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Novel method for the study of human epithelial ion transport
A606 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
3124
3126
HUMAN DUODENAL BICARBONATE ABSORPTION INVOLVES BOTH BRUSH BORDER NA+IH+ EXCHANGERS NHE2 AND NHE3. Dan Hogan, M. Repishti, V. Pratha, Mark Donowitz., Ming Tse, Jon Isenberg, Univ of CA Sch of Med, San Diego, CA; Johns Hopkins Univ Sch of Medicine, Baltimore, MD. Mammalian duodenum carries out net HC03 secretion with a steep proximal-to-distal gradient. The cellular mechanims involved in epithelial HC0 3 transport involve acidlbase transporters. The hypotheses of this study was that since the epithelial Na+/H + exchangers (NHE) are present on human villus epithelial cells, that inhibition of their activity will increase luminal HCOJ by decreasing HC0 3 absorption . Duodenal mucosal bicarbonate secretion (DMBS) was measured after an overnight fast in 5 healthy male subjects (ages 32-55 yrs). The proximal 3 em of duodenum were isolated between balloons and perfused with 154 mM NaCI (2 mUmin) containing 14C_PEG as a non-absorbable marker as validated previously . [HCO J ) was measured by pWPC02 using the HendersonHassalbalch equation and 15 min outputs calculated based upon volumes corrected for 14C_PEG recovery. Basal DMBS was measured for 45 min followed by luminal perfusion of graded doses of amiloride that inhibited NHE2 activity (10-5 and 1O-4M) and NHE3 activity (amiloride 1O-3M). Each dose was infused for 30 min in increasing sequential order. Also, proximal duodenal biopsies were obtained in subjects, coded, processed for immunohistochemistry using previously characterized rabbit polyclonal antibodies Ab 597 (NHE2) and Ab 1380 (NHE3). Basal DMBS was 355 (95% CI=300-41O) p,moUcm-h, Amiloride resulted in dose-dependent incremental net increases in DMBS [mean and 95% CI net .is=47 (11-82), 67 (32-103), and 111 (75-146) p,moUcm-h, respectively. DMBS in response to the highest amiloride dose was significantly greater than to the two lower doses. The total change in HC03 transport was similar for the amiloride concentration inhibiting NHE2 and that inhibiting NHE3. Thus amiloride increased net human duodenal HC03 secretion by inhibiting HC0 3 absorption . In each subject, NHE2 and NHE3 were localized in the brush border of duodenal villus epithelial cells, while neither occurred in the crypt epithelium . In conclusion (I) Both NHE2 and NHE3 are present in the human duodenal villus cell brush border ; (2) NHE2 and NHE3 contributed equally to basal duodenal HC0 3 absorption; and (3) Net DMBS consists of both HCO J absorptive and secretory processes.
ACTIVATION OF A CL-·SECRETORY RESPONSE IN NCM460 IMMORTALIZED HUMAN COLONIC CELLS, Heather M. Jones, Mary Pat Moyer, Peter Lance, Michael E. Duffey, State Univ of New York, Buffalo, NY; INCELL Corp, San Antonio, TX. Mechanisms of secretory diarrhea caused by active cr secretion can be studied in human colonic epithelial cell lines. Recently, a non-transformed immortalized epithelial secretory cell line, NCM460, was derived from normal adult human transverse colon . The response of NCM460 cells to agonists that cause active Clsecretion was investigated using the wholecell patch-clamp technique. Patch pipettes were filled with a simulated intracellular solution, and the cells were bathed in a standard NaCI solution. When membrane potential was voltage-clamped to the K+ equilibrium potential (-80 mY), the acetylcholine analog, carbachol (100 p,M),induced an inwardly directed cr current in 18 of 29 cells, with a peak value of 66:':16 pA (:':SEM). This current declined gradually to zero in less than 120 sec, demonstrating that Cl: channels are activated in NCM460 cells by cholinergic stimulation , as expected in Cl'-secretory cells. When membrane potential was clamped to the cr equilibrium potential (0 mY), carbachol induced a small outwardly directed K+ current in 3 of the 29 cells. This differs from our earlier results in a colon cancer cell line (T84) in which carbachol induced a K+ conductance without affecting CI- conductance (Am. 1. Physio!. 