High-performance liquid chromatographic analysis of monoamines and some of theirγ-glutamyl conjugates produced by the brain and other tissues ofHelix aspersa (gastropoda)

1. Earlier reports from this and other laboratories have indicated that wide variations exist in estimates of the concentrations of norepinephrine in the brain and heart of the snail Helix aspersa. This is a report on investigations of norepinephrine concentrations in Helix aspersa tissues using high-performance liquid chromatography with electrochemical detection. In addition, the effects of treatment with some amino acid precursors or enzyme inhibitors on the concentrations of norepinephrine, dopamine, 5-hydroxytryptamine, and some of their metabolites were investigated. 2. The levels of norepinephrine in the brain were low (46 ng/g) in comparison to dopamine (2.1) micrograms/g) and 5-hydroxytryptamine (2.6 micrograms/g). Epinephrine was not observed in either snail heart of snail nervous tissue. 3. Administration of L-3,4-dihydroxyphenylalanine resulted in elevated snail brain dopamine, while 3,4-dihydroxyphenylserine treatment increased norepinephrine. Treatment with blockers of tyrosine hydroxylase and aromatic-L-amino acid decarboxylase reduced dopamine concentrations without affecting 5-hydroxytryptamine. 4. The dopamine metabolite 3,4-dihydroxyphenylacetic acid was observed only after administration of L-3,4-dihydroxyphenylalanine or dopamine and then only in very small amounts. At no time was the dopamine metabolite homovanillic acid or the 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid observed in brain, heart, or whole-body extracts of the snail. 5. Incubation of nervous tissue with either dopamine or 5-hydroxytryptamine resulted in the production of electrochemically active metabolites which were identified by oxidation characteristics and cochromatography with synthesized standards as the gamma-glutamyl conjugates of the amines. Treatment of snails with 5-hydroxytryptamine or dopamine also resulted in the production of gamma-glutamyl conjugates. 6. The present experiments show that great care must be exercised when measuring monoamines and their metabolites in gastropod tissues by high-performance liquid chromatography with electrochemical detection.     

Transient accumulation of inositol (1,3,4,5)-tetrakisphosphate in response to alpha 1-adrenergic stimulation in adult cardiac myocytes

To investigate the response to catecholamine stimulation of adult cardiac myocytes and the metabolism of inositol (1,4,5)-trisphosphate (1,4,5-IP3) and inositol (1,3,4,5)-tetrakisphosphate (IP4), we have employed a procedure developed in our laboratory to directly measure the mass of inositol phosphates after separation of individual isomers of inositol phosphates by high performance liquid chromatography. Control, unstimulated myocytes, contained low levels of inositol (1,4)-bisphosphate (1,4-IP2), inositol (1,3)-bisphosphate (1,3-IP2), inositol (3,4)-bisphosphate (3,4-IP2), inositol (1,3,4)-trisphosphate (1,3,4-IP3), 1,4,5-IP3 and IP4. Stimulation with norepinephrine for 30 seconds produced peak 1,4,5-IP3 and IP4 levels which rapidly returned to basal values by 60 seconds of norepinephrine stimulation. 1,4-IP2, 1,3-IP2 and 1,3,4-IP3 were increased markedly but only after stimulation with norepinephrine for 60 seconds. These results indicate a rapid yet transient increase in 1,4,5-IP3 and IP4 in response to norepinephrine stimulation and are the first quantitative measurements of the isomers of inositol phosphates in cardiac tissue.     

Mass spectrometric measurements of norepinephrine synthesis in man from infusion of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine

The kinetics of stable isotope-labelled L-threo-3, 4-dihydroxyphenylserine(L-threo-DOPS), an immediate precursor of (-)-norepinephrine, was studied to investigate the pharmacologic mechanism of its therapeutic effect on orthostatic hypotension in familial amyloid polyneuropathy(FAP) and on akinesia and freezing in parkinsonism. [13C,D]-L-threo-DOPS was synthesized, and 100 mg of the compound was infused for 2 h into two normal subjects, two FAP patients and two patients with the degenerative diseases of the central nervous system. Labelled and endogenous norepinephrine in urine and plasma was assayed simultaneously by gas chromatography/mass spectrometry. The results indicate that the increase in norepinephrine in biological fluids after administration of L-threo-DOPS is attributable mostly to norepinephrine derived from L-threo-DOPS, not to pre-formed endogenous norepinephrine released by L-threo-DOPS.     

