Diarrhea is almost always associated with changes in. HCO 3 Secretion in the Rat Colonic Crypt Is Closely Linked to Cl Secretion

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1 GASTROENTEROLOGY 2000;118: HCO 3 Secretion in the Rat Colonic Crypt Is Closely Linked to Cl Secretion JOHN P. GEIBEL,*, SATISH SINGH,, VAZHAIKKURICHI M. RAJENDRAN, and HENRY J. BINDER, Departments of *Surgery, Internal Medicine, and Cellular and Molecular Physiology, Yale University, New Haven, Connecticut Background & Aims: The mechanism of colonic HCO secretion has not been established largely because of a lack of experimental methods for its detailed study. The present studies were designed to establish whether the isolated, perfused crypt of the rat distal colon is an excellent model to study HCO 3 movement and the mechanism of colonic HCO 3 secretion. Methods: HCO 3 secretion was determined in isolated, microperfused crypts by measuring [HCO 3 ] by microcalorimetry on nanoliter samples. Results: Net HCO 3 absorption was observed during lumen and bath perfusion with an HCO 3 -Ringer solution. Vasoactive intestinal polypeptide (60 nmol/l), acetylcholine (100 nmol/l), or dibutyryl adenosine 3,5 -cyclic monophosphate (DBcAMP, 0.5 mmol/l) induced active HCO 3 secretion that required bath but not lumen HCO 3 /CO 2. DBcAMPstimulated HCO 3 secretion was not affected by acetazolamide, an inhibitor of carbonic anhydrase. Removal of lumen Cl did not alter DBcAMP-stimulated HCO 3 secretion but reduced fluid secretion. DBcAMPstimulated HCO 3 secretion was closely linked to active Cl secretion because HCO 3 secretion was substantially reduced by removal of bath Cl, by addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl secretion, and by addition of lumen NPPB, a Cl channel inhibitor. Conclusions: These studies establish that colonic crypt HCO 3 secretion (1) is not a result of an apical membrane Cl - HCO 3 exchange, (2) is tightly associated with Cl secretion, and (3) primarily occurs via an apical membrane Cl channel. Diarrhea is almost always associated with changes in fluid and electrolyte movement because several agonists, e.g., cholera enterotoxin and vasoactive intestinal polypeptide (VIP), 1 induce fluid secretion secondary to a stimulation of active Cl secretion. 1,2 Active Cl secretion has been extensively studied in both in vivo and in vitro studies using both native intestinal epithelia, e.g., rabbit ileum, and intestinal cell lines, e.g., T 84 cells. 2 4 These latter studies have elegantly delineated the mechanism and regulation of active Cl secretion that includes the involvement of ion transport processes at both the apical and basolateral membranes. Thus, Na - K -2Cl cotransport, one or more K channels, and Na,K -adenosine triphosphatase (ATPase) at the basolateral membrane have been implicated in stimulation of active Cl secretion, while an apical membrane Cl channel is required. 1,4 Several observations indicate that bicarbonate secretion is also important in both normal individuals and those with diarrhea. First, stool water in most diarrheal disorders frequently has a high bicarbonate concentration, whereas the metabolic acidosis observed in some patients with severe diarrhea is caused by sustained stool bicarbonate losses. 5 Second, Cl -dependent bicarbonate secretion is an accepted transport process in the normal mammalian large intestine, including the human colon. 6 8 Third, bicarbonate concentrations are often high in experimentally induced fluid secretion in in vivo studies. 9 In contrast, the in vitro studies that have successfully dissected the mechanisms of active Cl secretion rarely provide evidence of bicarbonate secretion. An adequate explanation for the inability to observe bicarbonate secretion in in vitro studies has not evolved. The generally accepted model of absorptive and secretory processes in the large and small intestine is that absorptive processes are primarily located in surface/ villous epithelial cells, whereas secretory processes are in crypt epithelial cells. 