258: C318-C326, 1990). To determine whether a carbachol-induced rise in intracellular Ca 2+ concentration, [Cah)i, underlies the Cl current increase, as seen in other secretory cells, we used fluorescence microscopy and the 340 nm to 380 nm fluorescence ratio of the Ca2+ -sensitive dye, Fura-2, as an index of [Ca2+Ji. In resting cells this ratio averaged 0.13:':0.3 (N=198), corresponding to [Cah)i of about 35 nM. Exposure to carbachol (100 /-LM) caused a rapid rise in the fluorescence ratio in 96% of cells to 0.41 :':0.08, corresponding to [Cah)i of about 210 nM. This initial peak was followed by a gradual decline. A similar response to carbachol was seen when the bath was made Ca2+-free (N=23), demonstrating that the carbachol-induced rise is caused by the release of Ca2+ from intracellular stores and not Ca 2+ influx, as we found in T84 cells. These results demonstrate that the signalling mechanisms controlling Ca2+ in NCM460 cells in response to cholinergic stimulation are similar to the mechanisms in other secretory cells. However, since Ca2+ activates Cl channels in NCM460 cells but not in T84 cells, the NCM460 cell line may be a better model than tumor-derived cell lines for studying the mechanisms of diarrheas produced by active cr secretion.
3125 PERMEABLITY OF PROXIMAL AND DISTAL COLON CRYPT AND SURFACE EPITHELIUM TO HYDROPIllLIC MOLECULES. Mats Jodal, Britt-Marie Fihn, Ying Sun, Dept of Physiology, Gothenburg , Sweden. Aim. The colon epithelium is generally considered to be a moderately tight epithelium with a very low permeability to hydrophilic substances . Morphologically there is a difference in complexity of the zonulae occludentes within the crypt and surface epithelium but its functional implications are unknown. The aim of this study was therefore to investigate the sizes of the aqueous pores of different parts of the colonic epithelium. Methods. In anaesthetized Sprague Dawley rats proximal and distal colon were perfused for 4 hours in a recirculating system with a modified Kreb' s Henseleit solution. The appearance of the radio labelled probes Cr-EDTA, mannitol and urea (mol radii 5.25, 3.25 and 2.3 A respectively) into the perfusate from the plasma as well as there disappearance from the luminal perfusate into the intestinal tissue were measured and expressed as clearance. The localization of the fluorescence probes Evan blue labelled albumin and FITC-dextran 4000 along the colon epithelium was also investigated . Results. In the lumen to plasma experiments the proximal and distal colon segments exhibited a net fluid absorption as well a significant and similar disappearance rate of urea while the clearance of mannitol was not significantly different from zero. In the plasma to lumen experiments there was a significant appearance rate of Cr-EDT A, mannitol and urea in the proximal colon , the order of magnitude being urea > mannitol > Cr-EDTA. In the distal colon only the clearance of mannitol and urea were significant and lower than in the proximal colon. The clearance ratio mannitol/urea was around 0.23 in both colonic segments and the ratio Cr-EDTNmannitol in the proximal colon 0.71. Both fluorescence probes were found along the surface epithelium after 2 h luminal incubation in the both segments while no fluorescence was present in the crypt lumina . Conclusions. The results indicate that the surface epithelium of both colonic segments contain a single type of pore (radius about 3 -3.5 A) while the crypt epithelium , particularly in the proximal colon, is a heteroporou s membrane probably containing at least two pores, one large (radius above 35-40) and one small A (radius 4-5 A). Furthermore, in vivo, substance s dissolved in a fluid solution do not have access to the crypt lumina and can therefore not be absorbed via the crypt epithelium .
