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1 The modulatory effects of agonists and antagonists of prejunctional α[sub 2]-adrenoceptors on the electrical field stimulation (EFS, 0.3 ms, 12 V)-induced release of endogenous noradrenaline (NA) and the cotransmitter adenosine 5′ triphosphate (ATP) were measured in endothelium-denuded segments of canine inferior mesenteric artery and compared with effects in mesenteric vein. The overflow of NA and ATP was evoked by long-duration (2 min) EFS at low frequency (4 Hz) and high frequency (16 Hz) of stimulation and was analysed using HPLC techniques with electrochemical detection and fluorescence detection, respectively. 2 The EFS-evoked overflow of both NA and ATP was significantly reduced by tetrodotoxin (1 µM) and guanethidine (10 µM) in the artery and vein. Desipramine (10 µM), a blocker of neuronal uptake of NA, increased the EFS (4 and 16 Hz)-evoked overflow of NA in both artery and vein. EFS-evoked overflow of NA in vein exceeded the NA overflow in artery at both 4 and 16 Hz in control preparations as well as in the presence of desipramine. However, the EFS- evoked overflow of ATP was equal in the artery and vein. 3 Stimulation of α[sub 2]-adrenoceptors with clonidine (0.1 µM) and oxymethazoline (0.3 µM) reduced the EFS evoked overflow of NA in both artery and vein at 4 Hz, whereas the NA overflow at 16 Hz remained unchanged in both blood vessels. The overflow of ATP as well as of ADP (and hence ATP:ADP ratio) was unaffected by the α[sub 2]-adrenoceptor agonists in the artery and vein. 4 In artery, blockade of α[sub 2]-adrenoceptors with yohimbine at a concentration of 0.1 µM caused no effect on the NA overflow neither at 4 Hz nor at 16 Hz of EFS. Yohimbine at a concentration of 1 µM increased the overflow of NA at 4 Hz but not 16 Hz of EFS. In vein, however, yohimbine (0.1 and 1 µM) increased NA overflow at both 4 and 16 Hz of stimulation. Idazoxan (1 µM) increased the NA... [ABSTRACT FROM AUTHOR]

The main intention of this study was the investigation of impact of natural biologically active ligands of nuclear retinoid/retinoid X receptors (all- trans and 9- cis retinoic acid) on proteomic pattern in human estrogen receptor negative breast cancer cell line MDA-MB-231. For this purpose, proteomic strategies based on bottom-up method were applied. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and their characterization was achieved by MALDI-TOF/TOF. By employing PDQuest™ software, we identified more than 50 proteins affected by retinoic acid isomers. For more information, 9 proteins which are associated with tumor process were selected. We determined that derivatives of retinoic acid led to significantly reduced level of proteins belonging to metabolic pathway (e. g. glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase 2) or to other cellular processes as apoptosis, regulation of transcription process or epithelial–mesenchymal transition (e.g. annexins, nucleoside diphosphate kinase B, vimentin). On the other hand all-trans retinoic acid treatment indicates up-regulated effect for heterogeneous nuclear ribonucleoprotein A2/B1. [ABSTRACT FROM AUTHOR]

Retinoic acid (all- trans and 9- cis ) isomers represent important therapeutic agents for many types of cancers, including human breast cancer. Changes in protein composition of the MCF-7 human breast cancer cells were induced by all- trans retinoic acid, 9- cis retinoic acid, and their combination and subsequently proteomic strategies based on bottom-up method were applied. Proposed approach was used for the analysis of proteins extracted from MCF-7 human breast cancer cell line utilizing a commercially manufactured kit RIPA and separated on two dimensional (2D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after treatment with both retinoic acid isomers. We found significant differences in occurrence of proteins probably affecting the cell migration process in tumour cells. Heat shock protein 27, ribonucleoprotein SmD3, and cofilin-1 were significantly upregulated after treatment with combination of individual retinoic acid isomers. On the other hand, AP-5 complex subunit beta-1 shows the different response. Thus, the results might help to find the answer to important medical questions on (i) the identification of signaling pathways affected by retinoic acid isomers or (ii) how the observed proteomic pattern might reflect the effectiveness of retinoic acids treatment. [ABSTRACT FROM AUTHOR]