10,11 We recently established methods to isolate and to perfuse individual colonic crypts using microperfusion methodology that has been successfully used to study renal tubules for more than 25 years. 12,13 These recent studies in the rat distal colon established that Na -dependent fluid absorption is the constitutive transport process in the basal state, whereas fluid secretion was regulated by one or more neurohumoral agonists, probably released from lamina propria cells. 12 As renal physiologists have developed methods to Abbreviations used in this paper: ACh, acetylcholine; ACZ, acetazolamide; DBcAMP, dibutyryl adenosine 3,5 -cyclic monophosphate; DIDS, 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid; CFTR, cystic fibrosis transmembrane conductance regulator; JHCO, net HCO movement; Jv, net fluid movement; NMDG, N-methyl-Dglucamine-Cl ; NPPB, 5 -nitro-2-(3-phenylproplyamino)-benzoic acid by the American Gastroenterological Association /00/$10.00

2 102 GEIBEL ET AL. GASTROENTEROLOGY Vol. 118, No. 1 measure [HCO 3 ] in nanoliter samples, 13 the isolated, perfused colonic crypt provided an opportunity to study bicarbonate movement in vitro. The present study shows that bicarbonate absorption is present in the basal state in these isolated crypts but that active bicarbonate secretion is induced by both VIP and acetylcholine (ACh). These studies also establish the cellular mechanism of dibutyryl adenosine 3,5 -cyclic monophosphate (DBcAMP)- induced bicarbonate secretion and identify a close relationship between active bicarbonate and active Cl secretion. Materials and Methods Nonfasting Sprague Dawley rats weighing g were used in all experiments. Approval for these studies was provided by the Yale Animal Care and Use Committee. After the rats were dead, individual crypts were obtained from the distal colon by hand dissection. Net fluid movement ( Jv) was determined using the microperfusion methods previously described in detail for colonic crypts, renal tubules, and gastric glands Briefly, single, hand-dissected crypts were placed on a temperature-controlled chamber on the stage of an inverted microscope. An assembly of concentric glass micropipettes was used to hold the blind end of the crypt. The perfusion pipette was used to puncture this blind end and introduce the perfusate containing methoxy 3 H-inulin into the crypt lumen in an antegrade direction. A second set of micropipettes was used to cannulate the open end of the crypt and collect the effluent. After cannulation, lumen and bath solutions flowed continuously at 10 and 5 nl/min, respectively. The effluent was sampled with a volume-calibrated pipette. Jv was determined from the length and diameter of the crypt, the rate at which the effluent accumulated in the collection pipette and from the concentration of methoxy 3 H-inulin in the perfusate and effluent. Methoxy 3 H-inulin concentration was determined in a scintillation counter. Experiments were discarded whenever bath methoxy 3 H-inulin concentration exceeded background. Jv was expressed as nl mm 1 min 1. HCO 3 movement was determined from Jv and [HCO 3 ]in both perfusate and effluent. [HCO 3 ] was measured in nanoliter samples using microcalorimetry 15 (Picapnotherm; World Precision Instruments, Sarasota, FL) that has been previously standardized in this laboratory. 16 Net HCO 3 movement (JHCO 3 ) is expressed as pmol mm 1 min 1. Each data point represents the average of three 5-minute collections of effluent. At least 5 crypts were studied in each experiment. At the end of each experiment, the viability of the crypt was assessed with trypan blue and experiments in which cells failed to exclude dye were discarded; fewer than 15% of crypts were discarded. Positive values represent net absorption, and negative ones net secretion. The composition of the standard Ringer solution (in mmol/l) was NaCl, 125; KCl, 5; CaCl 2, 1.2; MgSO 4, 1.2; NaH 2 PO 4,2; NaHCO 3, 22; and glucose In the Cl -free