Chronic ethanol exposure increases microtubule content in PC12 cells

BACKGROUND: Chronic ethanol exposure has been shown to result in changes in neuronal cyto-architecture such as aberrant sprouting and alteration of neurite outgrowth. In PC12 cells, chronic ethanol treatment produces an increase in Nerve Growth Factor (NGF)-induced neurite outgrowth that appears to require the epsilon, but not delta, isoform of Protein Kinase C (PKC). Neurites contain a core of microtubules that are formed from polymerization of free-tubulin. Therefore, it would be expected that an increase in neurite outgrowth would correlate with an increase in microtubule content. We examined the effect of chronic ethanol exposure on microtubule content in PC12 cells and the role of PKC epsilon and delta in ethanol's effect on microtubule levels. RESULTS: Chronic ethanol exposure of wild-type and vector control PC12 cells resulted in a significant increase in microtubule content and a corresponding decrease in free tubulin. There was also a significant increase in microtubule content in PC12 cells expressing a dominate-negative inhibitor of epsilon PKC; cells which have previously been shown to have no ethanol-induced increase in neurite outgrowth. In contrast, ethanol had no effect on microtubule content in PC12 cells expressing a dominate-negative inhibitor of delta PKC. CONCLUSION: These results suggest that chronic ethanol exposure alters the relative ratio of free tubulin to microtubule-associated tubulin, an important component of the cytoskeleton. Further, the data from the PKC dominant-negative cell lines suggest that the effects of ethanol on microtubule content do not correlate with the effects of ethanol on neurite outgrowth. The delta isoform of PKC appears to be necessary for the ethanol-induced increase in microtubule content. These studies demonstrate an effect of chronic ethanol exposure which may contribute to previously documented alterations of neuronal cyto-architecture

Multi-Level Communication of Human Retinal Pigment Epithelial Cells via Tunneling Nanotubes

Background: Tunneling nanotubes (TNTs) may offer a very specific and effective way of intercellular communication. Here we investigated TNTs in the human retinal pigment epithelial (RPE) cell line ARPE-19. Morphology of TNTs was examined by immunostaining and scanning electron microscopy. To determine the function of TNTs between cells, we studied the TNT-dependent intercellular communication at different levels including electrical and calcium signalling, small molecular diffusion as well as mitochondrial re-localization. Further, intercellular organelles transfer was assayed by FACS analysis. Methodology and Principal Findings: Microscopy showed that cultured ARPE-19 cells are frequently connected by TNTs, which are not attached to the substratum. The TNTs were straight connections between cells, had a typical diameter of 50 to 300 nm and a length of up to 120 µm. We observed de novo formation of TNTs by diverging from migrating cells after a short time of interaction. Scanning electron microscopy confirmed characteristic features of TNTs. Fluorescence microscopy revealed that TNTs between ARPE-19 cells contain F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of latrunculin-B, led to disappearance of TNTs. Importantly, these TNTs could function as channels for the diffusion of small molecules such as Lucifer Yellow, but not for large molecules like Dextran Red. Further, organelle exchange between cells via TNTs was observed by microscopy. Using Ca2+ imaging we show the intercellular transmission of calcium signals through TNTs. Mechanical stimulation led to membrane depolarisation, which expand through TNT connections between ARPE-19 cells. We further demonstrate that TNTs can mediate electrical coupling between distant cells. Immunolabelling for Cx43 showed that this gap junction protein is interposed at one end of 44% of TNTs between ARPE-19 cells. Conclusions and Significance: Our observations indicate that human RPE cell line ARPE-19 cells communicate by tunneling nanotubes and can support different types of intercellular traffic

Unilateral right-sided varicocele associated with pancreatic cancer: A cadaveric case report

A testicular varicocele is an abnormal dilation of the pampiniform plexus of veins within the spermatic cord. Due to the asymmetrical drainage pattern of the right and left testicular veins, the vast majority of varicoceles are found on the left side. Isolated right testicular varicoceles occur rarely and should raise clinical suspicion for underlying intra-abdominal pathology. During the dissection of a 67-year-old male cadaver a unilateral varicocele was discovered in the right spermatic cord. Upon dissection of the abdomen, a tumor was found in the head and body of the pancreas. The section of the inferior vena cava where the right testicular vein inserted laid directly posterior to the tumor and was markedly dilated. We hypothesize that this pancreatic tumor impeded venous return into the inferior vena cava from the right testicular vein and not the left renal vein (where the left testicular vein drains) resulting in a unilateral right varicocele (URV). To our knowledge, the relationship between URV and pancreatic cancer has not been previously described in the literature. Our findings suggest URV could be a clinical warning sign of cancer of the head and/or body of the pancreas and may be a source of discomfort to address in patients with pancreatic cancer in order to improve quality of life. Keywords: Testicular varicocele, Unilateral right-sided varicocele, Pancreatic cance

Variability in P-Glycoprotein Inhibitory Potency (IC 50

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC 50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells-Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLCPK1- MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratiobased equation generally resulted in severalfold lower IC 50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC 50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided. © 2013 by The American Society for Pharmacology

Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug- Drug Interaction Risk Assessment Decision Criterias

A P-glycoprotein (P-gp) IC(50) working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC(50) determinations. Each laboratory followed its in-house protocol to determine in vitro IC(50) values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells—Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK(1)-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC(50) values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC(50) values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC(50) values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC(50) values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC(50) values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC(50) determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided