Confirmation of an unexpected brain O-methylation pattern for the dopamine-derived tetrahydroisoquinoline,salsolinol

Using high resolution capillary gas chromatography, we have unequivocally separated two possible (6- and 7-)mono-O-methylated tetrahydroisoquinoline metabolites in rat brain after acute intraventricular administration of salsolinol, a cyclized dopamine/acetaldehyde derivative. 7-O-Methylsalsolinol (salsoline) constituted 94–98% of the two isomers in five brain regions examined. These results confirm the report by Bail $\text{et}$$\text{al}$. that salsolinol is largely O-methylated $\text{in}$$\text{vivo}$ (presumably by brain catechol-O-methyl transferase) on the hydroxyl situated “para” in the parent dopamine molecule. In comparison, dopamine itself, administered intraventricularly to pargyline-pretreated rats, was O-methylated exclusively on the “meta” hydroxyl group.     

Microdetermination of norepinephrine, 3,4-dihydroxyphenylethylamine, and 5-hydroxyyptamine from single extracts of specific rat brain areas

The conditions reported by Toru and Aprison (4) for extracting ACh in specific brain areas were tested to determine whether 5-HT, NE, and dopamine were also extracted quantitatively. It was found that the extraction solution used in brain ACh determinations, 15% 1N formic acid plus 85% acetone ($\text{v}\text{v}$), was also excellent for extraction of NE, 5-HT, and dopamine from different brain areas. Experimental conditions are given for the microdetermination of all three biogenic amines in such a single extract of a specific rat brain area. The methods are based on previously published fluorometric methods; these have been scaled down or modified slightly to permit analyses of small aliquots. The concentration of 5-HT, NE, and dopamine in the telencephalon, diencephalon plus mesencephalon, pons plus medulla oblongata, and cerebellum of the rat are also reported using the described micromethods after extraction with 15% 1 N formic acid plus 85% acetone ($\text{v}\text{v}$).     

Microbial transformations of natural antitumor agents. 23. conversion of withaferin-A to 12β- and 15β-hydroxy derivatives of withaferin-A.

Microbial transformation experiments were conducted with the antitumor lactone withaferin-A. $\text{Cunninghamella elegans}$ NRRL 1393 transformed withaferin-A ($\text{1a}$) to 15β-hydroxywithaferin-A ($\text{2a}$) and 12β-hydroxywithaferin-A ($\text{3a}$). The hydroxylated metabolites were isolated by solvent extraction and were purified by column and thin-layer chromatography. Structures of the hydroxylated metabolites were determined by protonand carbon-13 NMR, IR and mass spectral analyses, and by the preparation of acylated derivatives. Compounds $\text{2a}$ and $\text{3a}$ inhibited the growth and biochemical functions of $\text{in vitro}$ grown P-388 lymphocytic leukemic cells.     

Reconstitution of dopamine-sensitive adenylate cyclase from dissociated components in canine caudate nucleus

The catalytic component of adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were solubilized with 2% Lubrol PX in the presence of NaF from the synaptic membranes of canine caudate nucleus and were separated into distinct fractions by gel exclusion chromatography on a Sephadex G-200 column. The dissociated adenylate cyclase was no longer responsive to dopamine but was considerably stimulated by 10 mm NaF. Dissociated [${}^3\text{H}$]-dopamine binding protein possessed the apparent dissociation constant of 3.2 μm for dopamine, almost identical to that of the particulate preparations. The affinities of [${}^3\text{H}$]-dopamine binding protein to catecholamines and neuroleptics were also very similar to those of particulate preparations. After the adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were preincubated together at 4 °C for 30 min, the cyclase activity displayed a dose-dependent increase by dopamine with the K_a of 1.6 μm, the concentration of dopamine to stimulate half-maximally. Stimulation of the reconstituted adenylate cyclase by dopamine was maximally 2.7-fold and was strongly inhibited by neuroleptics such as chlorpromazine and haloperidol. These results suggest that [${}^3\text{H}$]dopamine binding protein is identical to the regulatory subunit of dopamine-sensitive adenylate cyclase in the synaptic membranes of canine caudate nucleus.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotreine B₄ has been developed. The assay is sensitive; 5 pg LTB₄ caused significant inibition of binding of [³H]-LTB₄ and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B₂ and arachidonic acid do not exhibit detectable cross-reactions (< 0.03%). Flowever, some non-cyclic dihydroxy- and monohydroxy-eicosatetraeonic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-$\text{trans}$-14-$\text{cis}$-elcosatetraenoic and 12-hydroxy-5,8,10,14-elcosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB₄ from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB₄ by reverse-phase high liquid chromatography follwing solvent extraction and silinic acid chromatography.     

