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Existence of functional beta1- and beta2-adrenergic receptors on microglia.

We examined the expression and function of beta-adrenergic receptor (beta-AR) subtypes in both isolated primary rat microglia and a rat microglial cell line. RT-PCR analyses revealed that microglia expressed beta(1)- and beta(2)-ARs but not beta(3)-ARs, whereas rat primary peritoneal macrophages expressed only beta(2)-ARs. Stimulation of beta-ARs on microglia by norepinephrine (NE) resulted in an increase in the level of intracellular cAMP and the subsequent expression of interleukin-1beta mRNA. These effects were prevented by propranolol. Similar results were obtained with other selective beta(1)-AR agonists and antagonists. beta(2)-ARs on microglia were also functional. It is possible that noradrenergic innervations participate in the control of microglial functions via beta(1)-ARs on microglia in the brain, because NE has high affinity for beta(1)- and beta(3)-ARs but little or no affinity for beta(2)-ARs. It seems physiologically significant that microglia can be controlled by NE, which predominates over epinephrine in the brain, whereas macrophages in peripheral tissues can be controlled by epinephrine, which is at higher levels in peripheral tissues.

Adrenergic beta-1 Receptor Agonists↗

The metabolic effects of sodium dichloroacetate in the suckling newborn rat.

Subcutaneous injection of sodium dichloroacetate (1 microgram/ g body wt every 3 h) in suckling newborn rats caused in 6 h a fall of 2.5 mmol/l in blood glucose concentrations, and a rise of 2.4 mmol/l in total blood ketone body levels, but no change in the high levels of plasma non esterified fatty acids. Glucose utilization, measured after intraperitoneal injection of D-glucose (2 microgram/g body wt), was not increased in newborns injected with dichloroacetate. The hypoglycaemia resulted from a decrease in gluconeogenic rate, secondarily to a lowering effect of dichloroacetate on blood levels of lactate, pyruvate and alanine. The hypoglycaemia induced by dichloroacetate was completely reversed by injecting newborn rats with a mixture of gluconeogenic precursors (lactate, pyruvate and alanine). It is concluded that the high rate of gluconeogenesis observed in suckling newborn rats in sustained by an increased release of lactate and, to a much smaller extent of pyruvate and alanine, by peripheral tissues. This probably resulted from the low pyruvate dehydrogenase activity found in peripheral tissues of the newborn rat. The hyperketonaemia induced by dichloroacetate could result from an increased ketogenesis and/or a decreased ketone body utilization.

Acetates↗

Steroid levels and steroid metabolism in the mussel Mytilus edulis: the modulating effect of dispersed crude oil and alkylphenols.

Significant amounts of oil and alkylphenols are released into the sea by petroleum installations as a result of discharges of produced water. Some of these pollutants elicit estrogenic responses in fish, but their effects on the endocrine system of molluscs are largely unknown. In this study, mussels Mytilus edulis were exposed to North Sea oil (O) and the mixture of North Sea oil+alkylphenols (OAP), and the effects on tissue steroid levels and steroid metabolism (P450-aromatase and estradiol-sulfotransferase) were monitored. Levels of free testosterone and free estradiol were much higher in gonad tissue than in peripheral tissue, whereas esterified steroids (released after saponification) were of the same order of magnitude in both tissues. Levels of free steroids determined in gonads were not affected by exposure, but esterified steroids significantly increased in OAP exposed mussels (up to 2.4-fold). The sulfation of estradiol was investigated as a conjugation pathway, and increased activities were observed in digestive gland cytosol of both O and OAP exposure groups (up to 2.8-fold). Additionally, increased P450-aromatase activity was determined in OAP exposed mussels (up to three-fold, both in gonad and digestive gland), but not in the O group. Altogether, the results indicate that North Sea oil leads to increased sulfation of estradiol, and that in combination with alkylphenols, additional alterations are observed: increased P450-aromatase, and increased levels of esterified-steroids in gonads. Nonetheless, mussels are able to maintain gonad concentrations of free steroids unaltered, possibly via homeostatic mechanisms such as the conjugation with fatty acid or the formation of sulphate conjugates.

Analysis of Variance↗

Novel evolutionary lineages of the invertebrate oxytocin/vasopressin superfamily peptides and their receptors in the common octopus (Octopus vulgaris).