Post-translational modifications (PTMs) of biomacromolecules can be useful for understanding the processes by which a relatively small number of individual genes in a particular genome can generate enormous biological complexity in different organisms. The proteomes of barley and the brewing process were investigated by different techniques. However, their diverse and complex PTMs remain understudied. As standard analytical approaches have limitations, innovative analytical approaches need to be developed and applied in PTM studies. To make further progress in this field, it is necessary to specify the sites of modification, as well as to characterize individual isoforms with increased selectivity and sensitivity. This review summarizes advances in the PTM analysis of barley proteins, particularly those involving mass spectrometric detection. Our focus is on monitoring phosphorylation, glycation, and glycosylation, which critically influence functional behavior in metabolism and regulation in organisms.

The knowledge of the structure, function, and abundance of specific proteins related to the EMT process is essential for developing effective diagnostic approaches to cancer with the perspective of diagnosis and therapy of malignancies. The success of all- trans retinoic acid (ATRA) differentiation therapy in acute promyelocytic leukemia has stimulated studies in the treatment of other tumors with ATRA. This review will discuss the impact of ATRA use, emphasizing epithelial-mesenchymal transition (EMT) proteins in breast cancer, of which metastasis and recurrence are major causes of death.

This work aimed to provide, in one isolation and separation step, an overview of the content of proteins with different solubility after treatment with all-trans retinoic acid, which is considered to be an important therapeutic agent, predominantly in acute promyelocytic leukemia. Breast, ovarian, bladder, and skin cancers have been demonstrated to be suppressed by retinoic acid, as well. The bottom-up proteomic strategies were applied for the analysis of proteins extracted from triple-negative breast cancer MDA-MB-231 cells utilizing a commercially manufactured kit. The gel electrophoresis followed by MALDI-TOF MS analysis was used for protein determination. By employing PDQuest™ software, we identified several proteins affected by all-trans retinoic acid. Two proteins, vimentin and CD44, which are associated with the epithelial-mesenchymal transition, were selected for a detailed study. We have found that all-trans retinoic acid results in significantly reduced levels of vimentin and CD44 in both the cytoplasmic and membrane fractions. A significant effect was particularly evident in CD44, where protein level in the cytoplasmic fraction was almost completely suppressed.

Changes in glycoprotein content, altered glycosylations, and aberrant glycan structures are increasingly recognized as cancer hallmarks. Because breast cancer is one of the most common causes of cancer deaths in the world, it is highly urgent to find other reliable biomarkers for its initial diagnosis and to learn as much as possible about this disease. In this Review, the applications of lectins to a screening of potential breast cancer biomarkers published during recent years are overviewed. These data provide a deeper insight into the use of modern strategies, technologies, and scientific knowledge in glycoproteomic breast cancer research. Particular attention is concentrated on the use of lectin-based affinity techniques, applied independently or most frequently in combination with mass spectrometry, as an effective tool for the targeting, separation, and reliable identification of glycoprotein molecules. Individual procedures and lectins used in published glycoproteomic studies of breast-cancer-related glycoproteins are discussed. The summarized approaches have the potential for use in diagnostic and predictive applications. Finally, the use of lectins is briefly discussed from the view of their future applications in the analysis of glycoproteins in cancer.

