Ralph Fritsch

Volume assessment in patients with necrotizing pancreatitis: a comparison of intrathoracic blood volume index, central venous pressure, and hematocrit, and their correlation to cardiac index and extravascular lung water index.

Objective:Volume depletion and/or increased hematocrit are associated with poor prognosis in necrotizing pancreatitis. Several studies suggest that intrathoracic blood volume index (ITBI) might be superior to central venous pressure (CVP) with regard to preload assessment. Therefore, the aim of our study was to evaluate the predictive value of CVP and hematocrit with regard to ITBI, and to correlate these parameters to cardiac index (CI). Design:Prospective study. Setting:Medical intensive care unit, university hospital. Patients and Interventions:Within 24 hrs of intensive care unit-admission, 96 hemodynamic measurements using the PiCCO system were performed in 24 patients with necrotizing pancreatitis. Main Results:Mean CVP (12.11 ± 5.97 mm Hg; median 11.5 normal: 1–9 mm Hg) was elevated, whereas mean ITBI (822.8 ± 157.0 mL/m2; median 836 mL/m2; normal: 850–1000 mL/m2) was decreased. Fifty-one of 96 ITBI values were decreased (prevalence of hypovolemia of 53%). No CVP value was decreased. Fifty-three CVP measurements were elevated despite simultaneous ITBI levels indicating a normal or decreased preload. Sensitivity, specificity, positive predictive value, and negative predictive value of CVP with regard to volume depletion (ITBI <850 mL/m2), were 0%, 100%, 0%, and 47%, respectively. An increase in hematocrit (hematocrit >40% [female] or >44% [male]) was found in 11 of 51 measurements with decreased ITBI. Sensitivity, specificity, positive predictive value, and negative predictive value of an increase in hematocrit with regard to volume depletion according to ITBI were 22%, 82%, 58%, and 48%, respectively. ITBI and &Dgr;-ITBI significantly correlated to CI and &Dgr;-CI (r = .566, p < 0.001; r = .603, p < 0.001), respectively. CVP and &Dgr;-CVP did not correlate to CI and &Dgr;-CI, respectively. There was a significant correlation between ITBI and extravascular lung water index (r = .392; p < 0.001), but no correlation between CVP and extravascular lung water index (r = .074; p = 0.473). Conclusions:Volume depletion according to ITBI was found in more than half the patients. The predictive values of CVP and hematocrit with regard to volume depletion were low. ITBI and its changes significantly correlated to CI and its changes, which was not observed for CVP and &Dgr;-CVP. Therefore, ITBI appears to be more appropriate for volume management in necrotizing pancreatitis than CVP or hematocrit.

IKKα controls p52/RelB at the skp2 gene promoter to regulate G1‐ to S‐phase progression

The IκB‐inducing kinase (IKK) is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit, IKKγ. IKK‐regulated signaling pathways are believed to promote the proliferation of normal cells as well as the aberrant proliferation of cancer cells. The molecular mechanisms linking the IKK signaling pathway components to the cell cycle machinery are not entirely understood. To study the function(s) of the catalytic subunits of the IKK complex, we used pancreatic cancer cells, with constitutive IKK activity. We show that the G1 phase of the cell cycle is specifically regulated by the IKKα subunit, which regulates the stability of the cyclin‐dependent kinase inhibitor p27Kip1. Increased p27Kip1 protein levels following the transfection of IKKα‐specific siRNAs are a result of the downregulation of the F‐box protein S‐phase kinase‐associated protein 2 (skp2). Additionally, we demonstrate that IKKα signaling regulates the transcription of the skp2 gene by controlling the composition of a RelB‐containing NF‐κB complex. Together, this work defines a novel IKKα‐regulated growth pathway involving the p52/RelB‐dependent transcriptional regulation of the skp2 gene.