experiments, Cl was replaced by gluconate or cyclomate; in the Na -free studies, N-methyl-D-glucamine-Cl (NMDG) was used to replace Na. The solution was equilibrated with 5% CO 2 and adjusted to an osmolality of mosm/kg. In the nominally CO 2 /HCO 3 -free solutions, 32 mmol/l HEPES replaced 22 mmol/l NaHCO 3 and the solution was air equilibrated. The ph of all solutions was 7.40 at 37 C. Results Basal Movement Perfusion of both the lumen and bath of isolated crypts with an HCO 3 -Ringer solution was associated with both net fluid absorption, as previously reported, 12 and net bicarbonate absorption (Figure 1). The HCO concentration of the perfusate was 22 mmol/l, and that of the effluent was 21.6 mmol/l. Perfusion of lumen and bath with a nominally HCO 3 /CO 2 -free solution reduced net bicarbonate absorption to zero. In contrast, perfusion of only the lumen with a nominally HCO 3 /CO 2 -free solution resulted in net HCO 3 secretion. In the absence of lumen Cl during perfusion with HCO 3 -Ringer solution, net bicarbonate secretion ( pmol mm 1 min 1 ) was also observed. The substitution of NMDG for Na in the lumen solution resulted in a significant reduction both in net fluid absorption, as previously reported, 12 and in net HCO 3 absorption (from to pmol mm 1 min 1 ). Agonist-Induced Bicarbonate Secretion The addition of 60 nmol/l VIP to the bath solution was associated with an increase in [HCO 3 ]in the effluent to mmol/l with a rate of bicarbonate secretion of pmol mm 1 min 1 (Figure 1). Because the concentration of bicarbonate in the perfusate solutions of both lumen and bath was 22 mmol/l, these studies establish that VIP stimulated active bicarbonate secretion. As reported previously, 12 VIP also reversed net fluid absorption to net fluid secretion. Figure 1. Effect of several secretogogues on net bicarbonate movement in isolated, perfused colonic crypt. After completion of a control period ( ), either 0.5 mmol/l DBcAMP (4), 60 nmol/l VIP ( ), or 100 nmol/l ACh (f) was added to the bath solution. Positive numbers represent net absorption, negative ones net secretion.

3 January 2000 COLONIC CRYPT HCO 3 SECRETION 103 Because ACh, a neurotransmitter that increases intracellular Ca 2, also induces active Cl secretion in in vitro studies with both intact colonic mucosa and T 84 cells, 17,18 the effect of 100 nmol/l ACh on bicarbonate movement was also assessed. In the presence of CO 2 /HCO 3 in both lumen and bath, addition of ACh to the bath solution resulted in stimulation of bicarbonate secretion ( pmol mm 1 min 1 ) (Figure 1) and, as previously reported, 12 net fluid secretion. The bicarbonate concentration in the effluent during the ACh perfusion was mmol/l, providing evidence that ACh also stimulated active bicarbonate secretion. Because VIP activates adenylate cyclase and increases mucosal camp, experiments were also performed in which 1 mmol/l DBcAMP was added to the bath solution. In these studies net bicarbonate absorption ( pmol mm 1 min 1 ) was observed during the control period (Figure 2B). Addition of DBcAMP to the bath solution in the presence of both HCO 3 and CO 2 resulted in stimulation of bicarbonate and fluid secretion ( pmol mm 1 min 1 and nl mm 1 min 1, respectively) (Figure 2B). Subsequent experiments to determine the cellular Figure 2. Bicarbonate movement in isolated, perfused colonic crypt. After completion of a control period ( ), (A) 60 nmol/l VIP ( )or (B) 0.5 mmol/l DBcAMP (4) was added to the bath solution. The experiments indicated as 22 mmol/l HCO 3 contained 22 mmol/l HCO 3 with 5% CO 2 ; those indicated as 0 mmol/l HEPES contained 32 mmol/l HEPES-replaced HCO 3. Positive numbers represent net absorption, negative ones net secretion. Numbers in parentheses represent numbers of crypts studied in each experiment. mechanism of bicarbonate secretion were performed in the presence of 1 mmol/l DBcAMP in the bath solution. Role of Bicarbonate Experiments were also performed to determine whether DBcAMP-induced transepithelial