Automated mass spectrometric analysis of urinary and plasma serotonin

Serotonin emerges as crucial neurotransmitter and hormone in a growing number of different physiologic processes. Besides extensive serotonin production previously noted in patients with metastatic carcinoid tumors, serotonin now is implicated in liver cell regeneration and bone formation. The aim was to develop a rapid, sensitive, and highly selective automated on-line solid-phase extraction method coupled to high-performance liquid chromatography–tandem mass spectrometry (XLC-MS/MS) to quantify low serotonin concentrations in matrices such as platelet-poor plasma and urine. Fifty microliters plasma or 2.5 μL urine equivalent were pre-purified by automated on-line solid-phase extraction, using weak cation exchange. Chromatography of serotonin and its deuterated internal standard was performed with hydrophilic interaction chromatography. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Serotonin concentrations were determined in platelet-poor plasma of metastatic carcinoid patients (n = 23) and healthy controls (n = 22). Urinary reference intervals were set by analyzing 24-h urine collections of 120 healthy subjects. Total run-time was 6 min. Intra- and inter-assay analytical variation were <10%. Linearity in the 0–7300 μmol/L calibration range was excellent (R2 > 0.99). Quantification limits were 30 and 0.9 nmol/L in urine and plasma, respectively. Platelet-poor serotonin concentrations in metastatic carcinoid patients were significantly higher than in controls. The urinary reference interval was 10–78 μmol/mol creatinine. Serotonin analysis with sensitive and specific XLC-MS/MS overcomes limitations of conventional HPLC. This enables accurate quantification of serotonin for both routine diagnostic procedures and research in serotonin-related disorders

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified

Inhibition of norepinephrine 3H release from sympathetic nerve endings in veins by acetylcholine

Experiments were designed to determine if the relaxation obtained with acetylcholine during electric stimulation of cutaneous veins results from inhibition of norepinephrine release. Helical strips of dog saphenous veins were incubated (4 hr) in Krebs Ringer solution which contained norepinephrine-7-3H (5 x 10-8 g/ml). The preparations were then rinsed and mounted for superfusion (3 ml/min) and isometric tension recording. The superfusate was collected at timed intervals for estimation of total radioactivity or for column chromatographic separation of norepinephrine and its metabolites. Electric stimulation increased the tension and the total radioactivity of the superfusate; it increased the amount of intact norepinephrine-3H present more than the metabolite fractions. Acetylcholine (5 x 10-8 to 5 x 10-7 g/ml) depressed the contractions and diminished the norepinephrine-3H efflux. Isoproterenol depressed the contractions but did not affect the efflux of radioactivity. Tyramine (2 x 10-6 g/ml) augmented both tension and efflux of radioactivity, but these actions were not depressed by acetylcholine. These experiments show that acetylcholine causes relaxation of venous smooth muscle constricted by sympathetic stimulation and does so by inhibiting the release of norepinephrine from the nerve endings. By contrast, acetylcholine does not inhibit the release of norepinephrine by tyramine.link_to_subscribed_fulltex

Norepinephrine metabolism in canine saphenous vein: Prevalence of glycol metabolites

To examine the disposition of [3H]norepinephrine ([3H]NE) in adrenergically innervated veins, helical strips of canine saphenous veins were incubated in Krebs-Ringer solution containing D,L[3H]NE (2 x 10-7 M) for 2 hr. [3H]NE and its metabolites were measured in extracts of veins and in superfusate (Krebs-Ringer) collected during basal conditions and during release of [3H]NE evoked by electrical stimulation (1-8 Hz), tyramine (5 x 10-6 x 10-4 M), or high concentrations of potassium (35-100 meq/liter). During basal conditions, the efflux from veins comprised mainly metabolites of [3H]NE, especially 3,4-dihydroxyphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG); this pattern was unchanged by cocaine treatment, and monoamine oxidase inhibition reduced the formation of DOPEG. During evoked release of NE, the major metabolites in the perfusate were DOPEG, MOPEG, and normetanephrine, and their proportions differed with the stimulus used: O-methylated metabolites in the perfusate always increased more than did the deaminated catechol compounds; DOPEG and MOPEG were released in greater amounts than the corresponding acids; and cocaine treatment caused a higher content of all metabolites except DOPEG. 3-Methoxy-4-hydroxymandelic acid was also formed by the vein but was retained in the tissue.link_to_subscribed_fulltex

Norepinephrine metabolism in canine saphenous vein: prevalence of glycol metabolites.

To examine the disposition of [3H]norepinephrine ([3H]NE) in adrenergically innervated veins, helical strips of canine saphenous veins were incubated in Krebs-Ringer solution containing D,L[3H]NE (2 X 10(-7) M) for 2 h. [3H]NE and its metabolites were measured in extracts of veins and in superfusate (Krebs-Ringer) collected during basal conditions and during release of [3H]NE evoked by electrical stimulation (1-8 Hz), tyramine (5 X 10(-6) to 5 X 10(-4) M), or high concentrations of potassium (35-100 meq/liter). During basal conditions, the efflux from veins comprised mainly metabolits of [3H]NE, especially 3,4-dihydroxphenylglycol (DOPEG) and 3-methoxy-4-hydroxyphenylglycol (MOPEG); this pattern was unchanged by cocaine treatment, and monoamine oxidase inhibition reduced the formation of DOPEG. During evoked release of NE, the major metabolites in the perfusate were DOPEG, MOPEG, and normetanephrine, and their proportions differed with the stimulus used: O-methylated metabolites in the perfusate always increased more than did the deaminated catechol compounds; DOPEG and MOPEG were released in greater amounts than the corresponding acids; and cocaine treatment caused a higher content of all metabolites except DOPEG. 3-Methoxy-4-hydroxymandelic acid was also formed by the vein but was retained in the tissue.link_to_subscribed_fulltex

Acetylcholine-inhibition of transmitter release from adrenergic nerve terminals mediated by muscarinic receptors

The evidence is reviewed for the presence of muscarinic receptors on the sympathetic nerves to blood vessels. Activation of these receptors by acetylcholine in doses that are too small to affect the smooth muscle cells directly inhibits the release of norepinpherine evoked by electric impulses or potassium ions. This inhibitory action of acetylcholine is prevented by muscarinic blocking agents and is probably due to hyperpolarization of the adrenergic nerve terminals.link_to_subscribed_fulltex