The mammalian neuroendocrine hormone norepinephrine supplies iron for bacterial growth in the presence of transferrin or lactoferrin

Norepinephrine stimulates the growth of a range of bacterial species in nutritionally poor SAPI minimal salts medium containing 30% serum. Addition of size-fractionated serum components to SAPI medium indicated that transferrin was required for norepinephrine stimulation of growth of Escherichia coli. Since bacteriostasis by serum is primarily due to the iron-withholding capacity of transferrin, we considered the possibility that norepinephrine can overcome this effect by supplying transferrin-bound iron for growth. Incubation with concentrations of norepinephrine that stimulated bacterial growth in serum-SAPI medium resulted in loss of bound iron from iron-saturated transferrin, as indicated by the appearance of monoferric and apo- isoforms upon electrophoresis in denaturing gels. Norepinephrine also caused the loss of iron from lactoferrin. The pharmacologically inactive metabolite norepinephrine 3-O-sulfate, by contrast, did not result in iron loss from transferrin or lactoferrin and did not stimulate bacterial growth in serum-SAPI medium. Norepinephrine formed stable complexes with transferrin, lactoferrin, and serum albumin. Norepinephrine-transferrin and norepinephrine-lactoferrin complexes, but not norepinephrine-apotransferrin or norepinephrine-albumin complexes, stimulated bacterial growth in serum-SAPI medium in the absence of additional norepinephrine. Norepinephrine-stimulated growth in medium containing (55)Fe complexed with transferrin or lactoferrin resulted in uptake of radioactivity by bacterial cells. Moreover, norepinephrine-stimulated growth in medium containing [(3)H]norepinephrine indicated concomitant uptake of norepinephrine. In each case, addition of excess iron did not affect growth but significantly reduced levels of radioactivity ((55)Fe or (3)H) associated with bacterial cells. A role for catecholamine-mediated iron supply in the pathophysiology of infectious diseases is proposed.     

Abnormal levels of urinary catecholamines in dystrophic mice and hamsters.

Twenty-four-hour urine was collected from normal and dystrophic mice and hamsters for catecholamine determinations. A new method of analysis was used whereby 3,4-dihydroxyphenylalanine (DOPA), dopamine (DA), norepinephrine (NE), and epinephrine (E) were measured simultaneously. The procedure is based on a combination of liquid-solid extraction, cation exchange chromatography, and controlled potential electrochemistry. The results of these experiments indicated that while DA levels were similar in both normal and pathological animal urine, DOPA levels decreased slightly in the dystrophic mouse but not the hamster, and NE and E levels in dystrophic groups were two and four times greater than normal in both species. The data supports the concept of biochemical alterations in tissue other than muscle. While not necessarily supportive to catecholamine abnormality as the primary cause of muscular dystrophy, the present data cast doubt that this disease is a primary muscle disease.     

PhenylethanolamineN-methyltransferase from the brain and adrenal medulla of the rat

Two different molecular forms of phenylethanolamineN-methyltransferase (PNMT, EC 2.1.1) have been isolated from the brain and adrenal glands of the rat as indicated by certain of their physicochemical properties, such as: molecular weight estimated on the basis of gel chromatography of Sephadex G-100; pH optima; electrophoretic mobility on acrylamide gel; and steady-state kinetic parameters. The highK _m value for the brain PNMT has been assumed to be responsible for the low methylation ratio between norepinephrine and epinephrine in the CNS.     

Role of sialic acid residues in crossed immuno-affinoelectrophoresis of alpha 1-proteinase inhibitor

We have investigated the importance of the degree of sialylation when an acute phase glycoprotein, alpha 1-proteinase inhibitor (alpha 1-Pi), was analysed both by affinity chromatography on concanavalin A (Con A)-Sepharose and by crossed immuno-affinoelectrophoresis (CIAE) using Con A in the first dimension. Human alpha 1-Pi was isolated by immunosorption chromatography and then more or less desialylated. On Con A-Sepharose chromatography no significant difference was observed in the percentage of the two fractions (retained or not retained) whatever the degree of desialylation. In contrast by CIAE this degree was largely involved in the separation of the different isoforms obtained in the first dimension.     

Isolation of an endogenous clonidine-displacing substance from rat brain

An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain alpha 2-adrenergic receptors, has been isolated. The new compound, designed clonidine-displacing-substance (CDS), has been partially purified by ion exchange chromatography, zone electrophoresis and high performance liquid chromatography (HPLC). CDS binds specifically to alpha 2-adrenergic receptors by competing with either alpha 2-adrenergic agonists or alpha 2-antagonists, but has no effect on the specific binding of [3H]prazosin to alpha 1-adrenergic receptors in rat brain membranes. In the course of isolation, CDS was shown to be neither the endogenous neurotransmitter (-)norepinephrine (NE) nor the guanyl nucleotide GTP which lowers the specific binding of alpha 2-agonists to the alpha 2-adrenergic receptors.     