3127 NOVEL METHOD FOR THE STUDY OF HUMAN EPITHELIAL ION TRANSPORT. Rune Larsen, Anette Mertz-Nielsen, Mark Berner Hansen, Steen Seier Poulsen, Niels Bindslev, Dept of Medicine CIF, Glostrup Hosp, Glostrup, Denmark ; Dept of Surg D, Glostrup Hosp, Glostrup, Denmark; Dept of Anatomy, Univ of Copenhagen, Copenhagen, Denmark ; Dept of Med Physiology, Univ of Copenhagen, Copenhagen, Denmark. Introduction. The study of epithelial ion transport has great importance for our understanding of the GI tract in health and disease. The Ussing chamber has been used for this purpose, but due to technical difficulties and limitations with the available small epithelial biopsies, these investigations in humans have been restricted. The aim of this study was to developed an Ussing chamber setup for the available biopsies taken at endoscopy . Methods. Routine biopsies were taken from the duodenum during endoscopy of patients with dyspepsia. Modified Ussing chambers were constructed, exposing an area of 5 mrrr'. Biopsies were mounted in these mini-chambers by continous air-suction in an atraumatic manner, verified by histological procedures . Furthermore , a clamp unit and software program were developed allowing automatic measurements of short circuit current (SCC, resolution 25 nA) , voltage (PD, resolution 50 p,V) and conductance (G, resolution> 12.5 p,S), all with a time resolution of2 sec. To determine basic functional parameters, biopsies were examined for viability by measuring electrogenic absorption with d-glucose and electrogenic secretion with acetylcholine (ACh). Results. Basal SCC and G were in the range of20-70 p,Ncm2 and 30-60 mS/cm2 (n=32), respectively. The biopsies were fully functional for up to 8 hrs to mucosally applied dglucose (Jrnax about 400 p,Ncm 2 and Kd around 10 nM, n=32) and serosally applied ACh (Jmax about 200 p,Ncm2 and Kd around 35 /-LM, n=20). We conclude, that this mini-Ussing air-suction (MUAS) chamber is a promising and reliable novel gadget for functional characterization of electrogenic ion transport in human (duodenal) epithelial biopsies.
GASTROENTEROLOGY Vol. 118, No.4
3124
3126
HUMAN DUODENAL BICARBONATE ABSORPTION INVOLVES BOTH BRUSH BORDER NA+IH+ EXCHANGERS NHE2 AND NHE3. Dan Hogan, M. Repishti, V. Pratha, Mark Donowitz., Ming Tse, Jon Isenberg, Univ of CA Sch of Med, San Diego, CA; Johns Hopkins Univ Sch of Medicine, Baltimore, MD. Mammalian duodenum carries out net HC03 secretion with a steep proximal-to-distal gradient. The cellular mechanims involved in epithelial HC0 3 transport involve acidlbase transporters. The hypotheses of this study was that since the epithelial Na+/H + exchangers (NHE) are present on human villus epithelial cells, that inhibition of their activity will increase luminal HCOJ by decreasing HC0 3 absorption . Duodenal mucosal bicarbonate secretion (DMBS) was measured after an overnight fast in 5 healthy male subjects (ages 32-55 yrs). The proximal 3 em of duodenum were isolated between balloons and perfused with 154 mM NaCI (2 mUmin) containing 14C_PEG as a non-absorbable marker as validated previously . [HCO J ) was measured by pWPC02 using the HendersonHassalbalch equation and 15 min outputs calculated based upon volumes corrected for 14C_PEG recovery. Basal DMBS was measured for 45 min followed by luminal perfusion of graded doses of amiloride that inhibited NHE2 activity (10-5 and 1O-4M) and NHE3 activity (amiloride 1O-3M). Each dose was infused for 30 min in increasing sequential order. Also, proximal duodenal biopsies were obtained in subjects, coded, processed for immunohistochemistry using previously characterized rabbit polyclonal antibodies Ab 597 (NHE2) and Ab 1380 (NHE3). Basal DMBS was 355 (95% CI=300-41O) p,moUcm-h, Amiloride resulted in dose-dependent incremental net increases in DMBS [mean and 95% CI net .is=47 (11-82), 67 (32-103), and 111 (75-146) p,moUcm-h, respectively. DMBS in response to the highest amiloride dose was significantly greater than to the two lower doses. The total change in HC03 transport was similar for the amiloride concentration inhibiting NHE2 and that inhibiting NHE3. Thus amiloride increased net human duodenal HC03 secretion by inhibiting HC0 3 absorption . In each subject, NHE2 and NHE3 were localized in the brush border of duodenal villus epithelial cells, while neither occurred in the crypt epithelium . In conclusion (I) Both NHE2 and NHE3 are present in the human duodenal villus cell brush border ; (2) NHE2 and NHE3 contributed equally to basal duodenal HC0 3 absorption; and (3) Net DMBS consists of both HCO J absorptive and secretory processes.