HCO 3 movement required either lumen or bath HCO 3 or HCO that had been generated intracellularly by carbonic anhydrase. In these experiments the effect of acetazolamide (ACZ) and the removal of HCO 3 /CO 2 on net fluid movement and net bicarbonate movement was established. Neither 1 µmol/l nor 1 mmol/l ACZ altered either DBcAMP-stimulated fluid secretion or DBcAMPstimulated HCO 3 secretion (data not shown). This observation suggests that HCO 3 endogenously generated via carbonic anhydrase has, at best, a minimal role in cyclic nucleotide stimulated bicarbonate secretion. The importance of HCO 3 /CO 2 in the bath and lumen solutions on DBcAMP-stimulated and VIP-stimulated HCO 3 and fluid secretion was established in experiments in which HCO 3 and CO 2 were nominally absent in both the lumen and bath solutions or in only the lumen solution (Figure 2A and B). In the nominal absence of HCO 3 /CO 2 from both lumen and bath, net fluid absorption was approximately 25% less than that observed in the presence of HCO 3 /CO 2 ( vs nl mm 1 min 1 ; P 0.01) (Figure 3). Net HCO 3 movement was zero in this experiment (Figure 2B). Although both DBcAMP and VIP reversed fluid absorption to fluid secretion in the nominal absence of HCO 3 /CO 2 (Figure 3), the rate of either DBcAMPinduced or VIP-induced HCO 3 secretion was minimal (Figure 2). The secretion that was observed possibly reflects the transport of HCO 3 already present in the air-equilibrated solutions. The effect of both DBcAMP and VIP on fluid movement and on HCO 3 movement in the nominal absence of HCO 3 /CO 2 in the lumen solution (but with HCO 3 /CO 2 present in the bath solution) was also examined (Figures 2 and 3). In resting conditions, net fluid absorption was not affected by the nominal absence of HCO 3 /CO 2 from the lumen solution. In contrast, in experiments in which HCO 3 /CO 2 was present only in the bath solution, the crypts secreted small amount of HCO 3, possibly reflecting a steep bath-to-lumen HCO gradient (Figure 2). The rate of both fluid secretion and HCO 3 secretion stimulated by DBcAMP or by VIP was identical when HCO 3 /CO 2 was present in both lumen and bath solutions or only in the bath solution (Figures 2 and 3). Thus, the nominal absence of HCO 3 /CO 2 only from the lumen is not required for either DBcAMPinduced or VIP-induced fluid secretion. DBcAMP-

4 104 GEIBEL ET AL. GASTROENTEROLOGY Vol. 118, No. 1 Figure 3. Effect of HCO 3 /CO 2 on net fluid movement in isolated, perfused colonic crypt. The experiments indicated as 22 mmol/l HCO 3 contained 22 mmol/l HCO 3 with 5% CO 2 ; in those indicated as 0 mmol/l HCO 3, 32 mmol/l HEPES replaced 22 mmol/l HCO 3. In the top panel, 0.5 mmol/l DBcAMP (4) was added to bath solution; in the bottom panel, 100 nmol/l VIP ( ) was added to bath solution., Control. Positive numbers represent net absorption, negative ones net secretion. Numbers in parentheses represent numbers of crypts studied in each experimental group. induced and VIP-induced HCO 3 secretion requires the presence of HCO 3 /CO 2 in the bath solution. Role of Chloride Most existing models of HCO 3 secretion include either an apical membrane Cl -HCO 3 exchange or an apical membrane Cl -HCO 3 exchange coupled to an apical membrane Cl channel. 6 8,19 To determine whether DBcAMP-stimulated HCO 3 secretion in the colonic crypt is coupled to Cl absorption or to Cl secretion, experiments were performed in which either lumen Cl or bath Cl was selectively replaced by cyclomate. The absence of lumen Cl modestly reduced DBcAMPstimulated net fluid secretion ( nl mm 1 min 1 ) but did not alter HCO 3 secretion ( pmol mm 1 min 1 ) (Figure 4). In limited studies the same results were obtained using gluconate to replace Cl (data not presented). This observation suggests that an apical membrane Cl -HCO 3 exchange is not solely required for camp-induced active bicarbonate secretion and is consistent with recent studies of the spatial distribution Cl -anion exchange in apical membranes isolated from surface and crypt cells. 