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On behalf of the Canadian Association of Gastroenterology (CAG) Board, I am pleased to provide you with this report summarizing the activities and directions of the organization on behalf of its members. It is an honour to participate in the affairs of the organization and interact with groups and individuals from across the country dedicated to advancing science and care in the field of digestive health and disease. This is a challenging time in medicine, and the organization has been working hard to enhance the benefits, programs and services available to its members. The goal is to provide the highest level of services possible to meet your needs.

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 +/- 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 +/- 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium (Cai2+) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in Cai2+ was unaffected, but the initial response and overall time to Cai2+ peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.

We have previously demonstrated that the airway serous cell line Calu-3 employs a number of pH regulatory mechanisms required for bicarbonate secretion by these cells. The aim of the present study was to investigate the pH regulatory mechanisms of serous cells of freshly isolated submucosal glands (SMG). Porcine SMG were dissected out of pig tracheas obtained from a local slaughterhouse. Single glands were transferred into the chamber of an inverted microscope, immobilized by two holding pipettes and the serous cells loaded with the fluorescent pH probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Fluorescence was monitored from small areas consisting of up to 20 cells. The fluorescence ratio of the emission after excitation at 488 nm and 436 nm respectively was used to estimate cytosolic pH (pH(i)). Resting pH(i) of SMG cells in the absence of HCO(3)(-)/CO(2) was 7.1 +/- 0.16 (n=24). Addition of a solution buffered with HCO(3)(-)/CO(2) to the bath transiently acidified the cells by 0.18 +/- 0.03 (n=18). pH(i) rapidly recovered to a slightly more alkaline value than baseline pH(i). Removal of the HCO(3)(-)/CO(2) buffer strongly alkalinized SMG cells by 0.2 +/- 0.03 (n=18). To challenge pH regulatory mechanisms we exposed the cells to 20 mmol/L NH4(+) in the absence and presence of HCO(3)(-)/CO(2). In both cases we observed a rapid increase in pH(i) followed by a slight recovery. Washout of NH4(+) strongly acidified the cells. Realkalinization of pH(i) could only be observed in the presence of Na(+). This effect was inhibited by the addition of the specific Na(+)/H(+) exchanger isoform 1 (NHE1) blocker 3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride (HOE 694, 10-100 micromol/L) with an half maximal inhibitory concentration (IC(50)) of approximately 20 micromol/L. Full recovery of pH(i) in the presence of HOE 694 was observed when the cells were bathed in HCO(3)(-)/CO(2) solution. Addition of forskolin (5 micromol/L) in the presence of HCO(3)(-)/CO(2) did not significantly alter pH(i) or change pH(i) recovery after acid loading. We conclude that SMG cells possess both HCO(3)(-) dependent and HCO(3)(-) independent pH(i); regulatory mechanisms that require the presence of extracellular Na(+). Further studies are required to understand whether bicarbonate is only transported to regulate pH(i) or whether this transport determines the overall secretory capacity of SMG serous cells.

To test the hypothesis that Na+ transport in human bronchial epithelial (HBE) cells is regulated by a protease-mediated mechanism, we investigated the effects of BAY 39-9437, a recombinant Kunitz-type serine protease inhibitor, on amiloride-sensitive short-circuit current of normal [non-cystic fibrosis (CF) cells] and CF HBE cells. Mucosal treatment of non-CF and CF HBE cells with BAY 39-9437 decreased the short-circuit current, with a half-life of approximately 45 min. At 90 min, BAY 39-9437 (470 nM) reduced Na+ transport by approximately 70%. The inhibitory effect of BAY 39-9437 was concentration dependent, with a half-maximal inhibitory concentration of approximately 25 nM. Na+ transport was restored to control levels, with a half-life of approximately 15 min, on washout of BAY 39-9437. In addition, trypsin (1 microM) rapidly reversed the inhibitory effect of BAY 39-9437. These data indicate that Na+ transport in HBE cells is activated by a BAY 39-9437-inhibitable, endogenously expressed serine protease. BAY 39-9437 inhibition of this serine protease maybe of therapeutic potential for the treatment of Na+ hyperabsorption in CF.

