Repeated immobilization stress induces catecholamine production in rat mesenteric adipocytes.

Catecholamines (CATs), the major regulator of lipolysis in adipose tissue, are produced mainly by the sympathoadrenal system. However, recent studies report endogenous CAT production in adipocytes themselves. This study investigated the effects of single and repeated (7-14 times) immobilization (IMO) stress on CAT production in various fat depots of the rat. Single IMO quickly induced a rise of norepinephrine (NE) and epinephrine (EPI) concentration in mesenteric and brown adipose depots. Adaptive response to repeated IMO included robust increases of NE and EPI levels in mesenteric and subcutaneous adipose tissue. These changes likely reflect the activation of sympathetic nervous system in fat depots by IMO. However, this process was also paralleled by an increase in tyrosine hydroxylase gene expression in mesenteric fat, suggesting regulation of endogenous CAT production in adipose tissue cells. Detailed time-course analysis (time course 10, 30, and 120 min) clearly showed that repeated stress led to increased CAT biosynthesis in isolated mesenteric adipocytes resulting in gradual accumulation of intracellular EPI during IMO exposure. Comparable changes were also found in stromal/vascular fractions, with more pronounced effects of single than repeated IMO. The potential physiological importance of these findings is accentuated by parallel increase in expression of vesicular monoamine transporter 1, indicating a need for CAT storage in adipocyte vesicles. Taken together, we show that CAT production occurs in adipose tissue and may be activated by stress directly in adipocytes.

Adipocytes as a new source of catecholamine production.

Catecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.

Development and validation of a high throughput direct radioimmunoassay for the quantitative determination of serum and plasma melatonin (N-acetyl-5-methoxytryptamine) in mice.

A simple, sensitive and specific high throughput radioimmunoassay (RIA) for the quantitative determination of total melatonin (N-acetyl-5-methoxytryptamine) was developed. This method allows the analysis of melatonin in different biological fluids with small sample volumes (e.g. mice and rats), and wide working range. With the preparation of matrix-specific calibrators called "Equalizing Reagent" the influence of results due to different composition between standards and sample matrix was reduced. This reagent is produced by use of the respective biological liquid. Endogenous melatonin is removed by adsorption to activated charcoal. The melatonin-free biological liquid is then used to equalize the assay matrix of standards and untreated samples. Finally, all samples including the standards are digested by use of a protease to reduce non-specific binding, for example to albumin or albumin-like molecules. High-affinity specific antibodies were produced by immunization of rabbits with 5-methoxytryptamine-bovine serum albumin. The review of cross reactions to ten structurally similar compounds showed that the antibody has a high specificity for melatonin. This direct RIA uses a [(125)I]-melatonin tracer for the determination of melatonin. 5-methoxytryptamine was synthesized by direct iodination with [(125)I]-Bolton-Hunter-Reagent. The required acceptance criteria for validation parameters were fulfilled. The flexible standards cover a working range from 12 to 4000 pg/mL with a sample volume of 50 microL (e.g. working range from 3 to 1000 pg/mL with a sample volume of 200 microL). The limit of detection in mouse serum and mouse plasma was 9 pg/mL and 7 pg/mL, respectively. The recovery of melatonin in mouse serum was 108% and in mouse plasma 99%. The variation coefficients of the assay, within and between runs, ranged between 7 and 13% in mouse serum and between 5 and 8% in mouse plasma. Based on the determination of a 24-h profile of melatonin in mouse samples a characteristic diurnal rhythm of melatonin was observed. The wide working range makes it possible to analyse low and high melatonin concentrations. The validated direct RIA was compared with established sample preparation methods such as liquid-liquid- and solid-phase-extraction followed by RIA. The correlation for methanol extraction is y=1.1x-0.9, R(2)=0.98, P<0.001 and C(18)-extraction y=0.8x-0.03, R(2)=0.99, P<0.001. The distinct advantage of the direct assay of melatonin is that complicated extraction steps can be avoided. The realized advantages of the direct RIA when compared to a commercially available melatonin RIA are its low sample volume and ease of implementation. Exemplary, plasma melatonin was determined at C3H, C57BL, wild-type and melatonin receptor (MT1-/- and MT2-/-) knockout mice kept under L:D=12:12 cycles. The results have indicated that at all mouse strains have plasma melatonin content, with higher levels during the night and lower levels during the day. Because of different melatonin concentrations the direct RIA convince by its low detection limit and wide working range. The determination of serum and plasma melatonin in mice by ELISA or HPLC-technique previously failed due to the required use of a high sample volume and the low sensitivity. In summary, it can be concluded that the developed and validated direct RIA meets the requirements for the determination of melatonin and is especially suitable for the analysis of melatonin in different mouse strains.