20 Although separate and distinct Cl -HCO 3 and Cl -OH exchanges are present in apical membranes of surface cells, only a Cl -OH exchange was identified in crypt apical membranes. 20 In contrast to the effect of the removal of Cl from the lumen solution, the removal of Cl from the bath solution completely inhibited DBcAMP-stimulated HCO 3 secretion. In these experiments, HCO 3 secretion was effectively abolished ( pmol mm 1 min 1 ). As expected, the presence of a Cl -free Ringer s solution in the bath resulted in inhibition of DBcAMPinduced fluid secretion ( nl mm 1 min 1 ) (Figure 4). This observation indicates a close association between Cl secretion and HCO 3 secretion at either the basolateral or apical membranes or both. Effect of Transport Inhibitors The effect of 3 inhibitors of Cl transport was determined in another series of experiments that included 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid (DIDS), an inhibitor of anion exchangers; bumetanide, an inhibitor of basolateral Na -K -2Cl cotransport; and 5 -nitro- 2-(3-phenylproplyamino)-benzoic acid (NPPB), an inhibitor of apical Cl channels. Lumen perfusion of the crypt with 100 µmol/l DIDS in the presence of DBcAMP in the bath solution resulted in a 40% decrease in crypt HCO 3 secretion (Figure 5). Net fluid secretion was modestly (but not significantly) reduced in this experiment. Because only Cl -OH exchange and not Cl - HCO 3 exchange is present in the apical membranes of Figure 4. Role of lumen and bath Cl on DBcAMP-induced net fluid and bicarbonate secretion in isolated, perfused colonic crypt. After completion of a control period ( ) with a lumen solution containing mmol/l Cl, 0.5 mmol/l DBcAMP (4) was added to bath solution; Cl was replaced by gluconate in both the lumen Cl ( ) and bath Cl (f) experiments. Positive numbers represent net absorption, negative ones net secretion. Numbers in parentheses represent numbers of crypts studied in each experiment.

5 January 2000 COLONIC CRYPT HCO 3 SECRETION 105 Figure 5. Effect of transport inhibitors on HCO and fluid movement. Fluid and HCO secretion was stimulated by bath 0.5 mmol/l DBcAMP ( ). In the lumen DIDS experiment (4), 100 µmol/l DIDS was added to lumen; in the bath bumetanide experiment ( ), 100 nmol/l bumetanide was added to the bath solution; and in the lumen NPPB experiment (f), 10 µmol/l NPPB was added to lumen solution. Positive numbers represent net absorption, negative numbers net secretion. Numbers in parentheses represent numbers of crypts studied in each experiment. crypt cells, 20 these results suggest that a small fraction of HCO 3 secretion may be mediated by apical membrane Cl -OH exchange. To explore further the relationship between Cl and bicarbonate secretion, the effect of bumetanide and NPPB, in the presence of bath Cl, was determined (Figure 5). Similar to the effect of the removal of Cl from the bath solution, the addition of 50 µmol/l bumetanide, an Na -K -2Cl cotransport inhibitor, to the bath solution reversed DBcAMP-induced fluid secretion to net fluid absorption ( nl mm 1 min 1 ). DBcAMP-induced HCO 3 secretion was also inhibited by bumetanide in the bath solution. In parallel studies fluid and bicarbonate movement was also assessed in the presence of a Cl channel blocker, NPPB, in the lumen solution. Similar to the bumetanide results, 10 µmol/l NPPB inhibited DBcAMP-induced fluid secretion ( nl mm 1 min 1 ) and markedly reduced DBcAMP-induced HCO 3 secretion ( pmol mm 1 min 1 ). Together, these experiments indicate a close and critical relationship between active Cl secretion and active HCO 3 secretion; that is, active Cl secretion is required for HCO 3 secretion. Discussion These experiments examined the mechanism of HCO 3 secretion in the crypt of the rat distal colon. HCO 3 secretion has been studied in the small and large intestine intermittently during the past 3 decades by various methods. 