Resistant starch and SCFA: adjunct to ORS?
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Diarrhea↗
Biomedical subjects
H J Binder
Publications and source records attributed to H J Binder.
Differential localization of colonic H(+)-K(+)-ATPase isoforms in surface and crypt cells.
Two distinct colonic H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) isoforms can be identified in part on the basis of their sensitivity to ouabain. The colonic H(+)-K(+)-ATPase alpha-subunit (HKc alpha) was recently cloned, and its message and protein are present in surface (and the upper 20% of crypt) cells in the rat distal colon. These studies were performed to establish the spatial distribution of the ouabain-sensitive and ouabain-insensitive components of both H(+)-K(+)-ATPase activity in apical membranes prepared from surface and crypt cells and K(+)-dependent intracellular pH (pHi) recovery from an acid load both in isolated perfused colonic crypts and in surface epithelial cells. Whereas H(+)-K(+)-ATPase activity in apical membranes from surface cells was 46% ouabain sensitive, its activity in crypt apical membranes was 96% ouabain sensitive. Similarly, K(+)-dependent pHi recovery in isolated crypts was completely ouabain sensitive, whereas in surface cells K(+)-dependent pHi recovery was insensitive to ouabain. These studies provide compelling evidence that HKc alpha encodes the colonic ouabain-insensitive H(+)-K(+)-ATPase and that a colonic ouabain-sensitive H(+)-K(+)-ATPase isoform is present in colonic crypts and remains to be cloned and identified.
Animals↗
The pendulum of medical progress and intestinal fluid secretion: 1890-1997.
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Body Fluids↗
Novel transport properties of colonic crypt cells: fluid absorption and Cl-dependent Na-H exchange.
Colonic ion transport is heterogeneous including the long-accepted spatial separation of absorptive and secretory processes between surface and crypt cells. We recently described the isolation of individual crypts from the rat distal colon that were studied using microperfusion technology. Na-dependent fluid absorption was consistently demonstrated in these crypts during perfusion with a Ringer-like solution; dibutyryl cyclic AMP, VIP and acetylcholine, when added to the bath solution, all induced net fluid secretion. As several morphologic techniques, including immunocytochemistry, failed to provide evidence for the presence of myofibroblasts in the isolated crypt preparation, we propose that a Na-dependent absorptive process is a constitutive transport mechanism in crypt cells, while secretory processes are regulated by the release of one or more neurohumoral agonists from lamina propria cells including myofibroblasts. The mechanism of Na-dependent fluid movement was also studied by determining [H] gradient stimulation of 22Na uptake in isolated apical membrane vesicles (AMV) from crypt cells. In contrast to Na-H exchange in surface cell AMV, Na-H exchange in crypt cells is Cl-dependent. Intracellular pH determined in crypt cells using video-imaging fluorescence microscopy established that the response to an acid load requires both lumen Na and Cl. As a result, these studies have identified a novel Cl-dependent Na-H exchange in crypt AMV that may mediate apical membrane Na uptake and regulate pHi.
Animals↗
Regulation of colonic H-K-ATPase in large intestine and kidney by dietary Na depletion and dietary K depletion.
Active K absorption in the rat distal colon is energized by an apical membrane H-K-ATPase, whereas K absorption in the distal collecting duct is generally believed to be modulated by a related renal H-K-ATPase. Experiments were performed to establish the mechanism(s) by which dietary Na depletion (with resulting elevated aldosterone levels) and K depletion stimulate K absorption. A colonic H-K-ATPase-specific cDNA probe and a polyclonal antibody were utilized to measure mRNA (Northern blot analyses) and protein (Western blot and immunofluorescence studies) abundance in the distal and proximal colon and renal collecting ducts and cortex of dietary Na- and K-depleted rats. Dietary Na depletion, but not K depletion, upregulated H-K-ATPase-specific mRNA and protein expression in the distal and proximal colon; Na depletion also stimulated H-K-ATPase activity in the distal colon. In contrast to the distal colon, H-K-ATPase-specific protein level in the outer medulla was enhanced by dietary K depletion, but not by Na depletion. This study establishes that 1) dietary Na depletion stimulates colonic H-K-ATPase activity most likely by a transcriptional process and 2) the regulation of colonic H-K-ATPase expression by dietary Na depletion and dietary K depletion is not identical in the large intestine and differs in the kidney from the colon, suggesting the presence of two (or more) H-K-ATPase isoforms in the rat colon.