ACTIVATION OF A CL-·SECRETORY RESPONSE IN NCM460 IMMORTALIZED HUMAN COLONIC CELLS, Heather M. Jones, Mary Pat Moyer, Peter Lance, Michael E. Duffey, State Univ of New York, Buffalo, NY; INCELL Corp, San Antonio, TX. Mechanisms of secretory diarrhea caused by active cr secretion can be studied in human colonic epithelial cell lines. Recently, a non-transformed immortalized epithelial secretory cell line, NCM460, was derived from normal adult human transverse colon . The response of NCM460 cells to agonists that cause active Clsecretion was investigated using the wholecell patch-clamp technique. Patch pipettes were filled with a simulated intracellular solution, and the cells were bathed in a standard NaCI solution. When membrane potential was voltage-clamped to the K+ equilibrium potential (-80 mY), the acetylcholine analog, carbachol (100 p,M),induced an inwardly directed cr current in 18 of 29 cells, with a peak value of 66:':16 pA (:':SEM). This current declined gradually to zero in less than 120 sec, demonstrating that Cl: channels are activated in NCM460 cells by cholinergic stimulation , as expected in Cl'-secretory cells. When membrane potential was clamped to the cr equilibrium potential (0 mY), carbachol induced a small outwardly directed K+ current in 3 of the 29 cells. This differs from our earlier results in a colon cancer cell line (T84) in which carbachol induced a K+ conductance without affecting CI- conductance (Am. 1. Physio!. 258: C318-C326, 1990). To determine whether a carbachol-induced rise in intracellular Ca 2+ concentration, [Cah)i, underlies the Cl current increase, as seen in other secretory cells, we used fluorescence microscopy and the 340 nm to 380 nm fluorescence ratio of the Ca2+ -sensitive dye, Fura-2, as an index of [Ca2+Ji. In resting cells this ratio averaged 0.13:':0.3 (N=198), corresponding to [Cah)i of about 35 nM. Exposure to carbachol (100 /-LM) caused a rapid rise in the fluorescence ratio in 96% of cells to 0.41 :':0.08, corresponding to [Cah)i of about 210 nM. This initial peak was followed by a gradual decline. A similar response to carbachol was seen when the bath was made Ca2+-free (N=23), demonstrating that the carbachol-induced rise is caused by the release of Ca2+ from intracellular stores and not Ca 2+ influx, as we found in T84 cells. These results demonstrate that the signalling mechanisms controlling Ca2+ in NCM460 cells in response to cholinergic stimulation are similar to the mechanisms in other secretory cells. However, since Ca2+ activates Cl channels in NCM460 cells but not in T84 cells, the NCM460 cell line may be a better model than tumor-derived cell lines for studying the mechanisms of diarrheas produced by active cr secretion.