Changes in adrenal dopamine concentration after metyrapone or ACTH administration: implications for the in vivo regulation of dopamine beta-hydroxylase by glucocorticoids.

$\text{In vitro}$ studies have demonstrated that rat adrenal dopamine beta-hydroxylase activity is controlled by neural input and by glucocorticoid production. However, beta-hydroxylation of dopamine $\text{in vivo}$ is a first-order reaction and may be considerably slower than the maximal rate determined by $\text{in vitro}$ methods. To estimate the $\text{in vivo}$ reaction rate the concentrations of dopamine (substrate) and of beta-hydroxylated catecholamine (product) were measured as a function of endogenous glucocorticoid production. Beta-hydroxylated catecholamine changed little but dopamine was increased 2-fold or more 17.5 h after the inhibition of steroidogenesis with metyrapone. Dopamine was also increased by metyrapone in animals with pre-existing adrenal denervation. ACTH 17.5 h before sacrifice caused only slight changes in normal rats but reduced the increase in dopamine caused by stress. The results indicate that adrenal dopamine concentration is inversely related to glucocorticoid production at a given level of neural input and provide $\text{in vivo}$ evidence that glucocorticoids maintain dopamine beta-hydroxylase activity in the adrenal gland.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

Effects of morphine on dopamine-stimulated adenylate cyclase and on cyclic GMP formation in primate brain amygdaloid nucleus.

In homogenates of $\text{Macaca}$$\text{mulatta}$ (Rhesus) or $\text{Cebus}$$\text{apella}$ amygdaloid nuclear complex, adenylate cyclase activity was approximately doubled by either 10μ$\text{M}$ dopamine or 8m$\text{M}$ NaF. In the presence of morphine, the stimulation by dopamine was reduced. A 90–100% inhibition of the dopamine stimulation was obtained with 20μ$\text{M}$, and a 50% inhibition, with 5μ$\text{M}$ morphine. The effects of 10μ$\text{M}$ morphine on dopamine stimulation were reversed by 10μ$\text{M}$ naloxone. Morphine itself did not significantly affect the basal adenylate cyclase activity, but in the presence of 10μ$\text{M}$ morphine the stimulation by 8m$\text{M}$ NaF was reduced approxiamtely 50%. The data suggest an action of morphine at a receptor site which is distinct from the dopamine receptor, but which inhibits the dopamine-stimulated adenylate cyclase. In addition, the cyclic GMP content of $\text{Cebus}$ amygdala slices was reduced by 50–75% during incubation for 5–20 minutes with morphine. Maximum effects on cyclic GMP were obtained with 10μ$\text{M}$, and half-maximum effects, with 0.1μ$\text{M}$ morphine. The effect of morphine on amygdala cyclic GMP was not reversed by naloxone. Thus, this action of morphine may not be receptor mediated, or may involve the interaction of morphine with receptors other than the opiate receptor.     

In vivo receptor binding: attempts to improve specific/non-specific ratios.

$\text{In vivo}$ receptor binding was examined using ³H-spiperone and ³H-pimozide for dopamine receptors and ³H-LSD for serotonin receptors. Two strategies for improving total: nonspecific binding ratios were tested. The first was to deplete endogenous ligands by various pharmacological treatments prior to ³H-ligand administration in an attempt to increase specific receptor binding; the second was to perfuse the brain with ice-cold saline after ³H-ligand administration in an attempt to reduce nonspecific binding. Alteration of dopamine and serotonin by administering d-amphetamine, reserpine, alpha-methyl-paratyrosine or parachlorophenylalanine did not significantly elevate striatal: cerebellar or cortical: cerebellar (measures of total: nonspecific) bonding ratios. However, perfusion with ice-cold saline significantly improved the ratios for both dopamine and serotonin receptors. Thus, cold saline perfusion may be of value in reducing blank values in autoradiographic and other studies requiring $\text{in}$$\text{vivo}$ labelling of receptors.     

Detection of endogenous salsolinol in neonatal rat tissue by a radioenzymatic—thin-layer chromatographic assay

A sensitive radioenzymatic—thin-layer chromatographic assay for the quantitative analysis of the tetrahydroisoquinoline alkaloid, salsolinol, in plasma and neonatal rat tissue is described. The assay involves the enzymatic O-methylation of salsolinol by catechol-O-methyltransferase in presence of [³H] S-adenosylmethionine, and subsequent separation by thin-layer chromatography of the resultant [³H] O-methyl-salsolinol from the O-methylated derivatives of dopamine, epinephrine and norepinephrine. The method allows the detection of as little as 100 pg salsolinol per g tissue, and the accurate quantitation of as little as 100 pg/ml plasma and 500 pg/g tissue. This assay permitted the detection of trace amounts of endogenous salsolinol in neonatal rat tissue (< 500 pg/g tissue).     