Interferences by anti-TB drugs in a validated HPLC assay for urinary catecholamines and their successful removal

A validated high performance liquid chromatographic assay for urinary catecholamines is presented. After addition of 3,4-dihydroxybenzylamine as internal standard (IS) to urine, norepinephrine (NE), epinephrine (E), dopamine (DA) are extracted by ion exchange chromatography and eluted with boric acid. After paired ion separation, quantitation is by electrochemical (coulometric) detection after correction of internal standard recovery. Novel interferences by anti-TB drugs on norepinephrine assay are discussed. A simple method for their removal using alumina is presented.     

Stereospecific (--)-[3H]norepinephrine binding to bovine hypothalamus. Possible identification of the catecholamine uptake site in synaptic vesicles

A (--)-[3H]norepinephrine binding site was identified in a crude synaptosomal fraction isolated from bovine hypothalamus which bound norepinephrine rapidly, reversibly, and stereospecificially. The results were most consistent with binding of (-)-[H]norepinephrine to the carrier molecule used to translocate biogenic amines into synaptic vesicles. The binding studies indicated that specific binding of (--)-[3H]norepinephrine to the crude synaptosomal fraction was greatly enhanced by 4 mM MgCl2 pand 1 mM ATP. The increased binding of (--)-[3H5norepinephrine also occurred in the presence of MgCl2 and GTP, but AMP, adenosine and adenyl-5'-yl imidodiphosphate would not substitute for ATP. Neither CaCl2 nor ZnSO4 could be substituted for the MgCl2. In the presence of MgCl2 and ATP, the dissociation constant for (--)-[3H]norepinephrine was 280 nM with a specific binding site density of 4.8 pmol/mg protein. Binding was stereospecific with ratios of 15, 4, and 6.5 for the affinities of (--)-isomers to (+)-isomers for norepinephrine, epinephrine and isoproterenol, respectively. Drug competition studies, conducted in the presence of Mg2+ and ATP, indicated that (--)-epinephrine, (--)-norepinephrine, dopamine and serotonin had inhibitory constants ranging from 0.25 to 0.8 micron with (--)-isoproterenol and tyramine having inhibitory constants around 2 micron. Reserpine was the most potent inhibitor having an inhibition constant of 8.6 +/- 0.3 nM. The binding data were not consistent with the specific site being the alpha- or beta-receptors for norepinephrine, the Uptake1 Site for norepinephrine into synaptosomes or the metabolizing enzymes for norepinephrine.     

RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A).

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.     

Subcellular localization of phospholipase C isoforms in vascular smooth muscle

The phospholipase C (PLC) isoform most important during agonist-activated IP(3) production in vascular smooth muscle is still unknown. When PLC activity in rat tail artery homogenate was determined, this activity was shown to be inhibited by an antibody directed against PLCbeta2. Antibodies directed against the gamma1, beta1, beta3 and delta1 isoforms of PLC failed to inhibit PLC activity in this tissue. Both PLCbeta2 and PLCgamma1 were isolated from rat tail artery by DEAE column chromatography and PLCbeta2 activity was shown to be 3-fold greater than PLCgamma1 activity. When rat tail artery was treated with norepinephrine (10 mM), PLCbeta2 was shown to translocate from cytosol to membranes. When subcellular fractions of rat tail artery were isolated by sucrose density gradient centrifugation, including nuclei, plasma membrane, and cytosol, PLCbeta2 was detected in the plasma membrane and the cytosol but not in the nuclei. PLCdelta1 and PLCgamma1 were found only in cytosol. This evidence is consistent with the model wherein an agonist such as norepinephrine can activate smooth muscle contraction via interaction with a plasma membrane receptor which can easily interact with a plasma membrane-associated isoform of PLC, such as PLCbeta2.     