Animals↗
Passing the torch: a look back at our editorship.
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Gastroenterology↗
An apical permeability barrier to NH3/NH4+ in isolated, perfused colonic crypts.
Fermentation of nonabsorbed nutrients in the colon generates high concentrations of NH3/NH4+ in the colonic lumen. NH3 is a small, lipophilic neutral weak base that readily permeates almost all cell membranes, whereas its conjugate weak acid NH4+ generally crosses membranes much more slowly. It is not known how colonocytes maintain intracellular pH in the unusual acid-base environment of the colon, where permeant acid-base products of fermentation exist in high concentration. To address this issue, we hand dissected and perfused single, isolated crypts from rabbit proximal colon, adapting techniques from renal-tubule microperfusion. Crypt perfusion permits control of solutions at the apical (luminal) and basolateral (serosal) surfaces of crypt cells. We assessed apical- vs. basolateral-membrane transport of NH3/NH4+ by using fluorescent dyes and digital imaging to monitor intracellular pH of microvacuolated crypt cells as well as luminal pH. We found that, although the basolateral membranes have normal NH3/NH4+ permeability properties, there is no evidence for transport of either NH3 or NH4+ across the apical borders of these crypt cells. Disaggregating luminal mucus did not increase the transport of NH3/NH4+ across the apical border. We conclude that, compared to the basolateral membrane, the apical border of crypt colonocytes has a very low permeability-area product for NH3/NH4+. This barrier may represent an important adaptation for the survival of crypt cells in the environment of the colon.
Absorption↗
Chloride-dependent Na-H exchange. A novel mechanism of sodium transport in colonic crypts.
The mechanism of sodium movement across apical membrane of colonic crypt cells of rat distal colon was examined in studies of both 22Na uptake by apical membrane vesicles (AMV) and the rate of intracellular pH (pHi) recovery from an acid load by the addition of lumen sodium. In the presence of chloride but not in its absence, 22Na uptake in crypt AMV was stimulated by an outward gradient of either [H+] or [Na+]. 22Na uptake stimulated by an outward [Na+] gradient was also observed in the presence of other halides in the order of chloride > bromide > fluoride > iodide. pHi recovery from an acid load was both lumen sodium- and chloride-dependent, and the rate of pHi recovery by lumen sodium in the presence of chloride was 65-fold greater than that in the absence of chloride (dpH/dt is 655.4 and 10.2 in the presence and absence of chloride, respectively). One mM amiloride inhibited both [H+] gradient-stimulated 22Na uptake in the presence of chloride in crypt AMV (80%) and lumen sodium- and chloride-dependent pHi recovery in crypt cells (96%). [H+] gradient stimulation of 22Na uptake by crypt AMV in the presence of chloride was less sensitive to amiloride than amiloride inhibition of Na-H exchange in colonic surface AMV. These studies provide compelling evidence that a chloride-dependent Na-H exchange that is relatively amiloride-resistant is present in the apical membrane of colonic crypt cells. As prior studies have not identified a chloride-dependent Na-H exchange, the molecular and functional basis of this novel transport process is not known.
Amiloride↗
Diagnostic utility of contrast echocardiography and lung perfusion scan in patients with hepatopulmonary syndrome.
BACKGROUND & AIMS: Two modalities, contrast echocardiography and lung perfusion scan, are used to identify intrapulmonary vascular dilatation and diagnose hepatopulmonary syndrome (HPS), but a comparison of these two procedures has not been performed. The aim of this study was to compare the use of these diagnostic modalities in detecting intrapulmonary vascular dilatation and diagnosing HPS. METHODS: Forty consecutive outpatients with biopsy-proven cirrhosis had contrast echocardiography, a lung perfusion scan, and arterial blood gases analyzed. RESULTS: Fifteen of 40 cirrhotics (38%) had positive contrast echocardiogram results. Seven patients with positive echocardiogram results had gas exchange abnormalities and could be considered to have HPS (7 of 40 [17.5%]). Three of these patients were hypoxemic and had no concurrent cardiopulmonary disease, and each had positive contrast echocardiogram and lung perfusion scan results and were readily diagnosed as having HPS. The other 4 patients (3 hypoxemic and 1 normoxemic with an elevated alveolar-arterial gradient) had coexisting intrinsic lung disease and/or chest radiograph abnormalities complicating the diagnosis of HPS, and each had positive echocardiogram and negative lung scan results. The remaining 8 patients with positive echocardiogram results had normal lung scan and normal gas exchange results. No patient had positive lung scan and negative contrast echocardiogram results. CONCLUSIONS: Contrast echocardiography is the most useful screening test for intrapulmonary vasodilatation and may be positive more frequently than lung perfusion scans in patients with HPS.