An attempt has been made to delineate the role of natural and synthetic retinoid receptor ligands on vimentin expression in the human triple-negative breast cancer cells. The effects of currently synthesized triorganotin derivatives of the general formula R 3 SnX (R is butyl or phenyl, X is isothiocyanate), which are considered RXR ligands, were investigated in the human MDA-MB-231 breast cancer cell line. Studies were evaluated in the presence and absence of all-trans retinoic acid (ATRA), a natural RAR ligand. Vimentin represents the major protein associated with epithelial-mesenchymal transition (EMT), an essential process when the primary tumour transforms into a malignant one. mRNA and proteomic data obtained in this study, based on the PDQuest software protein evaluation and further quantification of proteins by iTRAQ analysis, suggest that vimentin was significantly reduced in the combination of RAR ligand and RXR ligand treatment. Both tested triorganotin compounds showed similarly reduced expression of vimentin, but tributyltin isothiocyanate (TBT-ITC) proved to be more effective than triphenyltin isothiocyanate (TPT-ITC). Furthermore, the effect of natural (9cRA) and synthetic RXR ligands, both chloride and isothiocyanate derivatives, on vimentin expression was compared., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Trialkyltins and triaryltins function as nuclear retinoid X receptors (RXR) agonists due to their affinity to the ligand-binding domain of RXR subtypes and function as transcriptional activators. We present the data on combined effects of all-trans retinoic acid (ATRA), retinoic acid receptor (RAR) ligand and tributyltin chloride or triphenyltin chloride (RXR ligands) on protein pattern in MDA-MB-231 cells. Proteomic strategies based on bottom-up method were applied in this study. The total cell proteins were extracted, separated on 2D SDS-PAGE and their characterization was achieved by MALDI-TOF/TOF MS/MS. By employing PDQuest™ software, we identified more than 30 proteins differently affected by the above compounds. For further studies, we selected specific proteins associated either with metabolic pathway (glyceraldehyde-3-phosphate dehydrogenase) or to cellular processes as apoptosis, regulation of gene transcription or epithelial-mesenchymal transition (annexin 5, nucleoside diphosphate kinase B and vimentin). We have found that treatment of MDA-MB-231 cells with triorganotins reduced the expression of studied proteins. Moreover, the treatment of MDA-MB-231 cells with triorganotin compounds together with ATRA resulted in an additional reduction of annexin 5, vimentin and nucleoside diphosphate kinase B. These results demonstrate that RXR/RAR heterodimer may act under this experimental design as permissive heterodimer allowing activation of RXR by triorganotins.

Transdermal administration of drugs that penetrate, in this case directly into the blood circulation, has many advantages and is promising for many drugs thanks to its easy application and good patient compliance. ( S )-8-Methyl-6,9-diazaspiro[4.5]decan-7,10-dione (alaptide), has been studied as a potential chemical permeation enhancer. Based on its structure, four selected piperazine-2,5-diones were synthesized by means of multi-step synthetic pathways. All the compounds were investigated on their ability to enhance the permeation of the model drug theophylline from the hydrophilic medium propylene glycol:water (1:1). In vitro experiments were performed using vertical Franz diffusion cells at constant temperature 34 ± 0.5 °C and using full-thickness pig ( Sus scrofa f. domestica ) ear skin. Withdrawn samples were analyzed by RP-HPLC for determination of the permeated amount of theophylline. All the compounds were applied in ratio 1:10 ( w / w ) relative to the amount of theophylline. One hour after application, the permeated amount of theophylline from formulations with alaptide and (3 S ,6 S )-3,6-dimethylpiperazine-2,5-dione, was ca. 15- and 12-fold higher, respectively, than from the formulation without the tested compounds. Despite the enhancement ratio of both enhancers in a steady state was ca. 2.3, the pseudo-enhancement ratio in the time range from 1 to 3 h was 4.4. These enhancement ratios indicate that the compounds are able to enhance the permeation of agents through the skin; however, the short-term application of both compound formulations seems to be more advantageous. In addition, the screening of the cytotoxicity of all the prepared compounds was performed using three cell lines, and the compounds did not show any significant toxic effect.

Sphingosine kinase 1 (SphK1), the enzyme that produces the bioactive sphingolipid metabolite, sphingosine-1-phosphate, is a promising new molecular target for therapeutic intervention in cancer and inflammatory diseases. In view of its importance, the main objective of this work was to find new and more potent inhibitors for this enzyme possessing different structural scaffolds than those of the known inhibitors. Our theoretical and experimental study has allowed us to identify two new structural scaffolds (three new compounds), which could be used as starting structures for the design and then the development of new inhibitors of SphK1. Our study was carried out in different steps: virtual screening, synthesis, bioassays and molecular modelling. From our results, we propose a new dihydrobenzo[b]pyrimido[5,4-f]azepine and two alkyl{3-/4-[1-hydroxy-2-(4-arylpiperazin-1-yl)ethyl]phenyl}carbamates as initial structures for the development of new inhibitors. In addition, our molecular modelling study using QTAIM calculations, allowed us to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analyses indicate that the cationic head of the different compounds must be refined in order to obtain an increase in the binding affinity of these ligands., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

In the present study, we analyzed the cell lysates of human tumour cell lines representing two major clinically different types of breast cancer. Our main goal was to show the differences between them on proteomic level. Gel electrophoresis followed by MALDI-TOF MS analysis was used for proteins determination. Exactly 98 proteins were unequivocally identified and 60 of them were expressed differentially between MDA-MB-231 and MCF-7 cell lines. Among the proteins reported here, some well-known breast cancer markers (e.g., annexin A1, annexin A2 and vimentin) were identified in the MDA-MB-231 cell line and thus we were able to distinguish both cell lines sufficiently.