The common octopus, Octopus vulgaris, is the first invertebrate species that was shown to possess two oxytocin/vasopressin (OT/VP) superfamily peptides, octopressin (OP) and cephalotocin (CT). Previously, we cloned a GPCR (G-protein-coupled receptor) specific to CT [CTR1 (CT receptor 1)]. In the present study, we have identified an additional CTR, CTR2, and a novel OP receptor, OPR. Both CTR2 and OPR include domains and motifs typical of GPCRs, and the intron- exon structures are in accord with those of OT/VP receptor genes. CTR2 and OPR expressed in Xenopus oocytes induced calcium-mediated inward chloride current in a CT- and OP-specific manner respectively. Several regions and residues, which are requisite for binding of the vertebrate OT/VP receptor family with their ligands, are highly conserved in CTRs, but not in OPR. These different sequences between CTRs and OPR, as well as the amino acid residues of OP and CT at positions 2-5, were presumed to play crucial roles in the binding selectivity to their receptors, whereas the difference in the polarity of OT/VP family peptide residues at position 8 confers OT and VP with the binding specificity in vertebrates. CTR2 mRNA was present in various peripheral tissues, and OPR mRNA was detected in both the nervous system and peripheral tissues. Our findings suggest that the CT and OP genes, similar to the OT/VP family, evolved through duplication, but the ligand-receptor selectivity were established through different evolutionary lineages from those of their vertebrate counterparts.

Amino Acid Sequence↗

45Ca2+ uptake into rat whole brain synaptosomes unaltered by dihydropyridine calcium antagonists.

Voltage-dependent 45Ca2+ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca2+ concentration of 1.2 mM, nitrendipine, in concentrations ranging from 0.1 nM to 10 microM, had no effect on 45Ca2+ uptake. When the Ca2+ concentration was lowered to 0.06 and 0.12 mM, nitrendipine, 10 microM, inhibited 45Ca2+ uptake in response to 109 mM KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 nM to 10 microM, failed to alter the uptake of 45Ca2+ (0.06 mM Ca2+) into 30 mM KCl-depolarized synaptosomes. The high concentrations of these agents required to depress 45Ca2+ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.

Animals↗

The rat corticotropin-releasing hormone gene.

In this paper we have described the isolation and characterization of the rat corticotropin releasing hormone gene. Nucleotide sequence comparisons with the human CRH gene have demonstrated several interesting regions of homology and suggest that the gene was highly conserved through evolution. Additionally we have demonstrated the tissue-specific expression of the rat CRH gene. The regional distribution of expression parallels previously documented immunocytochemical demonstrations and supports the hypothesis that CRH peptides have multiple roles in different tissues. In the peripheral tissues that express CRH mRNA it will be very interesting to document the specific cell type of synthesis by using combined immunocytochemical and in situ histochemical techniques. Additionally we have described initial studies using gene transfer techniques to examine the cAMP responsiveness of the rat CRH gene. We are presently constructing other fusion genes (CRHCAT plasmids) in order to more carefully localize the DNA sequence in the rat CRH gene which mediates this effect, and compare it to the previously reported cAMP-responsive "consensus sequence." Similarly, we also plan to utilize the CRHCAT constructs to examine regulation of the rat CRH gene by glucocorticoids and several other hormone-mediated regulatory pathways. Through these CAT fusion studies we hope to gain a better understanding of the role of certain conserved sequences in the 5' flanking DNA for transcriptional control of the rat (and human) CRH genes.

Animals↗

Postnatal ontogenesis of the circadian clock within the rat liver.

In mammals, the circadian oscillator within the suprachiasmatic nuclei (SCN) entrains circadian clocks in numerous peripheral tissues. Central and peripheral clocks share a molecular core clock mechanism governing daily time measurement. In the rat SCN, the molecular clockwork develops gradually during postnatal ontogenesis. The aim of the present work was to elucidate when during ontogenesis the expression of clock genes in the rat liver starts to be rhythmic. Daily profiles of mRNA expression of clock genes Per1, Per2, Cry1, Clock, Rev-Erbalpha, and Bmal1 were analyzed in the liver of fetuses at embryonic day 20 (E20) or pups at postnatal age 2 (P2), P10, P20, P30, and in adults by real-time RT-PCR. At E20, only a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Cry1 but no clear circadian rhythms in expression of other clock genes were detectable. At P2, a high-amplitude rhythm in Rev-Erbalpha and a low-amplitude variation in Bmal1 but no rhythms in expression of other genes were detected. At P10, significant rhythms only in Per1 and Rev-Erbalpha expression were present. At P20, clear circadian rhythms in the expression of Per1, Per2, Rev-Erbalpha, and Bmal1, but not yet of Cry1 and Clock, were detected. At P30, all clock genes were expressed rhythmically. The phase of the rhythms shifted between all studied developmental periods until the adult stage was achieved. The data indicate that the development of the molecular clockwork in the rat liver proceeds gradually and is roughly completed by 30 days after birth.