Dopamine and norepinephrine in the alimentary tract changes after chemical sympathectomy and surgical vagotomy

The aim of this study was to examine the distribution of dopamine and norepinephrine in the proximal alimentary tract of the rat and to assess the contributions of sympathetic and vagal fibers to the tissue concentrations of both catecholamines. Tissues were extracted in perchloric acid and the catecholamines were separated by high pressure liquid chromatography and detected electrochemically. In untreated rats (controls) both catecholamines were concentrated in the gastric muscle but norepinephrine levels were 6-8 times higher (corpus, dopamine 35 +/- 7 ng . g-1, norepinephrine 265 +/- 50 ng . g-1, mean +/- SE, n = 6). In the mucosa norepinephrine concentrations were 10-12 times higher (corpus, dopamine 12 +/- 3 ng . g-1, norepinephrine 140 +/- 26 ng . g-1). Chemical sympathectomy (6 hydroxydopamine, 100 mg . kg-1 ip 3 days) significantly reduced dopamine concentrations in muscle and norepinephrine in muscle, mucosa, pylorus and duodenum. In all tissues the effects on norepinephrine were greater. Surgical vagotomy significantly reduced dopamine concentrations in the gastric muscle, but not the mucosa. Norepinephrine concentrations in the stomach of vagotomized rats were significantly reduced only in the pylorus. Differences in the relative concentrations of dopamine and norepinephrine in gastric tissues of the normal rat and differences in the effects of sympathectomy and vagotomy suggest that dopamine and norepinephrine exist, to an extent, in separate populations of cells and that dopamine is not merely a precursor of norepinephrine. Gastric mucosal dopamine, which was mainly unaffected by either treatment, may exist in APUD cells.     

Plasma catecholamines in resting rainbow trout,Salmo gairdneri Richardson,by high pressure liquid chromatography*

Plasma norepinephrine and epinephrine from cannulated trout were measured by high pressure liquid chromatography with electrochemical detection. The catecholamines were extracted with acid-washed alumina using a microfilter assembly which permitted analysis of small volumes of plasma. Mean (± S.D.) values for plasma norepinephrine and epinephrine were 1.83 (0.97) pmol ml^?1 and 8.95 (4.94) pmol ml^?1, respectively. These values are compared with catecholamine levels from other vertebrate species.     

Nicotine's attenuation of body weight involves the perifornical hypothalamus

Previously we showed that intermittent administration of nicotine (NIC) in the dark phase decreased food intake and body weight and this could be blocked when the NIC receptor antagonist mecamylamine was infused into the fourth ventricle. Catecholaminergic neurons adjacent to the fourth ventricle contain NIC receptors and directly innervate the perifornical hypothalamus (PFH) which has been shown to be involved in regulation of feeding. This study explored whether NIC regulates feeding behavior by modulating catecholaminergic input to the PFH. Epinephrine and norepinephrine neuronal input was ablated within the PFH by infusion of 6-hydroxydopamine hydrobromide (6-OHDA), while bupropion was infused to protect dopaminergic neurons. After recovery of body weights to pre-surgery levels, food intake, meal size, meal number and body weight were measured after intermittent NIC injections. The results showed the PFH lesioned animals did not exhibit the typical prolonged drop in food intake, meal size and body weight normally associated with NIC administration. High performance liquid chromatography analyses demonstrated that compared to control rats, 6-OHDA administration significantly reduced PFH norepinephrine and epinephrine levels, but not dopamine levels. These results are consistent with NIC reducing food intake in part by acting through catecholaminergic neurons within or extending through the PFH.     

Inhibition of norepinephrine re-uptake by angiotensin in brain

—A method for perfusion of rat brain ventricles accompanied by electrical stimulation of right vagus nerve was used in the study of the re-uptake mechanism of norepinephrine in rat brain. Tritiated norepinephrine was injected into the left lateral brain ventricle. After a 1-hr equilibration period, the brain was perfused, and effluent was collected and assayed for norepinephrine, normetanephrine and acid metabolites by means of column chromatography and liquid scintillation counting. Electrical stimulation of central end of right vagus nerve produced a significant release of norepinephrine and decrease of acid metabolites; this indicates a shift of catecholamines to extracellular sites. Angiotensin (200 ng/min) added to the perfusion fluid potentiated the effect of nerve stimulation. Cocaine, desmethylimipramine and phenoxybenzamine greatly reduced the release of norepinephrine from brain tissue. None on these drugs potentiated the effect of angiotensin. It is concluded that angiotensin prevents re-uptake of norepinephrine released by nerve stimulation. As a naturally occurring, physiologically active peptide, angiotensin seems to be highly specific in modulation of adrenergie neurotransmission, allowing increased extraneuronal accumulation of neurotransmitter.     