Adult↗
Functional expression and segmental localization of rat colonic K-adenosine triphosphatase.
A putative cDNA for the colonic K-ATPase has recently been cloned (Crowson, M.S., and G. E. Shull. 1992. J. Biol. Chem. 267:13740-13748). Considerable evidence exists that there are two K-ATPases and active K absorptive processes in the rat distal colon: one that is ouabain sensitive and the other ouabain insensitive. The present study used the baculovirus expression system to express K-ATPase activity in insect Spodoptera frugiperda (Sf 9) cells and a polyclonal antibody (M-1), developed against a fusion protein produced from the 327 nucleotide fragment from 5' coding region of the putative K-ATPase cDNA, to identify the specific localization of the K-ATPase protein. K-ATPase activity (28.7 +/- 1.2 nmol inorganic phosphate/mg protein min) was expressed in plasma membranes isolated from Sf 9 cells infected with baculovirus containing recombinant DNA with the putative K-ATPase cDNA. Km for K for the K-ATPase was 1.2 mM. The expressed K-ATPase activity was not inhibited by ouabain (1 mM); while the Ki for vanadate inhibition was 8.3 microM. Western blot analysis with the M-1 antibody identified a 100-kD protein in apical membranes prepared from distal, but not proximal, rat colon. Immunohistochemical studies with M-1 antibody localized K-ATPase only in the apical membrane of surface cells, while an mAb (c464.6) against Na,K-ATPase localized basolateral membranes of both surface and crypt cells of rat distal colon. In conclusion, the putative K-ATPase cDNA encodes an ouabain-insensitive K-ATPase that is present only in the apical membrane of surface cells of rat distal colon.
Adenosine Triphosphatases↗
Fluid absorption in isolated perfused colonic crypts.
A spatial segregation of ion transport processes between crypt and surface epithelial cells is well-accepted and integrated into physiological and pathophysiological paradigms of small and large intestinal function: Absorptive processes are believed to be located in surface (and villous) cells, whereas secretory processes are believed to be present in crypt cells. Validation of this model requires direct determination of fluid movement in intestinal crypts. This study describes the adaptation of techniques from renal tubule microperfusion to hand-dissect and perfuse single, isolated crypts from rat distal colon to measure directly fluid movement. Morphologic analyses of the isolated crypt preparation revealed no extraepithelial cellular elements derived from the lamina propria, including myofibroblasts. In the basal state, crypts exhibited net fluid absorption (mean net fluid movement = 0.34 +/- 0.01 nl.mm-1.min-1), which was Na+ and partially HCO3- dependent. Addition of 1 mM dibutyryl-cyclic AMP, 60 nM vasoactive intestinal peptide, or 0.1 mM acetylcholine to the bath (serosal) solution reversibly induced net fluid secretion (net fluid movement approximately -0.35 +/- 0.01 nl.mm-1.min-1). These observations permit speculation that absorption is a constitutive transport function in crypt cells and that secretion by crypt cells is regulated by one or more neurohumoral agonists that are released in situ from lamina propria cells. The functional, intact polarized crypt described here that both absorbs and secretes will permit future studies that dissect the mechanisms that govern fluid and electrolyte movement in the colonic crypt.
Animals↗
Brief report: factitious diarrhea detected by measurement of stool osmolality.
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Adult↗
Differential modulation of Na-HCO3 cotransport and Na-H exchange by pH in basolateral membrane vesicles of rat distal colon.