Salsolinol, a tetrahydroisoquinoline catechol neurotoxin, induces human Cu,Zn-superoxidie dismutase modificaiton

The endogenous neurotoxin, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), has been considered a potential causative factor for the pathogenesis of Parkinsonos disease (PD). In the present study, we examined the pattern of human Cu,Zn-superoxide dismutase (SOD) modification elicited by salsolinol. When Cu,Zn-SOD was incubated with salsolinol, some protein fragmentation and some higher molecular weight aggregates were occurred. Salsolinol led to inactivation of Cu,Zn-SOD in a concentration-dependent manner. Free radical scavengers and catalase inhibited the salsolinolmediated Cu,Zn-SOD modificaiton. Exposure of Cu,Zn-SOD to salsolinol led also to the generation of protein carbonyl compounds. The deoxyribose assay showed that hydroxyl radicals were generated during the oxidation of salsolinol in the presence of Cu,Zn-SOD. Therefore, the results indicate that free radical may play a role in the modification and inactivation of Cu,Zn-SOD by salsolinol.     

An automated procedure for the extraction and fluorimetric determination of 5-hydroxytryptamine in platelet subcellular fractions

A fully automated procedure for the determination of 5-hydroxytryptamine is described. The specificity and sensitivity of the assay depend upon a differential solvent extraction procedure to remove interfering 5-hydroxy-indoles and the measurement of the fluorescence of the amine after reaction with ninhydrin. The method is highly suitable for the analysis of tissue subcellular fractions since there is no interference by sucrose, and after a preliminary deproteinisation of the sample with $\text{ZnSO}{}_4\text{NaOH}$, performed manually, the rest of the procedure is fully automated. The sensitivity of the assay depends upon the quality of the fluorimeter used but is better than 5 ng/ml of sample with a double monochromator spectrofluorimeter. There is no interference by 5-hydroxytryptophan, 5-hydroxyindole-3-acetic acid, or 5-hydroxytryptophol. The suitability of the procedure for the routine analysis of 5-hydroxytryptamine in platelet subcellular fractions prepared by zonal gradient centrifugation has been explored and the soluble phase and particulate bound amine stores can be identified.     

The extraction of guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) from Escherichia coli using low pH reagents: a reevaluation.

It has been found that the most widely used method for the extraction of guanosine 5′-diphosphate, 3′-diphosphate (ppGpp) from $\text{E. coli}$ (1 M formic acid at 0°) results in its $\text{in vitro}$ degradation to ppGp and GDP. A comparison with several other extraction procedures indicated that this breakdown is due to the low pH of the reagents used during extraction. This degradation can largely be prevented by using a new extraction technique which involves freezing and thawing of the cells in the presence of lysozyme at a neutral pH followed by treatment with deoxycholate. With this method it is possible to recover from three to five times as much ppGpp from both unstarved and amino acid starved stringent strains of $\text{E. coli}$ as compared with the most widely used formic acid procedure. Consequently, it will be necessary to reevaluate the ppGpp values obtained from cells when formic acid or other low pH reagents were used during extraction.     

Effect of morphine, beta-endorphin and leu-enkephalin on 3H-spiroperidol binding to bovine pituitary membranes.

The ability of morphine, leu-enkephalin and β-endorphin to antagonize the binding of ³H-spiroperidol to bovine anterior pituitary membranes was studied. All three drugs were virtually inactive despite their ability to stimulate prolactin secretion $\text{in}$$\text{vivo}$ and the reported ability of morphine to antagonize the inhibitory effect of dopamine on prolactin release from rat hemi-pituitaries. These results suggest that opiates do not produce their direct effect on prolactin secreation at the pituitary level through an effect on the ³H-spiroperidol binding site. The opiates may antagonize the effect of dopamine at a component of the dopamine receptor which is independent of the ³H-spiroperidol binding site, or the opiates may stimulate prolactin secretion by an effect on the lactotrophes which is independent of dopamine.     

