Protein kinase C activates capacitative calcium entry in the insulin secreting cell line RINm5F

AbstractThis study examines the calcium store-regulated (capacitative) calcium influx pathway in the endocrine pancreatic cell line RINm5F, utilizing thapsigargin. After preincubation of the cells with the phorbol ester TPA, thapsigargin induced a sustained elevation of cytosolic calcium as well as a sustained stimulation of manganese entry, the latter being used to assess calcium influx. Thapsigargin given alone provoked a smaller and only transient elevation of cytosolic calcium and stimulation of manganese entry. The protein kinase C inhibitor staurosporine antagonized the effect of the phorbol ester. Verapamil, nifedipine, or measures to hyperpolarize the cells exerted no inhibitory action against this effect, which excludes an involvement of voltage-dependent calcium channels. In conclusion, our data shows for the first time that protein kinase C stimulation activates the capacitative calcium influx pathway of endocrine pancreatic insulin-producing cells

Untersuchung zur Beteiligung von Proteinkinase A und Tyrosinkinasen an der Calcium-abhängigen Signaltransduktion von Glucagon-like Peptide 1(7-36)amid in Insulin-sezernierenden Zellen des endokrinen Pankreas

Glucagon-like Peptide-1(7-37)/(7-36)amid, GLP-1, ist ein insulinotropes intestinales Peptid-Hormon, das vor allem wegen der Verstärkung der Glukose-abhängigen Insulinsekretion, ein großes Potential bei der Therapie des Diabetes mellitus hat. Die Signaltransduktion von GLP-1 ist in weiten Teilen noch nicht geklärt. Bisherige Studien ergaben, dass GLP-1 an den G-Protein-gekoppelten GLP-1-Rezeptor bindet. Über Aktivierung der Adenylatzyklase erhöht sich der cAMP-Spiegel. Letztlich kommt es durch unbekannte Schritte zu einem Anstieg der intrazellulären Calciumkonzentration und zu einer erhöhten Insulinsekretion. Oftmals sind bei der Signaltransduktion Proteinkinasen wie die cAMP-abhängige Proteinkinase A oder Tyrosinkinasen beteiligt, so dass dies auch bei der GLP-1-induzierten Calciumerhöhung und Insulinsekretion vermutet wurde. Bisherige Arbeiten wurden oft ungeeignete ß-Zellmodelle ausgeführt, verwendet zum Beispiel nur ein Proteinkinase A-Hemmstoff und kamen so zu unterschiedlichen Ergebnissen über die Bedeutung von Proteinkinasen bei der Signaltransduktion von GLP-1. Ziel dieser Arbeit war es, die Beteiligung von Proteinkinase A und Tyrosinkinasen an der Calcium-abhängigen Signaltransduktion von Glucagon-like Peptide 1(7-36)amid in Insulin-sezernierenden Zellen des endokrinen Pankreas zu untersuchen. Dazu wurde 1.) eines geeigneten ß-Zellmodell, hier die Glukose-sensible INS-1-Zellen sowie ß-Zellen in präparierten Langerhanssche Inseln der Maus, und 2.) verschiedene, angemessene Proteinkinasehemmstoffe verwendet. Mit den Fluoreszenzfarbstoffen Fura-2 bzw. Bisoxonol wurden die intrazelluläre Calciumkonzentrationen an beiden Zelltypen sowie Veränderungen im Membranpotential