Animals↗

Murine endothelia do not express MHC class II I-Ealpha subunit and differentially regulate I-Aalpha expression along the vascular tree.

Cellular elements of the vascular wall, such as endothelium (En) and smooth muscle cells/pericytes (SM/P) possess important immunologic properties. We have previously reported that murine brain microvessel En cells and SM/P express Major Histocompatibility (MHC) class II molecules and activate syngeneic CD4+ T cells in a class II dependent way. Herein we compare MHC class II expression on brain microvessel En to aorta large vessel En cells in order to explore the mechanisms of immune responses in brain tissue versus other peripheral tissues. Interestingly, we demonstrate that En cells from brain microvessel and large aortic vessel express the I-A but not the I-E subunit of MHC class II molecules. The expression of I-A class II molecules can be upregulated on brain microvessel and aortic En cells by interferon-gamma (IFN-gamma). Similarly, the expression of I-A, but not I-E, MHC class II molecules on brain microvessel endothelial cells was upregulated in the presence of activated T cells. Interleukin-10 (IL-10) was found to inhibit IFN-gamma-mediated upregulation of I-A class II molecule expression on aortic but not on microvessel En cells. Our data may indicate that some differences in organ-specific immune responses, are defined by local parameters, such as MHC distribution and regulation.

Animals↗

Nutritional support and cancer cachexia. Evolving concepts of mechanisms and adjunctive therapies.

Despite the widespread employment of intravenous feeding in patients with cancer cachexia to improve outcome, conflicting data exist regarding the efficacy of such therapy in addressing metabolic alterations secondary to immobility, malnutrition, and disease-specific processes. Persistence of distinct abnormalities of body and tissue integrity and peripheral tissue plasma inter-organ nitrogen flux imply the need for anabolic agents to enhance the therapeutic effects of parenteral nutrition. This article focuses upon potential anabolic agents such as chronic submaximal exercise, hormonal augmentation, pharmacologic intervention, and variations in substrate composition. Finally, the immunologic, metabolic, and endocrine consequences of bypassing the gastrointestinal tract are considered and related to the limited efficacy of current total parenteral nutrition regimens.

Cachexia↗

The Malpighi lecture. From 'Porositates carnis' to cellular microcirculation.

After Malpighi's discovery of the capillaries almost 200 years elapsed before clear concepts about their main function, solute transport between blood and tissue, were formulated. The German physiologist Carl Ludwig played a significant role in this development because of his will to seek physico-chemical explanations of physiological phenomena. Ludwig's pupil Christian Bohr formulated the first (and almost definitive) mathematical model of transcapillary diffusion, later extended by Bohr's pupil August Krogh, to describe blood-tissue exchange in peripheral tissues. In the 1950s the two-pore model of transcapillary transport became the main paradigm (Pappenheimer, Grotte). The morphological substrate for this concept is found in tortuous pathways in the interendothelial junctions together with a small number of 'leaks' or focal openings in the capillary wall. The concept makes transcapillary transport of hydrophilic molecules a paracellular phenomenon. Recent studies in the author's laboratory based on electrophysiological methods on single capillaries have disclosed the rapidity of changes in microvascular permeability in response to many different substances--indicating reactions elicited in the endothelial cells. It is almost certain that in many cases, these changes are mediated via a transitory increase in cytosolic free calcium concentration. The overlap between permeability-increasing substances and agents that induce formation of endothelial-derived relaxing factor, EDRF, is so striking that a possible connection between vasodilatation and subtle increase in permeability should be considered. It is likely that the endothelial system of plasmalemmal invaginations ('vesicles') plays a role in the control of cytosolic calcium concentration.

Biological Transport↗

[Clinical studies on various therapy for the intractable trauma of toes and fingers in cases of diabetes mellitus and peripheral ischemic diseases].

