Store-operated calcium entry (SOCE) and TRPC protein expression were investigated in the rat-derived hippocampal H19-7 cell line. Thapsigargin-stimulated Ba 2؉ entry and the expression of TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 mRNA and protein were observed in proliferating H19-7 cells. When cells were placed under differentiating conditions, a change in TRPC homolog expression profile occurred. The expression of TRPC1 and TRPC3 mRNA and protein dramatically increased, while the expression of TRPC4 and TRPC7 mRNA and protein dramatically decreased; in parallel a 3.4-fold increase in the level of thapsigarginstimulated Ba 2؉ entry was observed and found to be inhibited by 2-aminoethoxydiphenylborane. The selective suppression of TRPC protein levels by small interfering RNA (siRNA) approaches indicated that TRPC1 and TRPC3 are involved in mediating SOCE in proliferating H19-7 cells. Although TRPC4 and TRPC7 are expressed at much higher levels than TRPC1 and TRPC3 in proliferating cells, they do not appear to mediate SOCE. The co-expression of siRNA specific for TRPC1 and TRPC3 in proliferating cells inhibited approximately the same amount of SOCE as observed with expression of either siRNA alone, suggesting that TRPC1 and TRPC3 work in tandem to mediate SOCE. Under differentiating conditions, co-expression of siRNA for TRPC1 and TRPC3 blocked the normal 3.4-fold increase in SOCE and in turn blocked the differentiation of H19-7 cells. This study suggests that placing H19-7 cells under differentiating conditions significantly alters TRPC gene expression and increases the level of SOCE and that this increase in SOCE is necessary for cell differentiation.

Background Fine needle aspiration (FNA) biopsy plays a critical role in the diagnosis and staging of lung primary and metastatic lung carcinoma. Accurate subclassification of adenocarcinoma (ADC) and/or squamous cell carcinoma (SqCC) is crucial for the targeted therapy. However, the distinction between ADC and SqCC may be difficult in small FNA specimens. Here, we have retrospectively evaluated the utility of TTF-1, Napsin A, CK7, P63 and CK5/6 immunohistochemical (IHC) markers in the distinguishing and subclassification of ADC and SqCC. Methods A total of 246 FNA cases were identified by a computer search over a two-year period, including 102 primary NSCLC and 144 primary NSCLC which had metastasized to other sites. The immunostaining patterns of TTF-1, Napsin A, CK7, P63 and CK5/6 were correlated with the histological diagnosis of the tumor. Results In 72 primary ADCs, TTF-1, Napsin A and CK7 showed a sensitivity and specificity of 84.5%/96.4%, 92.0%/100%, and 93.8%/50.0%. In 30 primary SqCCs, CK5/6 and P63 showed a sensitivity and specificity of 100%/77.8% and 91.7%/78.3%. In 131 metastatic ADCs, Napsin A showed the highest specificity (100%), versus TTF-1 (87.5%) and CK7 (25%) but decreased sensitivity (67.8% versus 86.9% and 100%); whereas in 13 metastatic SqCCs, CK5/6 and P63 showed a sensitivity/specificity of 100%/84.6% and 100%/68.4%. Bootstrap analysis showed that the combination of TTF-1/CK7, TTF-1/Napsin A and TTF-1/CK7/Napsin A had a sensitivity/specificity of 0.960/0.732, 0.858/0.934, 0.972/0.733 for primary lung ADCs and 0.992/0.642, 0.878/0.881, 0.993/0.618 for metastatic lung ADCs. Conclusions Our study demonstrated that IHC markers had variable sensitivity and specificity in the subclassification of primary and metastatic ADC and SqCC. Based on morphological findings, an algorithm with the combination use of markers aided in the subclassification of NSCLCs in difficult cases.