The role of catecholamines in the prolactin release induced by salsolinol

Salsolinol (1,2,3,4-tetrahydro-6,7-dihydroxy-1-methylisoquinoline) is an endogenous prolactin releasing agent. Its action can be inhibited by another isoquinoline, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), which has a strong norepinephrine releasing activity. Salsolinol does not alter the dopamine release in median eminence in vitro, providing evidence for the lack of interaction with presynaptic D2 dopamine receptors. At the same time, lack of norepinephrine transporter abolishes salsolinol's action. Salsolinol decreases tissue level of dopamine and increases norepinephrine to dopamine ratio in organs innervated by the sympathetic nervous system indicating a possible decrease of norepinephrine release. Enzymes of catecholamine synthesis and metabolism are probably also not the site of action of salsolinol. In summary, based upon all of these observations a physiologically relevant interplay might exist between the sympatho-neuronal system and the regulation of prolactin release.     

Salsolinol, a tetrahydroisoquinoline-derived neurotoxin, induces oxidative modification of neurofilament-L protection by histidyl dipeptides

Salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline) is a compound derived from dopamine metabolism and is capable of causing dopaminergic neurodegeneration. Oxidative modification of neurofilament proteins has been implicated in the pathogenesis of neurodegenerative disorders. In this study, oxidative modification of neurofilament-L (NF-L) by salsolinol and the inhibitory effects of histidyl dipeptides on NF-L modification were investigated. When NF-L was incubated with 0.5 mM salsolinol, the aggregation of protein was increased in a time-dependent manner. We also found that the generation of hydroxyl radicals (?OH) was linear with respect to the concentrations of salsolinol as a function of incubation time. NF-L exposure to salsolinol produced losses of glutamate, lysine and proline residues. These results suggest that the aggregation of NF-L by salsolinol may be due to oxidative damage resulting from free radicals. Carnosine, histidyl dipeptide, is involved in many cellular defense processes, including free radical detoxification. Carnosine, and anserine were shown to significantly prevent salsolinol- mediated NF-L aggregation. Both compounds also inhibited the generation of ?OH induced by salsolinol. The results indicated that carnosine and related compounds may prevent salsolinol-mediated NF-L modification via free radical scavenging.     

Effect of Aspergillus flavus mycotoxin on Culex mosquito larvae

An isolate of Aspergillus flavus var. columnaris recovered from moribund larvae of the mosquito Culex peus produced metabolites which caused mortality in larvae of C. peus and C. tarsalis. Production of these toxins in vitro on solid substrate rice was based on culture techniques used in making Ang kak. The effect of incubation time on mycotoxin production was determined for cultures fermented from 3 to 13 days. Mycotoxins produced by isolate FU-051 were extracted by refluxing the spent medium with chloroform. Fractionation on a silicic acid column with a methanol-chloroform (3:97 $\text{v}\text{v}$) solvent mixture yielded two toxic fractions. Toxic activity was determined using a biological assay with mosquito larvae.     

Long-term amphetamine treatment attenuates or reverses the depression of neuronal activity produced by dopamine agonists in the ventral tegmental area

Neuronal activity was recorded from the ventral tegmental area (VTA) of immobilized, locally anesthetized rats on the day immediately following long-term treatment (twice daily for 6 consecutive days) with saline, 1.0 or 5.0 mg/kg $\text{d}$-amphetamine ($\text{d}$-AMPH). Each rat was challenged intravenously with $\text{d}$-AMPH (beginning with 0.0625 mg/kg) or with 0.005 mg/kg apomorphine. Treatment with $\text{d}$-AMPH significantly reduced the ability of this drug to inhibit VTA activity. In fact, nearly half of the neurons in the high-dose treatment group were excited by $\text{d}$-AMPH, whereas only 20% of control neurons showed this response. Moreover, apomorphine routinely accelerated firing rate in the VTA following treatment with 5.0 mg/kg $\text{d}$-AMPH but this response was never observed in control neurons, not even in those that were excited by $\text{d}$-AMPH. Thus, tolerance appears to develop to the ability of dopamine agonists to inhibit VTA activity and this effect may be mediated, at least in part, by a subsensitivity of inhibitory dopamine autoreceptors.     

N-acetylation of dopamine and tyramine by mosquito pupae (Aedes togoi)

The $\text{N-}\text{acetyltransferase}$ (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [¹⁴C]-, [³H]dopamine, [¹⁴C]tyramine or [¹⁴C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn²⁺ stimulated and Zn²⁺ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, $\text{893 ± 57}$ and $\text{861 ± 30}$ pmol [¹⁴C]NAcT formed/min per mg protein and $\text{21.9 ± 1.2}$ and $\text{22.0 ± 1.4}$ pmol [³H]NADA formed/min per mg protein.