide from the isolated alpha and beta chains (as well as the (alpha CO)2(beta + CN-)2 hybrid) is a simple process; (ii) the two chains display similar rate parameters, but show spectroscopic inequivalence, both in the Soret and the visible regions; (iii) cooperative effects are shown to control the rate of dissociation of cyanide from hemoglobin, similarly to what happens for oxygen; and (iv) allosteric effectors (typically inositol hexaphosphate) increase the overall rate of dissociation by stabilization of the T state. We have, therefore, shown for the first time that the dissociation of cyanide from ferrous hemoglobin is controlled by the quaternary state, thereby adding one more ligand to the analysis of the structure-function relationships in hemoglobin.     

Contribution of plasma homovanillic acid (HVA) to urine and cerebrospinal fluid HVA in the monkey and its pharmacokinetic disposition.

Homovanillic acid (HVA) labelled with two deuterium atoms (d₂-HVA) was used to determine the contribution of HVA in the blood to HVA in the urine and CSF of monkeys. During and after a six-hour intravenous infusion of d₂-HVA at a constant rate, the levels of both d₂-HVA and endogenous HVA (d₀-HVA) in plasma, urine, and CSF were determined by gas chromatography-mass spectrometry, and the relative enrichments of d₂-HVA in each of these fluids calculated. Results indicate that HVA in the urine is derived exclusively from the blood, with no contribution from renal metabolism of dopamine (DA). Furthermore, less than one percent of HVA in either lumbar or ventricular CSF is derived from circulating HVA. The plasma elimination curve of d₂-HVA was biexponential, with a terminal phase half-life ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) of 44 minutes and an apparent volume of distribution of 0.8 liters/kg.     

In Vivo Assessment of Dopamine and Norepinephrine Release in Rat Neocortex: Gas Chromatography-Mass Spectrometry Measurement of 3-Methoxytyramine and Normetanephrine

A new method with the sensitivity and specificity required to measure regional levels of 3-methoxytyramine (3-MT) and normetanephrine (NMN) in the rat cortex is described. The method utilizes a liquid ion exchanger to isolate the parent amines, dopamine (DA) and norepinephrine (NE), along with their methylated metabolites. These samples are derivatized and analyzed by negative ion gas chromatography-mass spectrometry. Using this method, we examined a number of drug actions on steady-state levels as well as pargyline-induced increases in 3-MT and NMN. In the prefrontal cortex, cingulate cortex, striatum, and olfactory tubercle, nomifensine was found to increase 3-MT steady-state levels and accumulation rates. Similar actions of this drug were observed in the cingulate and prefrontal cortices with NMN. In contrast, clonidine decreased cortical NMN levels and accumulation. A unique action was observed with haloperidol, in that both 3-MT levels and accumulation after pargyline were increased in the nigrostriatal and mesolimbic dopaminergic projections, whereas only the accumulation rates were accelerated in the mesocortical projections. In summary, our data indicate that this new assay is a useful approach for the in vivo evaluation of DA and NE release in cortical regions of the rat. This approach is unique in that no surgery, restraint, or anesthetic is required, thereby permitting more complicated experimental paradigms to be utilized.     

Protein dissociation from DNA in model systems and chromatin.

Salt induced dissociation of protamine, poly(L-lysine) and poly(L-arginine) from DNA was measured by relative light scattering at theta = 90 degrees and/or centrifugation. Dissociation of histones from DNA was studied using relative light scattering and intrinsic tyrosine fluorescence. Protamine was dissociated from DNA at 0.15 M MgCl2 (ionic strength mu = 0.45) or 0.53 M NaCl (mu = 0.53) based on light scattering data and at approximately 0.2 M MgCl2 (mu = 0.6) or 0.6 M NaCl based on centrifugation data. NaCl induced dissociation of poly(Lys) or poly(Arg) from natural DNAs measured by light scattering did not depend on the guanine plus cytosine content. To dissociate poly(Arg) from DNA higher ionic strength using NaCl, MgCl2, or CaCl2, similar ionic strength using NaClo4, and lower ionic strength using Na2SO4 was needed then to dissociated poly(Lys). Both the decrease in light scattering and the enhancement of tyrosine fluorescence of chromatin occurred between 0.5 and 1.5 M NaCl when histones were dissociated.     