Retinoids, acting through their cognate nuclear receptors, are crucial transcriptional regulators of many cellular processes such as differentiation, development, apoptosis, carbohydrate and lipid metabolism, homeostasis, etc. The aim of this study was the exploration of molecular mechanisms in relation to therapy of human breast cancer. One of the efficient strategies is identification of biomarkers as important tools in early cancer diagnosis and advisable treatment. Retinoids have been regarded as important therapeutic agents for many types of cancers, including human breast cancer. The effects of all-trans retinoic acid and 9-cis retinoic acid or their combination on proteomic pattern in human MCF-7 breast cancer line were investigated. The total cell proteins were extracted utilizing a commercially Radio-Immunoprecipitation Assay (RIPA) buffer and separated on 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D SDS-PAGE). The proteins were subsequently digested in-gel by trypsin and identified by matrix assisted laser desorption ionization technique with time of flight mass analyzer (MALDI-TOF/TOF). Our data offer novel information on the proteomic pattern of proteins evaluated after treatment of MCF-7 cells with retinoic acid isomers.

An efficient reduction of double bonds conjugated with nitrile groups and acid or base sensitive furan rings with 2-phenylbenzimidazoline generated in situ has been successfully accomplished with high yields and excellent selectivity. The employed reducing agent was prepared in one step from ordinary chemicals. The other advantages of the presented method include mild and convenient reaction conditions, a benign and cost effective reagent, simple work-up and separation of the products. As this process does neither affect cyano and nitro groups nor furan rings, it is a valuable alternative when metal-catalyzed hydrogenations or borohydride reductions have failed.

The major objective of proteomics is to identify and examine the large numbers of proteins extracted from complex biological systems. This is generally achieved by combining various techniques of protein separation with a mass spectrometric analysis of proteins that are digested enzymatically. Recently, several alternatives to this standard protocol have been developed for efficient and fast protein digestion. One option is the use of modified trypsin instead of native trypsin for the in-gel digestion of proteins. Microwave, ultrasonic-assisted protein enzymatic digestion and proteolysis accelerated by infrared radiation are other suitable alternatives. The application of the variable performance of the fast enzymatic digestion of proteins by using different techniques is reported here. The advantage of these methods is to have the ability to detect proteins in a shorter span of time. For example, using alternative protein digestion takes only minutes, in contrast to the several hours required by conventional methods. To demonstrate the suitability of this fast procedure, the digestion of carbonic anhydrase, bovine serum albumin, lysozyme and proteins extracted from plants (Hordeum vulgare, Arabidopsis thaliana) were used. Considering that the required reaction time for the conventional method is much longer, these applied methodic approaches tend to give in-gel digestion a much higher efficiency rating. This study examines the fast, efficient and low-cost proteolytic strategies for the digestion process, and for protein identification based on the use of ultrasound and infrared technology. In addition, comparisons of the applied techniques were studied. Several differences were found, suggesting the potential use of proteolysis accelerated by infrared radiation., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: In barley, starch occurs in the form of granules with bimodal size distribution. Enzymatic hydrolysis of the starch granule is one of the most important reactions occurring during malting and mashing. Previous studies revealed the discrepancies in the assumption that barley varieties with better malting qualities should have a higher A/B (large/small starch granules) ratio. This led us to focus our attention on detailed analysis of two barley varieties, Jersey and Tolar, both with high malting quality but significantly differing in A/B (1.28 and 0.66, respectively), were chosen for more detailed analysis in the actual work. In this study, the capacity of gravitational field flow fractionation (GFFF) to monitor amylolysis of the starch granules was investigated., Results: Isolated starch granules from these two barley cultivars were treated with amylases. The changes in starch granule size and bimodal distribution were studied by GFFF. Simultaneously, free sugars released during enzymatic digestion were observed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The changes in the fractogram and in the mass spectra reflect a correlation with the progress of enzymatic hydrolysis., Conclusion: The results show the interest in utilization of GFFF as a simple and cheap method for monitoring changes in the distribution of the starch granule size during amylolysis., (Copyright © 2011 Society of Chemical Industry.)