der INS-1-Zellen gemessen. Um Artefakte am Rezeptor auszuschließen, wurde neben GLP-1 durch Forskolin die Adenylatzyklase direkt aktiviert. Weder durch den selektiven Proteinkinase A-Hemmstoff KT5720 (bis zu 20µM) noch Rp-cAMPs (bis 1mM) konnte eine Hemmung der GLP-1- oder Forskolin-Wirkung erzielt werden. Der dritte Proteinkinase A-Inhibitor H-89 selbst führte konzentrationsabhängig (bis 40µM) zu einer deutlichen Reduktion der Glukose-induzierten Effekte, wie den Calciumanstieg oder den Calciumoszillationen der Langerhansschen Inseln. Erst durch lange Inkubationszeiten mit deutlich höheren als sonst verwendeten Konzentrationen ließen sich die GLP-1 bzw. Forskolin-induzierte Erhöhung des Calciumspiegels sowie die Membrandepolarisation leicht reduzieren, so dass es sich dabei eher um einen sekundären Effekt infolge des verminderten Glukoseeinflusses zu handeln scheint. Insgesamt zeigte die fehlende Inhibition durch die drei Hemmstoffe, dass die Proteinkinase A an der Signaltransduktion von GLP-1 bzw. Forskolin zur Erhöhung des intrazellulären Calciumspiegels nicht beteiligt ist. Die Tyrosinkinase-Hemmstoffe Genistein (bis 100µM) und Herbimycin A (bis 20µM) führten beide, Genistein mehr als Herbimycin A, zu konzentrationsabhängigen, massiven Veränderungen der Glukose-abhängigen Calciumerhöhung sowie der Calciumoszillationen. Die Ursache hierfür, ob es sich dabei zum Beispiel um eine spezifische Tyrosinkinase-Hemmung oder vielleicht einen unspezifischen toxischen Effekt handelt, werden in der Literatur kontrovers diskutiert. In Relation zu diesen starken Veränderungen der Glukosewirkung ließ sich bei keinem der beiden Hemmstoffe eine isolierte Inhibition des GLP-1 oder Forskolin-Effektes zeigen. Auch der potente, unspezifische Proteinkinasehemmstoff Staurosporin bei relevanter Dosierung zeigte keine Beeinflussung der durch GLP-1 und Forskolin bedingten Calciumerhöhung sowie der Membrandepolarisation. Insgesamt zeigt dies Arbeit, dass für den GLP-1-induzierten Anstieg der intrazellulären Calciumkonzentration und der Membrandepolarisation weder Proteinkinase A noch Tyrosinkinasen, eher sogar keine Proteinkinase eine entscheidende Rolle spielt. An der möglichen Signaltransduktion dieser GLP-1-Effekte könnten ein nicht selektiver Kationenkanal über cAMP alleinig (NSCC) oder in Kombination mit einer Hyperpolarisation (HCN) ebenso wie der cAMP-regulierte Ras Guanin-Nucleotid-Austausch-Faktor, Epac, beteiligt sein. Um den genauen Mechanismus, unter anderem welche Ionenkanäle beeinflusst werden, aufzuschlüsseln, sind weitere Untersuchungen notwendig. Zudem ist zu erwähnen, dass in der Literatur ebenfalls ein Calcium-unabhängiger Anteile der GLP-1-induzierten Insulinsekretion beschrieben ist. So ist eine Beteiligung von Proteinkinasen distal der Calciumerhöhung, zum Beispiel durch Vergrößerung des Reservepools der Insulingranula oder einer Tyrosinkinase-abhängig verstärkter Exozytose, durchaus mit den Ergebnissen dieser Arbeit vereinbar