Translational Repression of MCL-1 Couples Stress-induced eIF2α Phosphorylation to Mitochondrial Apoptosis Initiation

The integrated stress response (ISR) integrates a broad range of environmental and endogenous stress signals to the phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (eIF2α). Although intense or prolonged activation of this pathway is known to induce apoptosis, the molecular mechanisms coupling stress-induced eIF2α phosphorylation to the cell death machinery have remained incompletely understood. In this study, we characterized apoptosis initiation in response to classical activators of the ISR (tunicamycin, UVC, elevated osmotic pressure, arsenite). We found that all applied stress stimuli activated a mitochondrial pathway of apoptosis initiation. Rapid and selective down-regulation of the anti-apoptotic BCL-2 family protein MCL-1 preceded the activation of BAX, BAK, and caspases. Stabilization of MCL-1 blocked apoptosis initiation, while cells with reduced MCL-1 protein content were strongly sensitized to stress-induced apoptosis. Stress-induced elimination of MCL-1 occurred with unchanged protein turnover and independently of MCL-1 mRNA levels. In contrast, stress-induced phosphorylation of eIF2α at Ser51 was both essential and sufficient for the down-regulation of MCL-1 protein in stressed cells. These findings indicate that stress-induced phosphorylation of eIF2α is directly coupled to mitochondrial apoptosis regulation via translational repression of MCL-1. Down-regulation of MCL-1 enables but not enforces apoptosis initiation in stressed cells.

IkappaBbeta is an essential co-activator for LPS-induced IL-1beta transcription in vivo.

IkBβ forms a complex with the NF-κB subunits RelA and c-Rel that inhibits the transcription of IL-1β and other genes. Mice lacking IkBβ are protected against LPS-induced shock.

HMGA1 Controls Transcription of Insulin Receptor to Regulate Cyclin D1 Translation in Pancreatic Cancer Cells

The HMGA1 proteins act as architectural transcription factors and are involved in the regulation of genes important in the process of carcinogenesis. Although HMGA1 proteins are overexpressed in most types of cancer, signaling circuits regulated by HMGA1 are not clarified in detail. In this study, we show that HMGA1 proteins promote proliferation of pancreatic cancer cells by accelerating G(1) phase progression. Transfection of HMGA1-specific small interfering RNA (siRNA) activates the RB-dependent G(1)-phase checkpoint due to the impaired expression of cyclin D1. Down-regulation of cyclin D1 after the HMGA1 knockdown is due to translational control and involves the repressor of the eukaryotic translation initiation factor 4E (eIF4E) 4E-BP1. We show that 4E-BP1 and cyclin D1 act downstream of the insulin receptor (IR) in pancreatic cancer cells. At the molecular level transcription of the IR is controlled by a CAAT/enhancer binding protein beta (C/EBPbeta)/HMGA1 complex. Together, this work defines a novel pathway regulated by HMGA1, which contributes to the proliferation of pancreatic cancer cells.

InsP3R-associated cGMP Kinase Substrate (IRAG) Is Essential for Nitric Oxide-induced Inhibition of Calcium Signaling in Human Colonic Smooth Muscle

Nitric oxide (NO)-mediated relaxation of colonic smooth muscle is crucial for the maintenance of human gut function. The molecular mechanisms of NO-dependent smooth muscle relaxation involve cyclic GMP-mediated inhibition of store-dependent calcium signaling. Recently, IRAG (inositol 1,4,5-trisphophate receptor-associated cGMP kinase substrate) has been characterized as a novel target molecule of cGMP-dependent protein kinase (cGKI) mediating NO-/cGMP-dependent inhibition of inositol 1,4,5-trisphosphate (InsP3)-dependent calcium release in transfected COS cells. The aim of the present study was to characterize IRAG expression and its functional role in NO-dependent signaling in human colonic smooth muscle. Reverse transcriptase-PCR revealed IRAG mRNA expression in human colon, rectum, and cultured colonic smooth muscle cells. In cultured human colonic smooth muscle cells, bradykinin (BK) elicited InsP3-dependent calcium transients that were repeatable and independent of extracellular calcium. The NO donor sodium nitroprusside and the specific cGK activator 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic-monophosphate (8-pCPT-cGMP) significantly inhibited BK-induced increase in intracellular calcium. Cells transfected with antisense oligonucleotides raised against IRAG (IRAG-AS) showed strongly decreased IRAG protein expression. In these cells, sodium nitroprusside and 8-pCPT-cGMP both failed to modulate BK-induced calcium transients. Thus, endogenous IRAG appears to be essentially involved in the NO/cGK-dependent inhibition of InsP3-dependent Ca2+-signaling in colonic smooth muscle.

