Barrett's esophagus in the patients with familial adenomatous polyposis.

Familial adenomatous polyposis (FAP) is caused by germ line mutations in the APC gene. Barrett's esophagus (BE) and Barrett's adenocarcinoma are intestinal type lesions of the esophagus characterized by an early loss of heterozygosity at the APC locus. We hypothesized that patients with FAP are at risk for the early development of BE due to the inherited mutations in the APC gene (haploinsufficiency). Upper gastrointestinal (UGI) tract biopsies from 36 patients with FAP were reviewed to determine the incidence and characteristics of BE in these patients. Twenty-four patients were confirmed carriers of a deleterious germline APC mutation. The other 12 patients were from FAP families with known APC gene mutations and had clinical manifestations of FAP. The control group consisted of patients who did not have a personal or family history of FAP undergoing UGI endoscopic examination in our institution over a 30 month period of time. The difference in expression of Wnt pathway proteins (APC, ß-catenin, E-cadherin and cyclin D1) in BE between BE(+)/FAP(+), BE(-)/FAP(+) and age-matched BE(+)/FAP(-) groups was studied using immunohistochemistry. BE was found in 6 of 36 (6/36 or 16%) patients with FAP and in 266 of 1662 patients (16%) in the control group of symptomatic patients. The average age at the first diagnosis of BE in FAP patients was 37.8 versus 57.5 years in the control group (sporadic BE). When compared to age matched BE(+)/FAP- group (7/334), patients with FAP had a significantly (p = 0.005843, odds ratio 9.2; Fisher exact test) higher incidence of BE. Both classic FAP and attenuated FAP phenotypes were associated with BE .Two types of germ line mutations in APC gene were identified in BE(+)/FAP(+) patients: Five patients had 2-base deletion in exon 4 (426delAT) and one patient had 4-base deletion in exon 15 (3202del4). No difference in Wnt signaling pathway proteins expression was detected between BE(+)/FAP(+) and the age matched group of patients with sporadic BE (BE(+)/FAP(-)). Patients with FAP appear to have increased risk for the development of BE, which on average develops some 20 years earlier than in patients without FAP. This association needs to be taken in account when caring for the patients with FAP.

Brain oedema in focal ischaemia: molecular pathophysiology and theoretical implications.

Focal cerebral ischaemia and post-ischaemic reperfusion cause cerebral capillary dysfunction, resulting in oedema formation and haemorrhagic conversion. There are substantial gaps in understanding the pathophysiology, especially regarding early molecular participants. Here, we review physiological and molecular mechanisms involved. We reaffirm the central role of Starling's principle, which states that oedema formation is determined by the driving force and the capillary "permeability pore". We emphasise that the movement of fluids is largely driven without new expenditure of energy by the ischaemic brain. We organise the progressive changes in osmotic and hydrostatic conductivity of abnormal capillaries into three phases: formation of ionic oedema, formation of vasogenic oedema, and catastrophic failure with haemorrhagic conversion. We suggest a new theory suggesting that ischaemia-induced capillary dysfunction can be attributed to de novo synthesis of a specific ensemble of proteins that determine osmotic and hydraulic conductivity in Starling's equation, and whose expression is driven by a distinct transcriptional program.

EGF and HB-EGF modulate inward potassium current in human bladder urothelial cells from normal and interstitial cystitis patients.

Interstitial cystitis (IC) is an idiopathic condition characterized by bladder hyperalgesia. Studies have shown cytokine and purinergic signaling abnormalities in cultured bladder urothelial cells (BUC) from IC patients. We performed single-cell electrophysiological studies in both normal and IC BUC. A strongly inward rectifying potassium current with conductance of the Kir2.1 channel was identified in normal BUC. This current was significantly reduced in IC BUC. Kir2.1 protein and mRNA were detected in both IC and normal BUC. Epidermal growth factor (EGF) caused a dose-dependent decrease in the inward potassium current in normal BUC. EGF is secreted in higher amounts by IC BUC and is known to decrease Kir2.1 conductance by phosphorylation of Kir2.1. Genistein, a nonspecific phosphorylation inhibitor, increased the inward potassium current in IC BUC and blocked the effect of EGF on normal BUC. Treatment of IC BUC with heparin-binding epidermal growth factor-like growth factor (HB-EGF), previously shown to be secreted in lower amounts by IC BUC, significantly increased inward potassium current. These data show that the inward potassium current in BUC can be modulated by EGF and HB-EGF. Changes in BUC membrane potassium conductance caused by altered levels of EGF and HB-EGF may therefore play a role in the pathophysiology of IC.