Effects of LSD and 2-bromo LSD on striatal DOPAC levels.

Administration of d-lysergic acid diethylamide (LSD) and its analogue 2-bromo lysergic acid diethylamide (BOL) resulted in a shortlasting increase of 3,4-dihydroxyphenylacetic acid (DOPAC) levels in the rat striatum. BOL was more potent than LSD in the dose range 0.5–4.0 mg/kg. Since there was a concomitant increase in the striatal $\text{in}$$\text{vivo}$ tyrosine hydroxylation as measured by DOPA accumulation after decarboxylase inhibition, our findings suggest that LSD and BOL increase the impulse flow in the nigro-neostriatal pathway probably by central dopamine (DA) receptor antagonism. However, 4 hrs after LSD the DOPAC level was decreased, while the DOPA accumulation was not. Thus the effect of LSD on the dopaminergic system appears not to be limited to a pure receptor antagonism. The possibility also exists that the effect of LSD on the nigro-neostriatal DA pathway is secondary to its effect on the central 5-hydroxytryptaminergic system.     

Subunit dissociation in the allosteric regulation of Glycerol kinase from Escherichia coli. 3. Role in desensitization.

The mechanism of desensitization of glycerol kinase to allosteric inhibition by fructose 1,6-bisphosphate caused by salt, urea, and high pH has been examined in the light of the model proposed in an earlier paper [de Riel, J. K., and Paulus H. (1978), Biochemistry 17] relating subunit dissociation and ligand binding. KCl (0.4 M) causes a tenfold decrease in the affinity of tetrameric glycerol kinase for fructose, 1,6-bisphosphate but has no significant effect on the dissociation process itself. Urea (2 M) causes a large increase in the equilibrium constant for the dissociation of the glycerol kinase tetramer to dimer but has no effect on the affinity of the tetramer for the allosteric inhibitor. High pH (9--10) has only a small effect on the subunit dissociation constant but greatly reduces the rates of subunit association and dissociation. Desensitization of glycerol kinase to allosteric inhibition can thus occur by three different mechanisms, two of which are directly related to the polysteric nature of the enzyme.     

Reduction by beta-endorphin of efflux of dopamine and DOPAC from superfused medial basal hypothalamus

The effects of β-endorphin (β-EP) and naloxone on the efflux of dopamine (DA) and its specific deaminated metabolite, dihydroxyphenylacetic acid (DOPAC), from superfused rat medial basal hypothalamus (MBH) were determined. After an equilibration period of 2.5 hrs with Medium 199 at 37°C 0.5 ml fractions were collected. Injection of β-EP, but not medium, into the system significantly reduced (P<0.05) the effluent concentrations of both DA and DOPAC from female MBH's (N=5) by 71% and 55% respectively and from male MBH's (N=7) by 68% and 49% respectively. Conversely, administration of naloxone significantly increased DA and DOPAC efflux by 85% and 148% respectively in females and by 132% and 120% in males. When an equimolar mixture of β-EP and naloxone was injected, no detectable changes in DA and DOPAC efflux were found. The effects of β-EP and naloxone were demonstrable irrespective of the sequence of administration. We conclude that β-EP exerts a tonic inhibitory effect on tuberoinfundibular DA neurons by interaction with naloxone-sensitive opiate receptors. This action does not require the intermediation of brain centers outside the MBH.     

The dissociation of sigma-factor from ribonucleic acid polymerase.

The sigma-factor of Escherichia coli RNA polymerase was shown to dissociate from the core enzyme as a function of absolute concentration. The association constant is in the range 10(6)-10(8) litre/mol. This implies that the amount of holoenzyme, core enzyme and sigma-factor in RNA polymerase assays may vary according to the absolute concentration of the enzyme.