A study of complex protein mixtures obtained from biological samples by MS demands proper purification and separation technique. The method of divergent flow isoelectric focusing (DF IEF) promises improvement of sample preparation in proteomic studies. DF IEF was carried out in a separation channel with increasing width. The channel was cut out from a polyester nonwoven web. DC voltage (800 V) was brought to two pairs of electrodes situated on the channel sides. Amphoteric compounds, including proteins, drift through the channel carried by flow (18-25 ml/h) in streamlines given by their isoelectric points. The pH gradient (3-10) and its stability during analysis have been monitored with colored low-molecular mass pI markers. Separated fractions were collected in ten microvials and further analyzed by MS. The suggested method was used for separation and purification of crude protein extract from barley grain, malt, and beer. Collected fractions of separated proteins were characterized by MALDI-MS. Desalting during IEF enhanced significantly the quality of mass spectra. It also simplified monitoring of post-translational modifications and protein changes occurring during malting and brewing. Results have shown the real potential of the suggested DF IEF device lay-out as an efficient preparative tool for separation and purification of complex protein mixtures for further analyses.

Both top-down (combining protein separation with MS analysis of intact proteins) and bottom-up (MS analysis of digested proteins) proteomic approaches were used for detailed characterization of nonspecific lipid transfer protein from barley malt. The aim was obtaining high-coverage of the primary structure of the proteins and the determination of PTMs such as lipid adduction and glycation. Here we present an influence of 15 proteomic protocols (differing in applied separation technique, enzyme and digestion procedure) on the extent of the coverage of the protein primary structure. The most successful protocols were in-gel digestion with trypsin of alkylated protein and in-solution digestions with trypsin or trypsin/chymotrypsin mixture of the nonalkylated protein. Totally, full sequence coverage based on the PMF and 85% sequence coverage based on the peptide fragmentation including PTMs was obtained.

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.

Using high performance liquid chromatography techniques with fluorescence detection we demonstrate that overflow of beta-nicotinamide adenine dinucleotide evoked by electrical field stimulation (16 Hz, 0.3 ms) in the canine isolated mesenteric artery is increased by the activators of adenylyl cyclase (AC) forskolin and calcitonin gene-related peptide (CGRP), by dibutyryl cAMP, and by the inhibitors of phosphodiesterases III and IV milrinone and rolipram. The enhancing effect of forskolin is abolished by the AC inhibitor MDL 12,330A and by protein kinase A (PKA) inhibitors peptide 14-22 amide and 4-cyano-3-methylisoquinoline. Therefore, activation of the AC/cAMP/PKA pathway enhances the release of beta-NAD+ from perivascular nerve terminals.

Chemical signaling in autonomic neuromuscular transmission involves agents that function as neurotransmitters and/or neuromodulators. Using high performance liquid chromatography techniques with fluorescence and electrochemical detection we observed that, in addition to ATP and norepinephrine (NE), electrical field stimulation (EFS, 4-16 Hz, 0.1-0.3 ms, 15 V, 60-120 s) of isolated vascular and non-vascular preparations co-releases a previously unidentified compound with apparent nucleotide or nucleoside structure. Extensive screening of more than 25 nucleotides and nucleosides followed by detailed peak identification revealed that beta-nicotinamide adenine dinucleotide (beta-NAD) is released in tissue superfusates upon EFS of canine mesenteric artery (CMA), canine urinary bladder, and murine urinary bladder in the amounts of 7.1 +/- 0.7, 26.5 +/- 4.5, and 15.1 +/- 3.2 fmol/mg of tissue, respectively. Smaller amounts of the beta-NAD metabolites cyclic adenosine 5'-diphosphoribose (cADPR) and ADPR were also present in the superfusates collected during EFS of CMA (2.5 +/- 0.9 and 5.8 +/- 0.8 fmol/mg of tissue, respectively), canine urinary bladder (1.8 +/- 0.5 and 9.0 +/- 6.0 fmol/mg of tissue, respectively), and murine urinary bladder (1.4 +/- 0.1 and 6.2 +/- 2.4 fmol/mg of tissue, respectively). The three nucleotides were also detected in the samples collected before EFS (0.2-1.6 fmol/mg of tissue). Exogenous beta-NAD, cADPR, and ADPR (all 100 nm) reduced the release of NE in CMA at 16 Hz from 27.8 +/- 6.0 fmol/mg of tissue to 15.5 +/- 5.0, 12 +/- 3.0, and 10.0 +/- 4.0 fmol/mg of tissue, respectively. In conclusion, we detected constitutive and nerve-evoked overflow of beta-NAD, cADPR, and ADPR in vascular and non-vascular smooth muscles, beta-NAD being the prevailing compound. These substances modulate the release of NE, implicating novel nucleotide mechanisms of autonomic nervous system control of smooth muscle.