Newly expressed SUR1-regulated NC(Ca-ATP) channel mediates cerebral edema after ischemic stroke.

Pathological conditions in the central nervous system, including stroke and trauma, are often exacerbated by cerebral edema. We recently identified a nonselective cation channel, the NC(Ca-ATP) channel, in ischemic astrocytes that is regulated by sulfonylurea receptor 1 (SUR1), is opened by depletion of ATP and, when opened, causes cytotoxic edema. Here, we evaluated involvement of this channel in rodent models of stroke. SUR1 protein and mRNA were newly expressed in ischemic neurons, astrocytes and capillaries. Upregulation of SUR1 was linked to activation of the transcription factor Sp1 and was associated with expression of functional NC(Ca-ATP) but not K(ATP) channels. Block of SUR1 with low-dose glibenclamide reduced cerebral edema, infarct volume and mortality by 50%, with the reduction in infarct volume being associated with cortical sparing. Our findings indicate that the NC(Ca-ATP) channel is crucially involved in development of cerebral edema, and that targeting SUR1 may provide a new therapeutic approach to stroke.

Regulation by sulfanylurea receptor type 1 of a non-selective cation channel involved in cytotoxic edema of reactive astrocytes.

Hypoxia-ischemia and ATP depletion are associated with cytotoxic edema of glial cells, but mechanisms involved remain incompletely characterized. We examined morphologic and electrophysiological responses of freshly isolated native reactive astrocytes (NRAs) following exposure to NaN3, which depletes cellular ATP. NaN3 caused profound and sustained depolarization due to activation of a novel 35 pS Ca2+-activated, [ATP]i-sensitive non-selective cation (NCCa-ATP) channel found in >90% of excised membrane patches. This channel exhibited significantly different properties compared with previously reported NCCa-ATP channels, including different sensitivity to block by various adenine nucleotides (EC50=0.79 microM for [ATP]i, with no block by AMP or ADP), and activation by submicromolar [Ca]i. In addition, the channel was found to be regulated in a manner identical to that of SUR1-regulated KATP channels, including high affinity block by glybenclamide and tolbutamide, and opening by diazoxide. mRNA transcription and protein expression of SUR1 but not SUR2 were confirmed in reactive astrocytes both in situ and after isolation, whereas Kir6.x, which forms the pore-forming subunit of the KATP channel, was not expressed. Channel opening by [ATP]i depletion or exposure to diazoxide caused blebbing of the cell membrane, whereas [ATP]i depletion in the presence of glybenclamide did not. These findings are consistent with participation of this channel in cation flux involved in cell swelling. This novel channel may play an important role in the pathogenesis of brain swelling.

Functional coupling between sulfonylurea receptor type 1 and a nonselective cation channel in reactive astrocytes from adult rat brain.