The ability of muscarinic cholinergic receptors (mAChRs) to regulate the volume-sensitive efflux of two organic osmolytes, namely, taurine and D-aspartate, from human SH-SY5Y neuroblastoma cells has been examined. Incubation of the cells with hypoosmolar buffers resulted in an efflux of both osmolytes, with the threshold for release occurring at approximately 225 mOsM for taurine and D-aspartate. Inclusion of oxotremorine-M (Oxo-M), a muscarinic agonist, resulted in a marked enhancement of the volume-dependent efflux of both osmolytes and increased the threshold osmolarity for taurine and D-aspartate release to 340 (isotonic) and 320 mOsM, respectively. Maximum agonist stimulation of osmolyte release (350% of basal) was observed in the range of 225 to 250 mOsM. Oxo-M-stimulated osmolyte efflux was inhibited by muscarinic antagonists with a rank order of ,4]benzodiazepin-6-one, a pharmacological profile identical to that obtained for M 3 mAChRstimulated phosphoinositide hydrolysis. Agonist-stimulated efflux of both osmolytes could be inhibited by inclusion of either anion channel blockers known to inhibit the volume-sensitive organic anion channel (VSOAC) or by a tyrosine kinase inhibitor ␣-cyano-(3,4-dihydroxy)cinnamonitrile. The results indicate that the activation of M 3 mAChRs on SH-SY5Y neuroblastoma facilitates the ability of these cells to respond to very limited reductions in osmolarity via a release of osmolytes. mAChR-stimulated osmolyte efflux is mediated via a VSOAC and seems to require the activity of a tyrosine kinase.Although most cells possess homeostatic mechanisms for the maintenance of cell volume, these are particularly important to cells in the central nervous system (CNS) because of restrictions of the skull. Even modest alterations in brain volume can have profound effects on cell-cell signaling because the spatial relationship between neurons, astrocytes, and the extracellular space becomes compromised. Brain swelling, which can occur in response to conditions such as hyponatremia, inappropriate secretion of antidiuretic hormone, or after polydypsia, can lead to the compression of small blood vessels, and subsequently, cerebral anoxia and ischemia. Death can result from the displacement of brain parenchyma through the foramen magnum and the ensuing cardiac and respiratory arrest (Pasantes-Morales et al., 2000). To counter these deleterious changes, neural cells initially restore their osmotic balance via a loss of K ϩ and Cl Ϫ ions. However, because large changes in ion concentrations can adversely impact excitability, cells subsequently use "compatible" or nonperturbing organic osmolytes to counter changes in osmolarity without compromising cell function. In the CNS, the three quantitatively major organic osmolytes are taurine, glutamate, and myo-inositol. Organic osmolytes are released from neural cells via a volume-sensitive organic anion channel (VSOAC), a channel that has been extensively characterized both electrophysiologically and pharmacologically, although its molecular s...

Elevated endogenous cholecystokinin (CCK) release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated nuclear factor of activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5-1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo.

Although physiologic jaundice of neonates is common, persistent neonatal cholestasis is life-threatening and has multiple etiologies. Among these etiologies, biliary atresia (BA) requires rapid diagnosis and treatment. In diagnosing BA, the surgical pathologist must recognize subtle histologic changes, often with only a small core liver biopsy. To aid in the differential diagnosis of neonatal cholestasis, we investigated Yes-associated protein (YAP), a regulator of organ size and bile duct development. We examined whether a YAP immunostain can highlight emerging hepatobiliary epithelium in BA [n=28] versus other causes of persistent cholestasis (non-BA) [n=15] and thus serve as a useful diagnostic marker in persistent neonatal jaundice. We show significantly (p≤0.01) more high-grade (≤2) fibrosis and ductular proliferation among BA versus non-BA cases. Likewise, there was significantly more high-grade (2–3/3) cytoplasmic and nuclear YAP staining in BA (97% and 89%) versus non-BA (20% and 13%). High-grade nuclear YAP staining was both sensitive (88%) and specific (87%) for the diagnosis of BA. In contrast to neonatal cholestasis, the differences in YAP localization in cholestatic/obstructed vs. non-obstructed adult livers were not significant. Lastly, we found that pharmacological inhibition of the YAP complex in both cholangiocyte and cholangiocarcinoma cell lines blocked compensatory bile duct proliferation, an early marker of BA that requires nuclear YAP expression, in a time- and dose-dependent manner. In summary, we show that YAP expression modulates both bile duct proliferation and liver damage/fibrosis while acting as a sensitive and specific marker in the differential diagnosis of persistent neonatal cholestasis.