1. At least two enzymatic activities are proposed to degrade the extracellular ATP: (i) ubiquitously expressed membrane-bound enzymes (ecto-nucleotidases); and (ii) soluble (releasable) nucleotidases that are released during stimulation of sympathetic nerves and break down neuronal ATP. No quantitative data have placed the magnitude of these nucleotidase activities into a physiological perspective of neurovascular control. 2. We studied comparatively the membrane-bound and releasable nucleotidase activities in canine isolated inferior mesenteric arteries and veins using 1,N6-etheno(epsilon)-nucleotides (i.e. epsilon-ATP, epsilon-ADP, epsilon-AMP and epsilon-adenosine) as exogenous substrates. The enzymatic activities were estimated by measuring the disappearance of the epsilon-substrate and appearance of epsilon-products by means of HPLC-fluorescence detection during either stimulation of sympathetic perivascular nerves (releasable activity) or in the absence of nerve stimulation (ecto-nucleotidase activity). 3. Incubation of vascular segments with 50 nmol/L epsilon-ATP for 60 min resulted in a decrease of the epsilon-ATP substrate by 63.5 +/- 4.6 and 91.2 +/- 6.2% in the artery and vein, respectively. In contrast, the decrease of the epsilon-ATP during electrical field stimulation (EFS; 16 Hz, 0.3 msec, 2 min) was 39.8 +/- 4.2% in the artery and 13.1 +/- 7.3% in the vein. Therefore, the mesenteric arteries demonstrate a greater releasable ATPase activity and a weaker ecto-ATPase activity than mesenteric veins. 4. The degradation of epsilon-ADP and epsilon-AMP was similar in both blood vessels under either experimental protocol. The epsilon-adenosine was not significantly degraded in the absence or presence of EFS. 5. These data implicate a differential removal of extracellular ATP as a potential mechanism of serving resistance and capacitance in the splanchnic circulation.

Objective: Our major hypothesis is that cyclic adenosine-3',5'-monophosphate (cAMP)-mediated modulation of neurotransmitter release plays different roles at low and high activity of the sympathetic nervous system. We further hypothesize that cAMP-mediated neuromodulation might underlie disparate neurovascular control in mesenteric arteries and veins., Methods: Electrical field stimulation (EFS)-evoked overflow of noradrenaline (NA) was evaluated in the absence or presence of activators and inhibitors of cAMP-dependent pathway at low (4 Hz) and high (16 Hz) frequencies of stimulation of endothelium-denuded secondary and tertiary branches of the canine isolated inferior mesenteric arteries and veins. The content of NA in samples of the superfusates collected before and during nerve stimulation was assayed by high-performance liquid chromatography (HPLC) technique in conjunction with electrochemical detection. Student's t-test and ANOVA analyses were applied for statistical analysis., Results: Activation of cAMP-dependent pathway with either isoproterenol (ISO, 10 microM), forskolin (1 microM), dibutyryl cAMP (100 microM) or combined site-specific activators of cAMP-dependent protein kinase (PKA) [i.e. N(6)-phenyl-adenosine-3',5'-cyclic monophosphate, 8-(6-aminohexyl) aminoadenosine-3',5'-cyclic monophosphate, and the Sp-isomer of 5,6-dichloro-1-D-ribofuranosylbenzimidazole-3',5'-cyclic monophosphorothioate, each 100 microM] caused an enhancement of the EFS-evoked overflow of endogenous NA at 16 Hz of stimulation but was without an effect at 4 Hz of stimulation both in artery and vein. The EFS (16 Hz)-evoked overflow of NA in vein was also increased in the presence of inhibitors of phosphodiesterase (PDE) III and PDE IV (i.e. milrinone, 0.4 microM, and roilpram, 30 microM), whereas these inhibitors did not affect the overflow of NA in the artery. The facilitating effect of activators of cAMP-dependent pathway on the EFS-evoked release of NA at 16 Hz appears to be more pronounced in the vein than in artery. The increasing effect of ISO (10 microM) was inhibited with either propranolol (1 microM) or the adenylyl cyclase (AC) inhibitor [9-(tetrahydro-2'-furyl)adenine] (SQ 22,536, 100 microM) in both blood vessels. The ISO effect was inhibited by the PKA inhibitor 14-22 amide (PKI(14-22)), 1 microM, in the artery but not in vein. The enhancing effect of FSK was inhibited by pretreatment of the tissue with SQ 22,536, 100 microM, or the PKA inhibitors PKI(14-22), 1 microM, and 4-cyano-3-methylisoquinoline, 50 nM. However, the inhibitors alone did not significantly change the EFS-evoked overflow of NA in both blood vessels., Conclusions: Activation of AC-cAMP-PKA pathway appears to play a role in modulating NA release at higher stimulation frequencies as might be expected during stress, strenuous exercise, or hemorrhage. The AC-cAMP pathway plays a more pronounced role in the autonomic neural control of mesenteric veins than of the corresponding arteries, whereas the PKA contribution is more distinct in the arteries.