This study was designed to investigate the function of intravesicular proton on two pH gradient-dependent transport processes, a novel Na-HCO3 cotransport and a Na-H exchange, that are present in basolateral membrane vesicles of rat distal colon. Increasing intravesicular proton concentration saturated 22Na uptake via both Na-H exchange and Na-HCO3 cotransport; reduced the apparent Km for sodium for Na-H exchange from 94.9 to 35.6 mM, without alteration in the Vmax; but enhanced the Vmax for Na-HCO3 cotransport from 4.3 to 11.7 nmol/mg protein.6 s, while not changing the Km for sodium. The effect of a 10-fold proton concentration gradient at two different absolute proton concentrations on both systems was also determined. 22Na uptake via Na-HCO3 cotransport, but not via Na-H exchange, was enhanced at the higher proton concentration, indicating that the magnitude of the proton concentration gradient is primarily responsible for proton stimulation of Na-H exchange, whereas the absolute proton concentration is critical for proton enhancement of Na-HCO3 cotransport. These studies also demonstrate saturation of both transport systems as a function of intravesicular proton concentration without an exponential component. These results indicate that proton stimulated Na-H exchange and Na-HCO3 cotransport are regulated by distinct and separate mechanisms that may reflect their different cellular functions.
Animals↗
Apical membrane Cl-butyrate exchange: mechanism of short chain fatty acid stimulation of active chloride absorption in rat distal colon.
The cellular model of short chain fatty acid stimulation of electroneutral Na-Cl absorption in large intestine proposes that SCFA, following its uptake across the apical membrane, recycles and is coupled to functional Na-H and Cl-short chain fatty acid exchanges. To establish the presence of a Cl-butyrate exchange (used as a model short chain fatty acid), studies of 36Cl and 14C-butyrate uptake across apical membrane vesicles of rat distal colon were performed. An outward butyrate-gradient stimulated transient accumulation of 36Cl uptake that was not inhibited by pH clamping with valinomycin (a K ionophore) and FCCP (a proton ionophore). Outward butyrate-gradient-stimulated 36Cl uptake was inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) with a half-maximal inhibitory concentration (IC50) of 68.4 microM, and was saturated by both increasing extravesicular Cl concentration (Km for Cl of 26.8 +/- 3.4 mM and a Vmax of 12.4 +/- 0.6 nmol/mg protein x 9 sec) and increasing intravesicular butyrate concentration (Km for butyrate of 5.9 mM and a Vmax for Cl of 5.9 nmol/mg protein x 9 sec). 36Cl uptake was also stimulated by outward gradients of other short chain fatty acids (e.g., propionate, acetate and formate). In contrast, an outward Cl gradient failed to enhance 14C-butyrate uptake. Extravesicular Cl more than extravesicular butyrate enhanced 36Cl efflux from apical membrane vesicles. These studies provide compelling evidence for the presence of an electroneutral, pH-activated, Cl-butyrate exchange which in concert with Na-H exchange is the mechanism by which butyrate stimulates electroneutral Na-Cl absorption.
Animals↗
Bicarbonate-stimulated [14C]butyrate uptake in basolateral membrane vesicles of rat distal colon.
BACKGROUND: The mechanism of short-chain fatty acid (SCFA) absorption by the colon is not known. The aim of these experiments was to identify the transport mechanisms present in the basolateral membrane to develop an overall model of colonic SCFA absorption. METHODS: These studies determined the uptake of [14C]butyrate (used as a model SCFA) by basolateral membrane vesicles prepared from rat distal colonic mucosa. RESULTS: Significantly higher [14C]butyrate uptake under an acidic environment (extravesicular pH [pHo] = intravesicular pH [pHi] = 5.5) than that under alkaline environment (pHo = pHi = 7.5) indicates the presence of nonionic diffusion. In the absence of a pH gradient (pHo/pHi = 7.5/7.5), outward gradients of bicarbonate significantly stimulated [14C]butyrate uptake. Additional presence of a pH gradient (pHo/pHi = 6.0/7.5) further enhanced the bicarbonate gradient-stimulated [14C]butyrate uptake that was not inhibited by voltage clamping but was inhibited substantially by an anion exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) (82%). Both increasing extravesicular butyrate concentration and intravesicular bicarbonate concentration saturated bicarbonate/pH gradient-stimulated [14C]butyrate uptake with an apparent Michaelis constant (Km) for butyrate of 6.9 mmol/L and an apparent Km for bicarbonate of 27.4 mmol/L. CONCLUSIONS: Butyrate uptake by basolateral membrane vesicles represents both nonionic diffusion and a carrier-mediated SCFA-bicarbonate exchange process that differs from the SCFA-bicarbonate exchange recently identified in apical membrane vesicles. Thus, two distinct carrier-mediated anion exchange processes located in apical and basolateral membranes mediate transcellular SCFA transport in colonocytes.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
A putative H(+)-K(+)-ATPase is selectively expressed in surface epithelial cells of rat distal colon.