Functional coupling between nitric oxide synthesis and VIP release within enteric nerve terminals of the rat: involvement of protein kinase G and phosphodiesterase 5

1 The subcellular mechanisms involved in the effect of nitric oxide (NO) on the release of vasoactive intestinal polypeptide (VIP) were examined in synaptosomes isolated from rat small intestine. 2 VIP release was stimulated by the NO donor SNAP (10−7‐10−4m) in an oxyhaemoglobin‐sensitive manner. The presence of the guanylate cyclase inhibitor ODQ (10−5m), or inhibition of protein kinase G (PKG) by KT 5823 (3 × 10−6m) or Rp‐8Br‐PET‐cGMPS (5 × 10−7m), antagonized the SNAP‐induced VIP release, suggesting a regulatory role of PKG, confirming previously published data from enteric ganglia. This finding was further supported by the fact that direct PKG activation by the stable cGMP analogue 8‐pCPT‐cGMP stimulated VIP secretion to the same extent as SNAP. 3 Basal VIP secretion was enhanced in the presence of zaprinast, an inhibitor of cGMP‐dependent phosphodiesterase 5 (PDE 5), suggesting a functional role of PDE 5 in NO‐cGMP signalling. Supportive evidence for this finding was obtained by demonstration of the presence of PDE 5 using RT‐PCR. 4 Stimulation of endogenous NO production by l‐arginine was also effective in releasing VIP. The effect was abolished in the presence of KT 5823, but was insensitive to oxyhaemoglobin (10−3m), suggesting that an interaction between NO and VIP is likely to occur within the same nerve terminal rather than between terminals. 5 NO synthesis was not affected by VIP (10−8‐10−5m), suggesting that there is no feedback regulation between the NO and the VIP pathways. 6 These findings support the notion that an anatomical and functional interrelationship exists between NO and VIP in enteric nerve terminals and that complex signalling mechanisms involving PKG and PDE 5 contribute to NO‐induced VIP release.

HDAC3 is linked to cell cycle machinery in MiaPaCa2 cells by regulating transcription of skp2

Abstract.  Objective: Histone deacetylases (HDACs) have been linked to cell cycle control in various models, involving regulation of the cyclin‐dependent kinase inhibitor p27Kip1. Results: Here, we demonstrate that HDAC inhibition by trichostatin A reduces S‐phase kinase‐associated protein 2 mRNA and protein abundance. Furthermore, in contrast to HDAC1, recruited to the skp2 promoter in the G0 phase, HDAC3 is bound in early S phase. Activating function of HDAC3 towards the skp2 gene has been validated using RNA interference techniques. siRNAs, targeting HDAC3 specifically, reduced skp2 transcription. Conclusion: These findings propose that the skp2 gene is a novel target of HDAC3, mediating cell cycle control and oncogenesis.

Truncated IRAG variants modulate cGMP-mediated inhibition of human colonic smooth muscle cell contraction

Nitric oxide (NO) induces relaxation of colonic smooth muscle cells predominantly by cGMP/cGMP-dependent protein kinase I (cGKI)-induced phosphorylation of the inositol 1,4,5-trisphosphate receptor (IP(3)R)-associated cGMP kinase substrate (IRAG), to block store-dependent calcium signaling. In the present study we analyzed the structure and function of the human IRAG/MRVI1 gene. We describe four unique first exon variants transcribed from individual promoters in diverse human tissues. Tissue-specific alternative splicing with exon skipping and alternative splice donor and acceptor site usage further increases diversity of IRAG mRNA variants that encode for NH(2)- and COOH-terminally truncated proteins. At the functional level, COOH-terminally truncated IRAG variants lacking both the cGKI phosphorylation and the IP(3)RI interaction site counteract cGMP-mediated inhibition of calcium transients and relaxation of human colonic smooth muscle cells. Since COOH-terminally truncated IRAG mRNA isoforms are widely expressed in human tissues, our results point to an important role of IRAG variants as negative modulators of nitric oxide/cGKI-dependent signaling. The complexity of alternative splicing of the IRAG gene impressively demonstrates how posttranscriptional processing generates functionally distinct proteins from a single gene.