Analysis of endogenous nucleotides in biologic media is hampered by rapid degradation and low final concentrations that are difficult to detect. A reversed-phase high-performance liquid chromatographic (HPLC) technique is described that efficiently detects a stable fluorescence derivative of adenosine 5'-diphosphoribose (ADPR), 1,N6-etheno-ADPR (epsilon-ADPR), at low femtomolar concentration range in vascular tissue superfusates. epsilon-ADPR was formed by the reaction of ADPR with chloroacetaldehyde at 80 degrees C and pH 4.0. Gradient elution with 0.1 M KH2PO4 (pH 6.0), increasing methanol (0-35% over 18 min), and a 25-cm by 4.5-mm (5 microm) silica ODS-AM column were employed. epsilon-ADPR was detected by fluorescence at an excitation wavelength of 230 nm and an emission wavelength of 410 nm. The detection sensitivity for epsilon-ADPR was approximately 10 fmol. Linearity of the HPLC detection method was demonstrated in the range from 0.0125 to 1 pmol epsilon-ADPR. The method was validated in terms of within-day and between-day reproducibility of retention times and peak areas of standard nucleotide. Matrix-assisted laser desorption/ionization mass spectrometry measurements confirmed the presence of an etheno ring after reaction of ADPR with chloroacetaldehyde. The method was applied to quantitate the overflow of ADPR upon electrical field stimulation (8 Hz, 0.3 ms, 15 V, 1-2 min) of both canine and guinea-pig isolated mesenteric artery segments., (Copyright 2002 Elsevier Science (USA).)

1. The present study was designed to compare the overflow of sympathetic neurotransmitters of guinea-pig inferior mesenteric artery and mesenteric vein evoked by electrical field stimulation (EFS) with special emphasis on the simultaneous release of ATP and noradrenaline (NA). The stimulation-evoked overflow of ADP, AMP and adenosine was also evaluated. 2. Endothelium-denuded segments of inferior mesenteric arteries or veins were superfused in a small volume (200 microL)-chamber for EFS and subsequent detection of NA (HPLC- electrochemical detection) and adenine nucleotides and adenosine (HPLC-fluorescence detection) in samples of the superfusate. 3. Both arteries and veins responded to EFS (15 V, 4-16 Hz, 0.3 msec for 60 s) with overflow of ATP and NA in a tetrodotoxin (1 micromol/L)- and guanethidine (10 micromol/L)-sensitive manner. The EFS-evoked overflow of NA in veins exceeded the overflow of NA in arteries at all frequencies of stimulation, whereas the EFS-evoked overflow of ATP, ADP and AMP in veins exceeded the overflow of adenine nucleotides in arteries at 4 and 8 Hz but not at 16 Hz stimulation. The EFS-evoked overflow of adenosine was similar in arteries and veins. 4. Activation of alpha1-adrenoceptors with methoxamine (10 micromol/L) did not produce overflow of ATP. 5. Blockade of alpha1/alpha2-adrenoceptors with phentolamine (1 micromol/L) did not affect EFS-evoked overflow of ATP, ADP, AMP and adenosine. 6. It is concluded that overflow of ATP and NA from sympathetic nerves may constitute an effective mechanism in the complex balance between capacitance and resistance in splanchnic circulation.