Calu-3 cells secrete HCO(3)(-) in response to cAMP agonists but can be stimulated to secrete Cl(-) with K(+) channel activating agonists. Microelectrode and impedance analysis experiments were performed to obtain a better understanding of the conductances and driving forces involved in these different modes of anion secretion in Calu-3 cells. Microelectrode studies revealed apical and basolateral membrane depolarizations upon the addition of forskolin (V(ap) -52 mV vs. -21 mV; V(bl) -60 mV vs. -44 mV) that paralleled the hyperpolarization of the mucosal negative transepithelial voltage (V(T) -8 mV vs. -23 mV). These changes were accompanied by a decrease in the apical membrane fractional resistance (F(Rap)) from approximately 0.50 to 0.08, consistent with the activation of an apical membrane conductance. The subsequent addition of 1-ethyl-2-benzimidazolinone (1-EBIO), a K(+) channel activator, hyperpolarized V(ap) to -27 mV, V(bl) to -60 mV and V(T) to -33 mV. Impedance analysis revealed the apical membrane resistance (R(ap)) of the forskolin-stimulated cells was less than 20 ohm cm(2), indeed in most monolayers R(ap) fell to less than 5 ohm cm(2). The impedance derived estimate of the basolateral membrane resistance (R(bl)) was approximately 170 ohm cm(2) in forskolin treated cells and fell to 50 ohm cm(2) with the addition of 1-EBIO. Using these values for the R(bl) and the F(Rap) value of 0.08 yields a R(ap) of approximately 14 ohm cm(2) in the presence of forskolin and 4 ohm cm(2) in the presence of forskolin plus 1-EBIO. Thus, by two independent methods, forskolin-stimulated Calu-3 cells are seen to have a very high apical membrane conductance of 50 to 200 mS/cm(2). Therefore, we would assert that even at one-tenth the anion selectivity for Cl(-), this high conductance could support the conductive exit of HCO(3)(-) across the apical membrane. We further propose that this high apical membrane conductance serves to clamp the apical membrane potential near the equilibrium potential for Cl(-) and thereby provides the driving force for HCO(3)(-) secretion in forskolin-stimulated Calu-3 cells. The hyperpolarization of V(ap) and V(bl) caused by 1-EBIO provides a driving force for Cl(-) exit across the apical membrane, inhibits the influx of HCO(3)(-) on the Na(+):HCO(3)(-) cotransporter across the basolateral membrane, activates the basolateral membrane Na(+):K:2Cl(-) cotransporter and thereby provides the switch from HCO(3)(-) secretion to Cl(-) secretion.

A cytoprotective role for protease-activated receptor-2 (PAR2) has been suggested in a number of systems including the airway, and to this end, we have studied the role that PARs play in the regulation of airway ion transport, using cultures of normal human bronchial epithelial cells. PAR2 activators, added to the basolateral membrane, caused a transient, Ca2+-dependent increase in short-circuit current (I(sc)), followed by a sustained inhibition of amiloride-sensitive I(sc). These phases corresponded with a transient increase in intracellular Ca2+ concentration and then a transient increase, followed by decrease, in basolateral K+ permeability. After PAR2 activation and the addition of amiloride, the forskolin-stimulated increase in I(sc) was also attenuated. By contrast, PAR2 activators added to the apical surface of the epithelia or PAR1 activators added to both the apical and basolateral surfaces were without effect. PAR2 may, therefore, play a role in the airway, regulating Na+ absorption and anion secretion, processes that are central to the control of airway surface liquid volume and composition.