Background & Aims-Growth of exocrine pancreas is regulated by GI hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates NFATs (Nuclear Factor of Activated T-cells), but the genetic underpinnings and feedback mechanisms that regulate this response are not known.

Background Detection of BRAF mutations is an established standard of care to predict small-molecule inhibitor (vemurafenib) response in metastatic melanoma. Molecular assays should be designed to detect not only the most common p.V600E mutation, but also p.V600K and other non-p.V600E mutations. Objective The purpose of this study was to assess if tumor cellularity can function as a quality assurance (QA) measure in molecular diagnostics. Potential causes of discrepancy between the observed and predicted mutant allele percentage were also explored. Methods We correlated pathologist-generated estimates of tumor cellularity versus mutant allele percentage via pyrosequencing as a QA measure for BRAF mutation detection in formalin-fixed, paraffin-embedded melanoma specimens. Results BRAF mutations were seen in 27/62 (44 %) specimens, with 93 % p.V600E and 7 % non-p.V600E. Correlation between p.V600E mutant percentage and tumor cellularity was poor–moderate (r = −0.02; p = 0.8), primarily because six samples showed a low p.V600E signal despite high tumor cellularity. A QA investigation revealed that our initial pyrosequencing assay showed a false positive, weak p.V600E signal in specimens with a p.V600K mutation. A redesigned assay detected BRAF mutations in 50/131 (38 %) specimens, including 30 % non-p.V600E. This revised assay showed strong correlation between p.V600E BRAF mutant percentage and tumor cellularity (r = 0.76; p ≤ 0.01). Re-evaluation of the previously discordant samples by the revised assay confirmed a high level of p.V600K mutation in five specimens. Conclusions Pathologists play important roles in molecular diagnostics, beyond identification of correct cells for testing. Accurate evaluation of tumor cellularity not only ensures sufficient material for required analytic sensitivity, but also provides an independent QA measure of the molecular assays.

Endogenous CCK release induced by a synthetic trypsin inhibitor, camostat, stimulates pancreatic growth; however, the mechanisms mediating this growth are not well established. Early response genes often couple short-term signals with long-term responses. To study their participation in the pancreatic growth response, mice were fasted for 18 h and refed chow containing 0.1% camostat for 1–24 h. Expression of 18 early response genes were evaluated by quantitative PCR; mRNA for 17 of the 18 increased at 1, 2, 4, or 8 h. Protein expression for c-jun, c-fos, ATF-3, Egr-1, and JunB peaked at 2 h. Nuclear localization was confirmed by immunohistochemistry of c-fos, c-jun, and Egr-1. Refeeding regular chow induced only a small increase of c-jun and none in c-fos expression. JNKs and ERKs were activated 1 h after camostat feeding as was the phosphorylation of c-jun and ATF-2. AP-1 DNA binding evaluated by EMSA showed a significant increase 1–2 h after camostat feeding with participation of c-jun, c-fos, ATF-2, ATF-3, and JunB shown by supershift. The CCK antagonist IQM-95,333 blocked camostat feeding-induced c-jun and c-fos expression by 67 and 84%, respectively, and AP-1 DNA binding was also inhibited. In CCK-deficient mice, the maximal response of c-jun induction and AP-1 DNA binding were reduced by 64 and 70%, respectively. These results indicate that camostat feeding induces a spectrum of early response gene expression and AP-1 DNA binding and that these effects are mainly CCK dependent.