Vasoconstrictor responses to electrical field stimulation (EFS, 0.2-32 Hz, 0.1 ms, 12 V, for 1 min) were measured in endothelium-denuded segments of guinea-pig mesenteric vein and compared to responses in mesenteric artery. The distribution of both tyrosine-hydroxylase-like immunoreactivity (TH-LI) and neuropeptide Y-like immunoreactivity (NPY-LI) was also studied using anti-TH and anti-NPY antibodies. The effect of exogenous NPY (10 nM) on EFS (8 Hz, 0.3 ms, 12 V, for 1 min)-evoked overflow of noradrenaline (NA) was also studied using an HPLC technique with electrochemical detection. Veins responded with contractions at lower frequencies of stimulation than arteries. Prazosin (0.1 microM) abolished the EFS-evoked contractions in artery at 0.5-32 Hz and in vein at 0.2-1 Hz of stimulation. However, in vein, the contractile responses to EFS at 2-32 Hz of stimulation were only reduced by prazosin. Phentolamine (1 microM) abolished the responses to 0.5-4 Hz and reduced the responses to 8-32 Hz of EFS in artery. In vein, phentolamine (1 microM) abolished the responses to 0.2-1 Hz and facilitated the contractions elicited by 16-32 Hz. The NPY-receptor antagonist BIBP3226 (1 microM), in combination with phentolamine, abolished contractions in vein. Yohimbine (0.1 microM) abolished the responses to lower frequencies of stimulation in both artery (0.5-2 Hz) and vein (0.2-1 Hz). The responses to greater frequency stimulation were not affected by yohimbine in artery, and were facilitated in vein. Pre-treatment of animals for 24 h with reserpine abolished contractile responses to EFS in artery, whereas in vein, responses to 0.2-2 Hz were abolished while responses to 4-32 Hz were unchanged. Suramin (100 microM) or alpha,beta-methylene ATP (alpha,beta MeATP; 10-100 microM) treatment did not affect the contractile responses to EFS in either artery or vein. Pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS; 30 microM), even potentiated the responses to 2-16 Hz in vein. However, following resperine-treatment, both PPADS and suramin reduced the nerve-evoked contractions of vein. Either BIBP3226 (1 microM) alone or BIBP3226 in combination with PPADS or suramin abolished the contractile response to EFS in reserpine-treated veins. NPY (100 nM) produced significantly more contraction in vein than in artery (i.e., 93 +/- 2.5 versus 7 +/- 4% of the response to 70 mM KCl, respectively). NPY (10 nM) significantly reduced the NA overflow evoked by EFS at 8 Hz. Flat mount preparations and cryostat sections of both mesenteric artery and vein revealed that TH-LI and NPY-LI were co-localized in a dense network of fibers within the adventitial layer. In conclusion, NA exclusively mediates the contractile response to sympathetic nerve stimulation in guinea-pig mesenteric artery, whereas at least three neurotransmitters [i.e., NA, adenosine 5'-triphosphate (ATP) and NPY] are involved in the neural response of mesenteric vein.

Neuropeptide Y (NPY) is a cotransmitter with noradrenaline in guinea pig inferior mesenteric vein. Tyrosine hydroxylase-like immunoreactivity and NPY-like immunoreactivity were colocalized in a dense network of fibers within the adventitial layer of guinea-pig inferior mesenteric vein. Vasoconstrictor responses to electrical field stimulation (0.2-64 Hz, 0.1 ms, 12 V, for 10 s) appear to be mediated primarily by norepinephrine at 0.2 to 4 Hz and by NPY at 8 to 64 Hz. NPY Y1 receptors mediate the contractile responses to both endogenous and exogenous NPY. Norepinephrine and NPY are involved in neuromuscular transmission in guinea pig mesenteric vein suggesting that the sympathetic nervous system requires the coordinated action of norepinephrine and NPY to serve capacitance.