3125 PERMEABLITY OF PROXIMAL AND DISTAL COLON CRYPT AND SURFACE EPITHELIUM TO HYDROPIllLIC MOLECULES. Mats Jodal, Britt-Marie Fihn, Ying Sun, Dept of Physiology, Gothenburg , Sweden. Aim. The colon epithelium is generally considered to be a moderately tight epithelium with a very low permeability to hydrophilic substances . Morphologically there is a difference in complexity of the zonulae occludentes within the crypt and surface epithelium but its functional implications are unknown. The aim of this study was therefore to investigate the sizes of the aqueous pores of different parts of the colonic epithelium. Methods. In anaesthetized Sprague Dawley rats proximal and distal colon were perfused for 4 hours in a recirculating system with a modified Kreb' s Henseleit solution. The appearance of the radio labelled probes Cr-EDTA, mannitol and urea (mol radii 5.25, 3.25 and 2.3 A respectively) into the perfusate from the plasma as well as there disappearance from the luminal perfusate into the intestinal tissue were measured and expressed as clearance. The localization of the fluorescence probes Evan blue labelled albumin and FITC-dextran 4000 along the colon epithelium was also investigated . Results. In the lumen to plasma experiments the proximal and distal colon segments exhibited a net fluid absorption as well a significant and similar disappearance rate of urea while the clearance of mannitol was not significantly different from zero. In the plasma to lumen experiments there was a significant appearance rate of Cr-EDT A, mannitol and urea in the proximal colon , the order of magnitude being urea > mannitol > Cr-EDTA. In the distal colon only the clearance of mannitol and urea were significant and lower than in the proximal colon. The clearance ratio mannitol/urea was around 0.23 in both colonic segments and the ratio Cr-EDTNmannitol in the proximal colon 0.71. Both fluorescence probes were found along the surface epithelium after 2 h luminal incubation in the both segments while no fluorescence was present in the crypt lumina . Conclusions. The results indicate that the surface epithelium of both colonic segments contain a single type of pore (radius about 3 -3.5 A) while the crypt epithelium , particularly in the proximal colon, is a heteroporou s membrane probably containing at least two pores, one large (radius above 35-40) and one small A (radius 4-5 A). Furthermore, in vivo, substance s dissolved in a fluid solution do not have access to the crypt lumina and can therefore not be absorbed via the crypt epithelium .
3127 NOVEL METHOD FOR THE STUDY OF HUMAN EPITHELIAL ION TRANSPORT. Rune Larsen, Anette Mertz-Nielsen, Mark Berner Hansen, Steen Seier Poulsen, Niels Bindslev, Dept of Medicine CIF, Glostrup Hosp, Glostrup, Denmark ; Dept of Surg D, Glostrup Hosp, Glostrup, Denmark; Dept of Anatomy, Univ of Copenhagen, Copenhagen, Denmark ; Dept of Med Physiology, Univ of Copenhagen, Copenhagen, Denmark. Introduction. The study of epithelial ion transport has great importance for our understanding of the GI tract in health and disease. The Ussing chamber has been used for this purpose, but due to technical difficulties and limitations with the available small epithelial biopsies, these investigations in humans have been restricted. The aim of this study was to developed an Ussing chamber setup for the available biopsies taken at endoscopy . Methods. Routine biopsies were taken from the duodenum during endoscopy of patients with dyspepsia. Modified Ussing chambers were constructed, exposing an area of 5 mrrr'. Biopsies were mounted in these mini-chambers by continous air-suction in an atraumatic manner, verified by histological procedures . Furthermore , a clamp unit and software program were developed allowing automatic measurements of short circuit current (SCC, resolution 25 nA) , voltage (PD, resolution 50 p,V) and conductance (G, resolution> 12.5 p,S), all with a time resolution of2 sec. To determine basic functional parameters, biopsies were examined for viability by measuring electrogenic absorption with d-glucose and electrogenic secretion with acetylcholine (ACh). Results. Basal SCC and G were in the range of20-70 p,Ncm2 and 30-60 mS/cm2 (n=32), respectively. The biopsies were fully functional for up to 8 hrs to mucosally applied dglucose (Jrnax about 400 p,Ncm 2 and Kd around 10 nM, n=32) and serosally applied ACh (Jmax about 200 p,Ncm2 and Kd around 35 /-LM, n=20). We conclude, that this mini-Ussing air-suction (MUAS) chamber is a promising and reliable novel gadget for functional characterization of electrogenic ion transport in human (duodenal) epithelial biopsies.