Recently, a putative distal colon H(+)-K(+)-ATPase alpha-subunit has been identified and characterized (M. S. Crowson and G. E. Shull. J. Biol. Chem. 267:13740-13748, 1992). In the present study, we report the tissue and cell expression of this putative H(+)-K(+)-ATPase. The results indicate that, first, in the gut, the putative H(+)-K(+)-ATPase alpha-subunit is restricted to the distal part of the colon and is predominantly expressed in surface epithelial cells, in marked contrast to the alpha 1-subunit of Na(+)-K(+)-ATPase that is also expressed in glands. These data suggest that the H(+)-K(+)-ATPase alpha-subunit is a potential marker for terminal differentiation of distal colon. Second, in the uterus, the putative H(+)-K(+)-ATPase is restricted to the region of the myometrium between the inner and midmuscular zone that is very rich in vascular supply and nerve cells. This striking expression suggests that the H(+)-K(+)-ATPase may not be involved in the control of pH and potassium concentration of the uterine fluid but rather in distinct functions of vascular and/or nerve cells. Third, with the use of three independent and different approaches (Northern blot analysis, ribonuclease protection assay, and in situ hybridization), we were unable to detect any significant amount of H(+)-K(+)-ATPase transcripts in kidney tissue. Our data suggest that the putative distal colon H(+)-K(+)-ATPase is probably distinct from the kidney isoform. Finally, we report the sequence of a set of degenerate oligonucleotides that are useful to clone alpha-subunits of the Na(+)-K(+)-/H(+)-K(+)-ATPase gene family in different tissues and different species.
Animals↗
Inhibition of VIP-stimulated ion transport by a novel Y-receptor phenotype in rabbit distal colon.
Neurocrine, endocrine, and paracrine regulators are critical to the control of colonic secretion. These studies have investigated the inhibition of vasoactive intestinal polypeptide (VIP)-stimulated ion transport by peptide YY (PYY) and other Y-class effectors in rabbit distal colonic mucosa mounted in Ussing chambers. PYY decreased basal short-circuit current (Isc) but did not significantly change either basal Na+ or Cl- flux. PYY inhibited VIP-stimulated increases in Isc by up to 86% and abolished VIP-induced Cl- secretion. PYY decreased VIP-generated increases in Isc by a tetrodotoxin-insensitive mechanism. PYY inhibited cholera toxin-stimulated as well as forskolin-stimulated increases in Isc but failed to alter stimulation by 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP). PYY decreased VIP-stimulated increases in tissue cAMP by 88% and forskolin-stimulated increases by 84%. PYY, neuropeptide Y (NPY), (Leu31,Pro34)-NPY, and pancreatic polypeptide (PP) all demonstrated potent inhibition of VIP-stimulated increases in Isc. PYY-(13-36) demonstrated little effect on VIP stimulation. Thus the rabbit distal colon possesses a novel Y-class receptor phenotype that demonstrates high affinity for all three PP-fold peptides, NPY, PYY, and PP.
Animals↗
Cl-HCO3 and Cl-OH exchanges mediate Cl uptake in apical membrane vesicles of rat distal colon.
This study describes Cl-HCO3 and Cl-OH exchanges as the mechanism for Cl uptake by apical membrane vesicles (AMV) of rat distal colon. Although HCO3 gradient-stimulated 36Cl uptake was additionally stimulated by the additional presence of a pH gradient, pH gradient-stimulated 36Cl uptake was not further enhanced by a HCO3 gradient. HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake was not inhibited by voltage clamping, with K and its ionophore valinomycin, but was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor, with an apparent inhibitory constant of 7.8 and 106.0 microM, respectively. Increasing intravesicular OH concentration in the absence of HCO3 (with fixed extravesicular Cl concentration) yielded a sigmoidal curve for 36Cl uptake. In contrast, increasing intravesicular OH concentration in the presence of equimolar intra- and extravesicular HCO3 (25 mM) yielded a saturable hyperbolic curve. Increasing extravesicular Cl concentration saturated both HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake, with a kinetic constant for Cl of approximately 11.9 and 22.6 mM, respectively. We conclude that Cl uptake in AMV of rat distal colon occurs via two separate anion (Cl-HCO3 and Cl-OH) exchange processes. We speculate that one of these two anion exchanges may be responsible for transcellular Cl movement, while the other may be important in the regulation of intracellular pH homeostasis.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