Acute allograft rejection and immunosuppression: influence on endogenous melatonin secretion.

Melatonin displays a dose-dependent immunoregulatory effect in vitro and in vivo. Exogenous high-dose melatonin therapy exerted an immunosuppressive effect, abrogating acute rejection (AR), significantly prolonging transplant survival. Endogenous melatonin secretion, in response to heterotopic rat cardiac allograft transplantation (Tx), was investigated during the AR response and under standardized immunosuppressive maintenance therapy with cyclosporin A (CsA) and rapamycin (RPM). Recipients of syngeneic transplants, and recipients of allogeneic grafts, either untreated or receiving immunosuppressive therapy constituted the experimental groups. Endogenous circadian melatonin levels were measured at 07:00, 19:00, and 24:00 hr, using a novel radioimmunoassay (RIA) procedure, under standardized 12-hr-light/dark-conditions (light off: 19:00 hr; light on: 07:00 hr), before and after Tx. Neither the operative trauma, nor the challenge with a perfused allograft or the AR response influenced endogenous melatonin peak secretion. Immunosuppressive therapy with CsA led to a significant increase in peak secretion, measured for days 7 (212 +/- 40.7 pg/mL; P < 0.05), 14 (255 +/- 13.9 pg/mL; P < 0.001), and 21 (219 +/- 34 pg/mL; P < 0.01) after Tx, as compared with naïve animals (155 +/- 25.8 pg/mL). In contrast, treatment with RPM significantly decreased the melatonin peak post-Tx up to day 7 (87 +/- 25.2 pg/mL; P < 0.001), compared with naïve animals (155 +/- 25.8 pg/mL). These findings imply a robust nature of the endogenous circadian melatonin secretion kinetics, even against the background of profound allogeneic stimuli. Immunosuppressive maintenance therapy with CsA and RPM modulated early melatonin secretion, indicating a specific secondary action of these drugs. Further studies are necessary to disclose the long-term effect of immunosuppressive therapy on circadian melatonin secretion in transplant recipients.

Development of enantioselective immunoassays for free plasma metanephrines.

The development of an enantioselective radioimmunoassay (RIA) and enzyme immunoassay (EIA) for L-normetanephrine (NM) and L-metanephrine (M) were studied. Prior to the immunoassay, the protein matrix of the ethylenediaminetetraacetic acid (EDTA) plasma samples was removed by acid precipitation, followed by derivatization of the L-metanephrines to N-acyl-L-metanephrines. For the EIA, N-acyl-L-NM and N-acyl-L-M were bound to the surface of microtiter plates. Acylated L-metanephrines from the sample and solid-phase-bound N-acyl-L-NM or N-acyl-L-M competed for a fixed number of rabbit anti-N-acyl-NM or anti-N-acyl-M antibody binding sites. When the system was in equilibrium, free antigens and free antigen-antibody complexes were removed by washing. The antibodies bound to the respective solid-phase N-acyl-L-NM or N-acyl-L-M were detected by a goat anti-rabbit IgG-peroxidase conjugate using tetramethyl benzidine (TMB) as a substrate. The RIAs were conventional double antibody tests using the above rabbit antisera and specific (125)I-N-acyl-L-metanephrine tracers. Chiral recognition of the L-enantiomers was observed not only for the native molecules but for all N-acyl derivatives tested. The cross-reactivity to the corresponding D-enantiomers was always <1%. The detection limits were found to be approximately 0.04 nmol/L (7.5 pg/mL) for M and 0.08 nmol/L (15 pg/mL) for NM in RIA and EIA.