We previously identified a novel, nonselective cation channel in native reactive (type R1) astrocytes (NR1As) from injured rat brain that is regulated by cytoplasmic Ca2+ and ATP (NC(Ca-ATP)) and exhibits sensitivity to block by adenine nucleotides similar to that of sulfonylurea receptor type 1 (SUR1). Here we show that SUR1 is involved in regulation of this channel. NR1As within the site of injury and after isolation exhibited specific binding of FITC-tagged glibenclamide and were immunolabeled with anti-SUR1 antibody, but not with anti-SUR2, anti-Kir6.1 or anti-Kir6.2 antibodies, indicating absence of ATP-sensitive K+ (KATP) channels. RT-PCR confirmed transcription of mRNA for SUR1 but not SUR2. Several properties previously associated exclusively with SUR1-regulated KATP channels were observed in patch-clamp experiments using Cs+ as the charge carrier: (1) the sulfonylureas, glibenclamide and tolbutamide, inhibited NCCa-ATP channels with EC50 values of 48 nm and 16.1 microm, respectively; (2) inhibition by sulfonylureas was lost after exposure of the intracellular face to trypsin or anti-SUR1 antibody; (3) channel inhibition was caused by a change in kinetics of channel closing, with no change in channel amplitude or open-channel dwell times; and (4) the SUR activator ("KATP channel opener"), diazoxide, activated the NCCa-ATP channel, whereas pinacidil and cromakalin did not. Also, glibenclamide prevented cell blebbing after ATP depletion, whereas blebbing was produced by exposure to diazoxide. Our data indicate that SUR1 is functionally coupled to the pore-forming portion of the NC(Ca-ATP) channel, providing the first demonstration of promiscuity of SUR1 outside of the K+ inward rectifier family of channels.

Chlorotoxin-sensitive Ca2+-activated Cl- channel in type R2 reactive astrocytes from adult rat brain.

Astrocytes express four types of Cl(-) or anion channels, but Ca(2+)-activated Cl(-) (Cl(Ca)) channels have not been described. We studied Cl(-) channels in a morphologically distinct subpopulation ( approximately 5% of cells) of small (10-12 micro m, 11.8 +/- 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, -57.1 +/- 4.0 mV, polarized to -72.7 +/- 4.5 mV with BAPTA-AM, an intracellular Ca(2+) chelator, and depolarized to -30.7 +/- 6.1 mV with thapsigargin, which mobilizes Ca(2+) from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl(-) reversal potential, which was blocked by Cl(-) channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn(2+), by I(-) (10 mM), and by chlorotoxin (EC(50) = 47 nM). With conventional whole-cell clamp, NPPB- and Zn(2+)-sensitive currents became larger with increasing [Ca(2+)](i) (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca(2+) sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive Cl(Ca) channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive Cl(Ca) channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte.

Transforming growth factor-beta 1 regulates Kir2.3 inward rectifier K+ channels via phospholipase C and protein kinase C-delta in reactive astrocytes from adult rat brain.

The multifunctional cytokine, transforming growth factor beta(1) (TGF-beta(1)), exerts complex effects on astrocytes with early signaling events being less well characterized than transcriptional mechanisms. We examined the effect of TGF-beta(1) on the 14-pS Kir2.3 inward rectifier K(+) channel in rat primary cultured reactive astrocytes. Immunofluorescence study showed that cells co-expressed TGF-beta(1) receptors 1 and 2, Kir2.3, and glial fibrillary acidic protein (GFAP). Patch clamp study showed that TGF-beta(1) (0.1-100 ng/ml) caused a rapid (<5 min) depolarization because of dose-dependent down-regulation of Kir2.3 channels, which was mimicked by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (10-500 nm) and which was inhibited by the PKC inhibitor calphostin C (100 nm), by PKC desensitization produced by 3 h of exposure to phorbol 12-myristate 13-acetate (100 nm), and by the PKC-delta isoform-specific inhibitor rottlerin (50 microm). Immunoblot analysis and confocal imaging showed that TGF-beta(1) caused PKC-delta translocation to membrane, and co-immunoprecipitation experiments showed that TGF-beta(1) enhanced association between Kir2.3 and PKC-delta. Additional electrophysiological experiments showed that Kir2.3 channel down-regulation was blocked by the phospholipase C inhibitors, neomycin (100 microm) and D609 (200 microm). Given the commonality of signaling involving PLC-PKC-delta, we speculate that TGF-beta(1)-evoked depolarization may be an early signaling event related to gene transcription in astrocytes.