Effects of exercise on plasma catecholamine levels in the toad, Bufo paracnemis: role of the adrenals and neural control

Resting plasma epinephrine (E) and norepinephrine (N) concentrations for intact toads (Bufo paracnemis) were 5.57+/-1.0 and 0.88+/-0.38 ng/ml, respectively. Exercise induced a significant increase in heart rate, blood pressure and plasma epinephrine (about 4.3 times), whereas norepinephrine remained unchanged. The resting [E]/[N] ratio was 6.3 and increased to 32.9 during exercise. Adrenal denervation did not alter the basal plasma catecholamine or norepinephrine levels after exercise, but prevented the increase in epinephrine during exercise, suggesting that in the intact toad this increase is due to adrenal secretion whereas resting norepinephrine may be liberated by extra-adrenal chromaffin tissues. This also suggests that the adrenal glands can release selectively the two catecholamines. The increases in heart rate and blood pressure in denervated toads were not significantly different from those of intact animals, suggesting that during exercise the sympathetic nerves play the main role in inducing cardiovascular responses. Spinal transection induced a significant increase in basal norepinephrine levels, which remained elevated after exercise. Since spinal toads are unable to perform spontaneous movements it is possible that this increase may be caused by this stressful condition. The increases in heart rate and blood pressure observed in spinal toads during exercise may be due to direct mechanical effects of venous return on the heart.     

Improving enantiomeric resolutions by avoiding peak distortion effects in on‐line coupled liquid chromatography to gas chromatography

In this work, we study the effect of different variables affecting elution profile distortion on the enantiomeric resolution eventually achievable when working with on‐line coupled liquid chromatography to gas chromatography (LC‐GC). Specifically, the proposed configuration combines achiral reversed‐phase liquid chromatography (RPLC) and chiral gas chromatography (enantio‐GC), with heptakis‐(2,3,6‐tri‐O‐methyl)‐β‐cyclodextrin as enantioselective stationary phase to analyse target fractions transferred (from LC to GC) via the through oven transfer adsorption desorption (TOTAD) interface. The high degree of orthogonality resulting from the combination of two chromatographic columns having very different separation mechanisms (and also requiring mobile phases in distinct physical states), as well as integration of the sample preparation step in the first dimension of the system, significantly contributed to exploit the performance of the proposed two‐dimensional approach. Occasional adverse effects, which may result in severe peak distortions during LC‐GC analysis and could be explained by flow instabilities due to viscous fingering, are circumvented by using the outstanding capacity of the TOTAD interface for achieving effective elimination of the eluent arriving from the LC preseparation.     

Effect of various insecticides on the larval growth and biogenic amine levels of Tribolium castaneum Herbst

Various insecticides reduced larval growth of the red flour beetle (tribolium castaneum Herbst) and various biogenic amines, including octopamine (OA), dopamine (DA), serotonin (5-HT), epinephrine (E), norepinephrine (NE), their precursors and metabolites in the insects were measured by high-performance liquid chromatography coupled with electrochemical detection. Tyrosine occurred in the highest concentration followed by OA, tryptophan and 3,4-dihydroxymandelic acid (DOMA). Tyramine (a precursor of OA in the biosynthetic pathway), synephrine (N-methyl OA), DA, 5-HT, E, NE and their related substances occurred in extremely low quantities compared with OA. The insects were stressed by various insecticides, which resulted in a dramatic change of biogenic amine levels: the OA levels increased, whereas the levels of other biogenic amines and related substances decreased.     

Characterization of proteins in the human serum proteome.

This paper presents a multidimensional profile of the human serum proteome, produced by a two-dimensional protein fractionation system based on liquid chromatography followed by characterization with capillary electrophoresis (CE). The first-dimension separation was done by chromatofocusing over a pH range from 8.5 to 4.0, where proteins were separated by their isoelectric points (pI). In this dimension, fractions were collected based on pH. The first-dimension pI fractions were then resolved in the second dimension by high-resolution, reversed-phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. A selected protein fraction collected from the second dimension by time was characterized by CE for molecular-weight estimation and for presence of isoforms. Molecular-weight estimation was done by sodium dodecyl sulfate capillary gel electrophoresis, where proteins were separated in the range of 10,000-225,000 Da. Detection of isoforms was done by capillary isoelectric focusing over a pH range of 3-10. A selected second-dimension fraction that contained the putative serum iron-binding protein transferrin was analyzed by these two CE techniques for molecular-weight determination and the presence of isoforms. The combination of two-dimensional protein fractionation and CE characterization represents an advanced tool for proteomics.