6,8,21 27 During the past few years most studies of intestinal HCO 3 secretion have focused on HCO 3 movement in the proximal duodenum where the mucosa is exposed to a large acid load from the stomach. 25,26 Almost all prior studies have determined HCO movement via ph stat methods, calculation from ph measurements, or inferred from determination of the so-called residual flux In the present study total CO 2 concentrations were measured in infusate and effluent samples with the microcalorimetry method using a Picapnotherm that has a sensitivity of mmol/l for a 10-nL sample. This method has been previously used and validated in renal tubular studies 15,16 but has not been used in studies of HCO 3 movement in either the small or large intestine. Most in vitro studies of HCO 3 movement in the small or large intestine have studied sheets of intestinal mucosa mounted between Lucite chambers under voltage clamp conditions and have determined HCO 3 movement by ph stat In such preparations, HCO movement in both surface (or villous) and crypt epithelial cells are studied simultaneously. The present study used a recently developed method to isolate and perfuse individual crypts from the rat distal colon. 12 Although this model is devoid of neural and vascular input, the epithelium is intact and employs microperfusion techniques adapted from renal tubules. 13,16 This general methodological approach has also been used to study gastric glands. 14 The open end of the isolated crypts is cannulated, and the closed end is punctured with a micropipette. This method permits study of crypt cell function independent of that of surface cells, and HCO movement is studied under open circuit conditions. The general paradigm of fluid movement in the mammalian colon has been that absorptive processes are present in surface cells and secretory processes are exclusively located in crypt cells. 1,10 The initial studies of fluid movement in the isolated colonic crypt provided evidence that contradicted this concept: net fluid absorption was present in the basal state, whereas VIP and ACh induced fluid secretion. 12 As a consequence, these observations suggested that Na -dependent fluid absorption was the constitutive transport process in the crypt but that fluid secretion was regulated by one or more neurohumoral agonists. 12 Subsequent studies indicated that basal Na - dependent fluid absorption represented the sum of 2 different absorptive processes and a smaller secretory processes. These studies of fluid movement in isolated crypts provided the basis for these present studies of HCO 3 movement.

6 106 GEIBEL ET AL. GASTROENTEROLOGY Vol. 118, No. 1 Although lumen perfusion studies of the intact colon have shown HCO 3 secretion in the basal state, 6,8 these studies in the isolated crypt revealed net HCO 3 absorption when both the lumen and bath were perfused with an HCO 3 -Ringer solution in the basal state (Figure 1). Both VIP and ACh stimulated net HCO 3 secretion provided that HCO 3 /CO 2 was present in the bath solution (Figure 1 and 2A). Because the [HCO 3 ]inthe lumen in the presence of VIP and ACh was more than 30 meq/l, these observations indicate that active HCO secretion had been induced. These studies also provided evidence that HCO 3 secretion required the presence of HCO 3 /CO 2 in the bath solution because DBcAMPinduced HCO 3 secretion did not occur in the nominal absence of HCO 3 /CO 2 from the bath solution. Further, it is doubtful that HCO 3 is derived from endogenous CO 2 because ACZ, an inhibitor of carbonic anhydrase, did not affect DBcAMP-induced HCO 3 secretion. Most models of intestinal HCO 3 secretion have suggested a role for lumen Cl and the importance of an apical Cl -HCO 3 exchange. These present studies together with recent studies of apical membrane anion exchange 20 provide substantial evidence that HCO secretion does not primarily involve an apical membrane Cl -HCO 3 exchange. First, DBcAMP-induced HCO secretion was not reduced appreciably by the absence of Cl in the lumen perfusion solution (Figure 4). Second, recent studies with apical membrane vesicles prepared from crypt cells of rat distal colon failed to identify the presence of an apical membrane Cl -HCO 3 exchange, although a Cl -OH exchange was present in these membranes. 