In cases of ischemic extremities and diabetes mellitus, the trauma on finger and toe is very intractable. For such injuries amputation of extremity is indicated very often because of severe necrosis. The number of such cases has been increasing recently because many cases of these patients have arteriosclerotic arterial occlusion and diabetes mellitus, and these are correlated with the changes of aging. The number of cases of Buerger's disease has been also increasing and it is another etiology of intractable trauma in ischemic extremity. The repeated hyperbaric oxygenation, sympathetic block, warfarin therapy and insulin bath with bubbling of hyperbaric oxygen, were applied to has been of such necrosis. By these procedures, the rate of amputation of extremity decreasing. It was concluded that the surgical reconstruction of artery for ischemic extremity has never any meaning as the therapy of such intractable injuries, if blood flow in the peripheral tissue is not kept physiologically, before vascular reconstruction. In order to increase peripheral tissue circulation, the hyperbaric oxygenation, sympathetic block and warfarin therapy wer performed in many cases and these methods were very effective for intractable injuries with severe necrosis.

Adult↗

Flow cytometric single-cell analysis of cytokine production by CD4+ T cells in synovial tissue and peripheral blood from patients with rheumatoid arthritis.

OBJECTIVE: To determine the cytokine profile of CD4+ T cells in the synovial tissue (ST) of rheumatoid arthritis (RA) patients at the single-cell level. METHODS: Unseparated ST cells and paired CD4+ T cells separated from the peripheral blood (PB) and ST of RA patients were stimulated for 4 hours with phorbol myristate acetate (PMA) plus calcium ionophore A23187, or for 6 hours with immobilized anti-CD3 plus anti-CD28, in the presence of brefeldin A. Cells were stained for intracellular cytokines such as interferon-gamma (IFNgamma), interleukin-2 (IL-2), IL-4, IL-10, and IL-13, in combination with cell surface markers. The percentages of cytokine-producing T cells were analyzed by flow cytometry. RESULTS: When ST cells were stimulated with PMA plus A23187 in bulk culture, IFNgamma-producing T cells were more frequently detected in the CD8+ subset, but cells producing other cytokines were found in the CD4+ subset. Purified ST CD4+ T cells, after stimulation with PMA plus A23187, were able to produce higher levels of IFNgamma but lower levels of IL-4 and IL-13, by analysis at the single-cell level, as compared with the PB CD4+, CD45RO+ T cells. The majority of IL-4- or IL-13-producing ST CD4+ cells produced IFNgamma, although PB CD4+ T cells rarely showed this cytokine pattern. IL-10-producing CD4+ T cells were more frequently found in the ST than in the PB. Of interest, most of the IL-10-producing ST CD4+ T cells were able to produce IFNgamma. IL-2-producing CD4+ T cells were similarly present in both compartments. Similar intracellular cytokine patterns were observed with anti-CD3 plus anti-CD28 stimulation, although the number of detected cells was lower. CONCLUSION: These data indicate that CD4+ T cells present within the inflamed synovium have apparently distinct cytokine profiles from those of memory CD4+ T cells in the PB, as typified by their ability to secrete both IFNgamma and IL-10.

Aged↗

Distribution and characterization of pedal peptide immunoreactivity in Aplysia.

Pedal peptide (Pep) is a very abundant neuropeptide in Aplysia. A radioimmunoassay (RIA) was developed to quantify Pep-like immunoreactivity (IR-Pep) in tissue extracts. IR-Pep was present in very high concentrations in the central nervous system (CNS) and two peripheral tissues: the large hermaphroditic duct (LHD) and the foot. RIA of fractions from high-pressure liquid chromatography (HPLC) indicated that Pep itself was the predominant immunoreactive species in each of these tissues. Lower concentrations of Pep were found in a number of other peripheral tissues. Incorporation of labelled amino acid indicated that Pep was synthesized in the LHD, whereas Pep in the foot was synthesized primarily in central neurons and transported to the foot. IR-Pep was further localized by immunocytology. All peripheral IR-Pep appeared to be associated with neuronal fibers, most commonly varicose axons. Immunoreactive innervation of the LHD and foot was particularly dense but positive staining was also observed in other tissues including tegument, gill, gut, and heart, IR-Pep innervation in all tissues including the LHD appeared to be localized predominantly in muscular portions of the tissue. Spontaneous contractions of isolated LHD were accelerated by the application of Pep. Pep appears to act as a transmitter or neuromodulator at a number of different sites in Aplysia.

Animals↗