Corticotropin-releasing factor receptors couple to multiple g-proteins to activate diverse intracellular signaling pathways in mouse hippocampus: role in neuronal excitability and associative learning

Corticotropin-releasing factor (CRF) exerts a key neuroregulatory control on stress responses in various regions of the mammalian brain, including the hippocampus. Using hippocampal slices, extracts, and whole animals, we investigated the effects of human/rat CRF (h/rCRF) on hippocampal neuronal excitability and hippocampus-dependent learning in two mouse inbred strains, BALB/c and C57BL/6N. Intracellular recordings from slices revealed that application of h/rCRF increased the neuronal activity in both mouse inbred strains. Inhibition of protein kinase C (PKC) by bisindolylmaleimide I (BIS-I) prevented the h/rCRF effect only in hippocampal slices from BALB/c mice but not in slices from C57BL/6N mice. Inhibition of cAMP-dependent protein kinase (PKA) by H-89 abolished the h/rCRF effect in slices from C57BL/6N mice, with no effect in slices from BALB/c mice. Accordingly, h/rCRF elevated PKA activity in hippocampal slices from C57BL/6N mice but increased only PKC activity in the hippocampus of BALB/c mice. These differences in h/rCRF signal transduction were also observed in hippocampal membrane suspensions from both mouse strains. In BALB/c mice, hippocampal CRF receptors coupled to Gq/11 during stimulation by h/rCRF, whereas they coupled to Gs, Gq/11, and Gi in C57BL/6N mice. As expected on the basis of the slice experiments, h/rCRF improved context-dependent fear conditioning of BALB/c mice in behavioral experiments, and BIS-I prevented this effect. However, although h/rCRF increased neuronal spiking in slices from C57BL/6N mice, it did not enhance conditioned fear. These results indicate that the CRF system activates different intracellular signaling pathways in mouse hippocampus and may have distinct effects on associative learning depending on the mouse strain investigated

Mass spectrometric and quantum mechanical analysis of gas-phase formation, structure, and decomposition of various b2 ions and their specifically deuterated analogs

AbstractB ions represent an important type of fragment ions derived from protonated peptides by cleavage of an amide bond with N-terminal charge retention. Such species have also been discussed as key intermediates during cyclic peptide fragmentation. Detailed structural information on such ion types can facilitate the interpretation of multiple step fragmentations such as the formation of inner chain fragments from linear peptides or the fragmentation of cyclic peptides. The structure of different b2 ion isomers was investigated with collision-induced dissociations (CID) in combination with hydrogen/deuterium (H/D) exchange of the acidic protons. Special care was taken to investigate fragment ions derived from pure gas-phase processes. Structures deduced from the results of the CID analysis were compared with structures predicted on the basis of quantum chemical density functional theory (DFT) calculations to be most stable. The results pointed to different types of structures for b2 ion isomers of complementary amino acid sequences. Either the protonated oxazolone structure or the N-terminally protonated immonium ion structure were proposed on the basis of the CID results and the DFT calculations. In addition, the analysis of different selectively N-alkylated peptide analogs revealed mechanistic details of the processes generating b ions

The impact of an hematocrit of 20% during normothermic cardiopulmonary bypass for elective low risk coronary artery bypass graft surgery on oxygen delivery and clinical outcome – a randomized controlled study [ISRCTN35655335]

INTRODUCTION: Cardiopulmonary bypass (CPB) induces hemodilutional anemia, which frequently requires the transfusion of blood products. The objective of this study was to evaluate oxygen delivery and consumption and clinical outcome in low risk patients who were allocated to an hematocrit (Hct) of 20% versus 25% during normothermic CPB for elective coronary artery bypass graft (CABG) surgery. METHODS: This study was a prospective, randomized and controlled trial. Patients were subjected to normothermic CPB (35 to 36°C) and were observed until discharge from the intensive care unit (ICU). Outcome measures were calculated whole body oxygen delivery, oxygen consumption and clinical outcome. A nonparametric multivariate analysis of variance for repeated measurements and small sample sizes was performed. RESULTS: In a total of 54 patients (25% Hct, n = 28; 20% Hct, n = 26), calculated oxygen delivery (p = 0.11), oxygen consumption (p = 0.06) and blood lactate (p = 0.60) were not significantly different between groups. Clinical outcomes were not different between groups. CONCLUSION: These data indicate that an Hct of 20% during normothermic CPB maintained calculated whole body oxygen delivery above a critical level after elective CABG surgery in low risk patients. The question of whether a transfusion trigger in excess of 20% Hct during normothermic CPB is still supported requires a larger prospective and randomized trial

Corticotropin-releasing factor (CRF) rapidly suppresses apoptosis by acting upstream of the activation of caspases

The physiological role of the corticotropin-releasing factor (CRF) family of peptides has recently been extended by emerging evidence of their cytoprotective effects. To determine whether CRF-mediated cytoprotection is linked to caspase-dependent apoptosis, the effect of CRF on the activation of caspases was investigated in detail in Y79 human retinoblastoma cells. The results presented here demonstrate that the cytoprotective effect of CRF against the actions of camptothecin (CT) was mediated by CRF receptor subtype 1, but not subtype 2. The observed CRF-mediated cytoprotection involved rapid and pronounced suppression of proteolytic processing and activation of procaspase-3, exerted even when CRF was added hours after the application of the cytotoxic agent. Surprisingly, activation of procaspase-3 preceded activation of the initiator procaspases 2, 8, 9 and 10 during CT-induced apoptosis of Y79 cells. The mechanism of the effect of CRF was examined using inhibitors of signalling pathways such as Wortmannin (Akt), cyclic AMP-dependent protein kinase (PKA), extracellular signal-regulated kinase (ERK), protein kinase c (PKC), p38 mitogen-activated protein kinase (p38 MAPK), phospholipase c (PLC), nuclear factor-kappaB (NF-kappaB) and c-jun N-terminal kinase (JNK). The involvement of PKA in the mediation of the anti-apoptotic effect of CRF has been established. Taken together, these results demonstrate for the first time that the cytoprotective effect of CRF involved suppression of pro- apoptotic pathways at a site upstream of activation of procaspase-3