20 Third, the presence of DIDS in the lumen was associated with only a partial inhibition of DBcAMPinduced HCO 3 secretion (Figure 5), in contrast to the more profound inhibition of HCO 3 secretion produced by either bath bumetanide and lumen NPPB. Cl -OH exchange has a reduced affinity for HCO 3, and its K i for DIDS is substantially higher than that of Cl -HCO exchange. Because Cl -HCO 3 exchange is not present in apical membranes of crypt epithelial cells, it is likely that the portion of HCO 3 secretion that is inhibited by lumen DIDS (Figure 5) occurs via an apical Cl -OH exchange. These studies also establish that the source of HCO for agonist-induced HCO 3 secretion is extracellular and not related to intracellular HCO 3 production. Figure 2 demonstrates that neither VIP nor DBcAMP induced HCO 3 secretion in the absence of bath HCO 3. The presence of lumen HCO 3 alone was not sufficient to sustain agonist-induced HCO 3 secretion. Further, ACZ, a carbonic anhydrase inhibitor, also did not affect HCO secretion. Thus, these studies indicate that activation of transepithelial HCO 3 secretion requires its movement across both basolateral and apical membranes. These studies also establish a close association between Cl secretion and HCO 3 secretion and provide some suggestions regarding the mechanism of the movement of HCO 3 across apical and basolateral membranes. DBcAMP-induced active Cl secretion involves (1) the uptake of Cl across the basolateral membrane via Na,K -2Cl cotransport that is inhibited by bumetanide, (2) a basolateral K channel, (3) basolateral Na,K -ATPase, and (4) an apical Cl channel that is inhibited by NPPB. 2,4 When active Cl secretion (as measured by net fluid secretion) was inhibited either by the absence of bath Cl, by inhibition of its uptake across the basolateral membrane by bumetanide, or by blockade of the apical membrane Cl channel by NPPB, a Cl channel blocker, HCO 3 secretion was simultaneously inhibited (Figures 4 and 5). Two different but related explanations can explain these observations and suggest that HCO 3 movement across the apical and/or basolateral membranes is regulated by the cystic fibrosis transmembrane conductance regulator (CFTR). Shumaker et al. 28 recently provided evidence that CFTR and HCO secretion are linked in a human pancreatic duct cell line, CFPAC-1; their studies suggested that CFTR-driven Cl movement across the apical membrane results in an electrical potential that serves as the driving force for HCO 3 uptake across the basolateral membrane by NaHCO 3 cotransport. Such a model could explain the inhibition of HCO 3 secretion whenever active Cl secretion was blocked, because NaHCO 3 cotransport is present in basolateral membranes of rat distal colon. 29 An alternate possibility (that does not exclude the role of CFTR in basolateral HCO 3 uptake) is that HCO movement across the apical membrane is via CFTR. Support for this latter possibility was recently provided by the absence of HCO 3 secretion in proximal duodenum of CFTR knockout mice 25,26 and the demonstration of an HCO 3 conductance in a pancreatic cell line, PANC These studies are consistent with but do not provide unequivocal evidence for HCO 3 movement across the basolateral membrane via NaHCO 3 cotransport. In contrast, inhibition of HCO 3 secretion both by bath bumetanide and by the absence of bath Cl is the basis for the alternate speculation that the coupling of Na -K -2Cl cotransport with a basolateral Cl -HCO exchange could account for HCO 3 uptake. Alternatively, a decrease in CFTR s HCO 3 conductance when active Cl secretion is inhibited could explain the inhibition of HCO 3 secretion by bumetanide. These several possibilities can be tested experimentally.

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