Fronts between Hopf- and Turing-type domains in a two-component reaction-diffusion system

Propagating and standing fronts between Hopf- and Turing-type domains are observed experimentally on a one-dimensional array of resistively coupled nonlinear LC-oscillators describable by a two-component reaction-diffusion equation. Numerical and experimental results are compared in particular with respect to front velocities. In the neighbourhood of a codimension-two point two coupled Ginzburg-Landau equations, derived by multiple scale methods, are useful approximation

The Ratio of ADP- to TRAP-Induced Platelet Aggregation Quantifies P2Y(12)-Dependent Platelet Inhibition Independently of the Platelet Count

Objective This study aimed to assess the association of clinical factors with P2Y(12)-dependent platelet inhibition as monitored by the ratio of ADP-to TRAP-induced platelet aggregation and conventional ADP-induced aggregation, respectively. Background Controversial findings to identify and overcome high platelet reactivity (HPR) after coronary stent-implantation and to improve clinical outcome by tailored anti-platelet therapy exist. Monitoring anti-platelet therapy ex vivo underlies several confounding parameters causing that ex vivo platelet aggregation might not reflect in vivo platelet inhibition. Methods In a single centre observational study, multiple electrode aggregometry was performed in whole blood of patients after recent coronary stent-implantation. Relative ADP-induced aggregation (r-ADP-agg) was defined as the ratio of ADP-to TRAP-induced aggregation reflecting the individual degree of P2Y(12)-mediated platelet reactivity. Results Platelet aggregation was assessed in 359 patients. Means (+/- SD) of TRAP-, ADP-induced aggregation and r-ADP-agg were 794 +/- 239 AU* min, 297 +/- 153 AU* min and 37 +/- 14%, respectively. While ADP-and TRAP-induced platelet aggregation correlated significantly with platelet count (ADP: r = 0.302;p< 0.001;TRAP: r = 0.509 p< 0.001), r-ADP-agg values did not (r = -0.003;p = 0.960). These findings were unaltered in multivariate analyses adjusting for a range of factors potentially influencing platelet aggregation. The presence of an acute coronary syndrome and body weight were found to correlate with both ADP-induced platelet aggregation and r-ADP-agg. Conclusion The ratio of ADP-to TRAP-induced platelet aggregation quantifies P2Y(12)-dependent platelet inhibition independently of the platelet count in contrast to conventional ADP-induced aggregation. Furthermore, r-ADP-agg was associated with the presence of an acute coronary syndrome and body weight as well as ADP-induced aggregation. Thus, the r-ADP-agg is a more valid reflecting platelet aggregation and potentially prognosis after coronary stent-implantation in P2Y(12)-mediated HPR than conventional ADP-induced platelet aggregation

Genome-wide comparison between IL-17 and combined TNF-alpha/IL-17 induced genes in primary murine hepatocytes

<p>Abstract</p> <p>Background</p> <p>Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization.</p> <p>Results</p> <p>The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately up-regulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1beta proposed an "IL-1beta-like effect" of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase activity of the up-regulated Zc3h12a have to be considered.</p> <p>Conclusions</p> <p>Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signaling pathway. Gene expression profile suggests IL-17 alone and in concert with TNF-alpha a role in sustaining liver inflammatory processes. IL-17 might exceed this function by RNA stabilization.</p

Limited value of 18F-FDG PET/CT and S-100B tumour marker in the detection of liver metastases from uveal melanoma compared to liver metastases from cutaneous melanoma

Purpose: The objective of this study was to evaluate the value of 18F-FDG PET/CT and S-100B tumour marker for the detection of liver metastases from uveal melanoma in comparison to liver metastases from cutaneous melanoma. Methods: A retrospective evaluation was conducted of 27 liver metastases in 13 patients with uveal melanoma (UM) (mean age: 56.8, range: 30-77) and 43 liver metastases in 14 patients (mean age: 57.9, range: 40-82) with cutaneous melanoma (CM) regarding size and FDG uptake by measuring the maximum standardized uptake value (SUVmax). S-100B serum tumour markers were available in 20 patients. Cytology, histology, additional morphological imaging and follow-up served as reference standard. In nine patients liver metastases were further evaluated histologically regarding GLUT-1 and S-100 receptor expression and regarding epithelial or spindle cell growth pattern. Results: Of 27 liver metastases in 6 of 13 patients (46%) with UM, 16 (59%) were FDG negative, whereas all liver metastases from CM were positive. Liver metastases from UM showed significantly (p < 0.001) lower SUVmax (mean: 3.5, range: 1.5-13.4) compared with liver metastases from CM (mean: 6.6, range: 2.3-15.3). In four of six (66.7%) patients with UM and liver metastases S-100B was normal and in two (33.3%) increased. All PET-negative liver metastases were detectable by morphological imaging (CT or MRI). S-100B was abnormal in 13 of 14 patients with liver metastases from CM. S-100B values were significantly higher (p = 0.007) in the CM patient group (mean S-100B: 10.9μg/l, range: 0.1-115μg/l) compared with the UM patients (mean: 0.2μg/l, range: 0.0-0.5μg/l). Histological work-up of the liver metastases showed no obvious difference in GLUT-1 or S-100 expression between UM and CM liver metastases. The minority (36%) of patients with UM had extrahepatic metastases and the majority (86%) of patients with CM had extrahepatic metastases, respectively. There was a close to significant trend to better survival of UM patients compared with CM patients (p = 0.06). Conclusion: FDG PET/CT and serum S-100B are not sensitive enough for the detection of liver metastases from UM, whereas liver metastases from cutaneous melanoma are reliably FDG positive and lead regularly to increased S-100B tumour markers. The reason for the lower FDG uptake in UM liver metastases remains unclear. We recommend to perform combined contrast-enhanced PET/CT in order to detect FDG-negative liver metastases from U