Forskolin, UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen (Methoxsalen; 8-MOP), and genistein were evaluated for their effects on ion transport across primary cultures of human bronchial epithelium (HBE) expressing wild-type (wt HBE) and DeltaF508 (DeltaF-HBE) cystic fibrosis transmembrane conductance regulator. In wt HBE, the baseline short-circuit current (I(sc)) averaged 27.0 +/- 0.6 microA/cm(2) (n = 350). Amiloride reduced this I(sc) by 13.5 +/- 0.5 microA/cm(2) (n = 317). In DeltaF-HBE, baseline I(sc) was 33.8 +/- 1.2 microA/cm(2) (n = 200), and amiloride reduced this by 29.6 +/- 1.5 microA/cm(2) (n = 116), demonstrating the characteristic hyperabsorption of Na(+) associated with cystic fibrosis (CF). In wt HBE, subsequent to amiloride, forskolin induced a sustained, bumetanide-sensitive I(sc) (DeltaI(sc) = 8.4 +/- 0.8 microA/cm(2); n = 119). Addition of acetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4, 4'-dinitrostilben-2,2'-disulfonic acid further reduced I(sc), suggesting forskolin also stimulates HCO(3)(-) secretion. This was confirmed by ion substitution studies. The forskolin-induced I(sc) was inhibited by 293B, Ba(2+), clofilium, and quinine, whereas charybdotoxin was without effect. In DeltaF-HBE the forskolin I(sc) response was reduced to 1.2 +/- 0.3 microA/cm(2) (n = 30). In wt HBE, mucosal UTP induced a transient increase in I(sc) (Delta I(sc) = 15. 5 +/- 1.1 microA/cm(2); n = 44) followed by a sustained plateau, whereas in DeltaF-HBE the increase in I(sc) was reduced to 5.8 +/- 0. 7 microA/cm(2) (n = 13). In wt HBE, 1-EBIO, NS004, 8-MOP, and genistein increased I(sc) by 11.6 +/- 0.9 (n = 20), 10.8 +/- 1.7 (n = 18), 10.0 +/- 1.6 (n = 5), and 7.9 +/- 0.8 microA/cm(2) (n = 17), respectively. In DeltaF-HBE, 1-EBIO, NS004, and 8-MOP failed to stimulate Cl(-) secretion. However, addition of NS004 subsequent to forskolin induced a sustained Cl(-) secretory response (2.1 +/- 0.3 microA/cm(2), n = 21). In DeltaF-HBE, genistein alone stimulated Cl(-) secretion (2.5 +/- 0.5 microA/cm(2), n = 11). After incubation of DeltaF-HBE at 26 degrees C for 24 h, the responses to 1-EBIO, NS004, and genistein were all potentiated. 1-EBIO and genistein increased Na(+) absorption across DeltaF-HBE, whereas NS004 and 8-MOP had no effect. Finally, Ca(2+)-, but not cAMP-mediated agonists, stimulated K(+) secretion across both wt HBE and DeltaF-HBE in a glibenclamide-dependent fashion. Our results demonstrate that pharmacological agents directed at both basolateral K(+) and apical Cl(-) conductances directly modulate Cl(-) secretion across HBE, indicating they may be useful in ameliorating the ion transport defect associated with CF.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.

A rare case of Castleman's disease presenting as Crohn's disease is described. This 21-year-old male with chronic neutropenia for one year presented with recurrent right lower quadrant pain of two years' duration. Small bowel follow-through suggested Crohn's of the terminal ileum. Colonoscopy confirmed ulcerations in the terminal ileum and cecum, with biopsies showing necrosis and inflammation. Treatment was initiated with prednisone, 5-aminosalicylate and granulocyte colony-stimulating factor for neutropenia. Symptoms recurred one year later, and repeat colonoscopy showed a focal cecal ulceration. Two years after presentation a resection was planned. Laparotomy revealed a normal ileocecal region and a large retroperitoneal mass of lymphadenopathy. Biopsies confirmed reactive hyperplasia, consistent with the plasma cell variant of Castleman's disease. Chemotherapy has resulted in improvement of symptoms and decrease in mass size, but cecal ulceration persisted. This case illustrates a variant presentation of Castleman's disease with neutropenia and manifestations in the gastrointestinal tract.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.

Chloride channels are ubiquitously distributed, biophysically varied and functionally diverse. Despite the known contribution of chloride channels to the physiology of various cell types and the pathology of several diseases, high affinity ligands are not available to study these channels. Here we report the iterative and integrated use of ion channel kinetic analysis and computational chemical methods in the development of high affinity blockers of the outwardly rectifying chloride channel (ORCC). Kinetic analysis, with emphasis on estimation of the block time constant as determined from critical closed time plots, was used to guide the synthesis of new disulfonic stilbene derivatives. Computational chemical methods were used to deduce the important features of the disulfonic stilbene molecule necessary for potent blockade of ORCC and ultimately led to the discovery of the calixarenes. Para-sulfonated calixarenes were found to be potent blockers of ORCC with subnanomolar inhibition constants and exceptionally long block times.

Cystic fibrosis results from mutations in the gene encoding the CFTR Cl- channel. Although CFTR occurs as an integral component of the plasma membrane, recent studies implicate CFTR in endocytic recycling and suggest that the protein may also exist in intracellular vesicular compartments. To test this, we analyzed CFTR in clathrin-coated vesicles (CCV) purified from cells constitutively expressing CFTR at high levels. CFTR immunoreactivity was detected in CCV by immunoblot and was identified as CFTR based on labeling of immunoprecipitates with protein kinase A and by tryptic phosphopeptide mapping. Fusion of uncoated CCV with planar lipid bilayers resulted in the incorporation of kinase- and ATP-activated Cl- channel activity (7.8 pS at 20 degrees C; 11.9 pS at 37 degrees C), with a linear current-voltage relation under symmetrical conditions. Thus, functional CFTR occurs in CCV. Moreover, CFTR interacts with the plasma membrane specific adaptor complex during endocytosis through clathrin-coated pits. Therefore, the abundance of CFTR in the plasma membrane may be regulated by exocytic insertion and endocytic recycling, and these processes may provide an augmentation to protein kinase A activation as a mechanism for regulating CFTR Cl channels in the plasma membrane.

The gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) is defective in patients with cystic fibrosis. Although the protein product of the CFTR gene has been proposed to function as a chloride ion channel, certain aspects of its function remain unclear. The role of CFTR in the adenosine 3',5'-monophosphate (cAMP)-dependent regulation of plasma membrane recycling was examined. Adenosine 3',5'-monophosphate is known to regulate endocytosis and exocytosis in chloride-secreting epithelial cells that express CFTR. However, mutant epithelial cells derived from a patient with cystic fibrosis exhibited no cAMP-dependent regulation of endocytosis and exocytosis until they were transfected with complementary DNA encoding wild-type CFTR. Thus, CFTR is critical for cAMP-dependent regulation of membrane recycling in epithelial tissues, and this function of CFTR could explain in part the pleiotropic nature of cystic fibrosis.

Excitatory amino acids and their receptors play an important role in both normal synaptic transmission and excitotoxic-mediated neuronal death. In the present investigation we have prepared a series of glutamate analogs and examined the pharmacological specificity with which they interact with excitatory amino acid receptors. Included within this group of compounds is a potent excitotoxic amino acid, beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-L-ODAP). This excitotoxin is of particular interest because it has been identified as a major causative agent of human neurolathyrism, a disease characterized by permanent spastic paralysis. The site of action of beta-L-ODAP was delineated with both electrophysiological recordings in hippocampal slices and radioligand binding assays in synaptic plasma membranes. We report that beta-L-ODAP is a potent agonist at the non-N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. beta-L-ODAP interacts most potently with the quisqualate class of non-NMDA receptors (IC50 = 1.3 microM), less potently with the kainate receptor (IC50 = 17 microM), and very weakly with NMDA receptors. The specificity of this binding was consistent with physiological experiments that demonstrated that beta-L-ODAP-induced depolarizations were potently blocked by the newly identified non-NMDA receptor antagonist, CNQX, but were not affected by the NMDA antagonist D-AP5. These results extend recent studies that have focused on the contribution of NMDA receptors to excitotoxicity and highlight the potential involvement of non-NMDA receptors in excitotoxic-mediated cell death.

L-gamma(Threo-beta-methyl)glutamyl-L-alpha-aminobutyrate was was prepared and found to be an excellent substrate of gamma-glutamyl cyclotransferase; in contrast to gamma-glutamyl-glutamine and other good substrates of cyclotransferase, the new substrate is not acted upon by gamma-glutamyl transpeptidase. gamma-Glutamyl cyclotransferase converts the new substrate to alpha-aminobutyrate and 3-methyl-5-oxoproline; the latter compound is not a substrate of 5-oxoprolinase. These properties of L-gamma-(threo-beta-methyl)glutamyl-L-alpha-aminobutyrate facilitate its use in selectively determining cyclotransferase activity in biological materials that have transpeptidase activity. Thus, the new substrate was used here for the determination of the cyclotransferase activity of homogenates of various mouse tissues. The new substrate was also used to examine gamma-glutamyl cyclotransferase activity in vivo; thus, the rate of respiratory 14CO2 formation after administration of L-gamma-(threo-beta-methyl)glutamyl-L-alpha-amino[14C]butyrate to mice provides a valid measure of cyclotransferase activity. beta-Aminoglutaryl-L-alpha-aminobutyrate is a competitive inhibitor of cyclotransferase (apparent Ki, 0.6 mM). Administration of beta-amino-glutaryl-L-alpha-aminobutyrate to mice out only decreased the level of 5-oxoproline in the kidney of control mice, but also of mice in which kidney 5-oxoproline levels were increased by administration of methionine. Administration of beta-aminoglutaryl-L-alpha-aminobutyrate to mice decreased the in vivo metabolism of L-(threo-beta-methyl)glutamyl-L-alpha-amino[14C]butyrate as indicated by a marked decrease in the rate of respiratory 14CO2 formation. The findings indicate that gamma-glutamyl cyclo-transferase is a major in vivo catalyst for the formation of 5-oxoproline.

Colorectal cancer is a major health problem. Despite therapeutic advances, the overall five-year survival rate remains at 45%. At the present time the greatest potential for improved survival lies with early detection. The authors of this article have assessed a number of colorectal surveillance programs in an attempt to identify those individuals with potentially pre-malignant disease or early localized cancer. Differences in individual risks and the availability of numerous "screening tests" make it important to provide each patient with the most appropriate investigation. Preliminary evidence suggests that fecal occult blood tests, sigmoidoscopy, and colonoscopy with polypectomy can be effective in reducing the incidence of colon cancer.