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Effects of short chain fatty acids on colonic Na + absorption and enzyme activity

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Effects of short chain fatty acids on colonic Na + absorption and enzyme activity
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  Ž . Comparative Biochemistry and Physiology Part A 128 2001 335  347 Effects of short chain fatty acids on colonic Na  absorption and enzyme activity Valentin Zaharia a , Manuela Varzescu a , Ibrahim Djavadi a , Elliot Newman b ,Richard W. Egnor a , Jesline Alexander-Chacko a , Alan N. Charney a,  a  Department of Veterans Affairs Medical Center, Nephrology Section, New York Uni    ersity School of Medicine, New York, NY 10010, USA b  Department of Veterans Affairs Medical Center, Surgical Ser    ice, New York Uni    ersity School of Medicine, New York, NY 10010, USA Received 12 May 2000; received in revised form 17 October 2000; accepted 23 October 2000  Abstract Ž .    Short chain fatty acids SCFA stimulate colonic Na absorption and inhibit cAMP and cGMP-mediated Clsecretion. It is uncertain whether SCFA have equivalent effects on absorption and whether SCFA inhibition of Cl  secretion involves effects on mucosal enzymes. Unidirectional Na  fluxes were measured across stripped colonicsegments in the Ussing chamber. Enzyme activity was measured in cell fractions of scraped colonic mucosa. Mucosal 50mM acetate, propionate, butyrate and poorly metabolized isobutyrate stimulated proximal colon Na  absorption equally Ž . 300% . Neither 2-bromo-octanoate, an inhibitor of    -oxidation, nor carbonic anhydrase inhibition affected thisstimulation. All SCFA except acetate stimulated distal colon Na  absorption 200%. Only one SCFA affected proximal Ž . Ž . colon cGMP phosphodiesterase PDE 18% inhibition by 50 mM butyrate . All SCFA at 50 mM stimulated distal colon Ž . cAMP PDE 24  43% and decreased forskolin-stimulated mucosal cAMP content. None of the SCFA affectedforskolin-stimulated adenylyl cyclase in distal colon or ST -stimulated guanylyl cyclase in proximal colon. Na   K   - a Ž  ATPase in distal colon was inhibited 23  51% by the SCFA at 50 mM. We conclude that all SCFA except acetate in .   distal colon stimulate colonic Na absorption equally, and the mechanism does not involve mucosal SCFA metabolismor carbonic anhydrase. SCFA inhibition of cAMP-mediated secretion may involve SCFA stimulation of PDE andinhibition of Na   K   -ATPase.    2001 Elsevier Science Inc. All rights reserved.  Keywords:  Carbonic anhydrase inhibition; Human colon; Cl  secretion; Adenylyl cyclase; Guanylyl cyclase; Phosphodiesterase;Na   K   -ATPase  Corresponding author. Nephrology Section, VA Medical Center, 423 East 23 rd Street, New York, NY 10010, USA. Tel.:  1-212-263-6646; fax:   1-212-951-6842. Ž .  E-mail address:  alan.charney@med.va.gov A.N. Charney .1095-6433  01  $ - see front matter    2001 Elsevier Science Inc. All rights reserved. Ž . PII: S 1 0 9 5 - 6 4 3 3 0 0 0 0 3 1 8 - 4  ( )V. Zaharia et al.  Comparati    e Biochemistry and Physiology Part A 128 2001 335   347  336 1. Introduction The effects of various short chain fatty acids Ž .   SCFA on colonic Na absorption have beendescribed in several species including rats, rabbits Ž and humans Roediger and Moore, 1981; Binderand Mehta, 1989; Ramakrishna and Mathan, 1993; . Sellin and DeSoignie, 1998 . Although it is uncer-tain how SCFA enter the cell, whether in ex-change for HCO  or by non-ionic diffusion 3 Ž Bugaut, 1987; Mascolo et al., 1991; Charney et . al., 1998 , stimulation appears to involve an apicalmembrane Na   H  exchange process. There isgeneral agreement that this effect is rapid inonset, sizeable and reversible. What is uncertainis whether the various SCFA stimulate Na  ab-sorption equally. There are theoretical reasons why stimulation might differ including their rela-tive rates of absorption and propensity for mu- Ž cosal metabolism Bugaut, 1987; Halperin and . Kamel, 1996 . The importance of this informationrelates to the role of SCFA in the pathogenesisand treatment of fluid loss caused by diarrheal Ž disease Ramakrishna and Mathan, 1993; Ra- . makrishna et al., 2000 . In this regard, the totaland relative concentrations of the SCFA can beinfluenced by diet, the colonic flora and luminal Ž pH Halperin and Kamel, 1996; Velazquez et al., . 1996 .More recently, SCFA have been reported toreduce basal and 3  ,5  -cyclic adenosine Ž . monophosphate cAMP -mediated and 3  ,5  -cyclic Ž . guanosine monophosphate cGMP -mediated  Ž colonic Cl secretion Ramakrishna and Mathan,1993; Dagher et al., 1996b; Charney et al., 1999; . Krishnan et al., 1999 . Various SCFA have beencompared in this regard, and they appear to haveequal efficacy in inhibiting cGMP-mediated se- Ž . cretion in vitro Charney et al., 1999 but not in Ž .  vivo Ramakrishna et al., 1990 . By contrast, in-hibition of cAMP-mediated secretion appears toreflect the degree of cellular metabolism: bu-tyrate  acetate    propionate  isobutyrate Ž Bugaut, 1987; Dagher et al., 1996b; Ramakrishna . et al., 1990; Krishnan et al., 1999 . In addition,mucosal cAMP and cGMP concentrations werepartially reduced in association with a reductionin cholera toxin-induced and  E. coli  heat stable Ž . enterotoxin ST -induced secretion, respectively a Ž . Charney et al., 1999; Krishnan et al., 1999 . Thesefindings suggest that SCFAs may affect the enzy-matic production and  or degradation of cAMPand cGMP.SCFA also may affect intestinal electrolytetransport through alterations in Na   K   -ATPaseactivity. Although there are no data in the colon,this enzyme is inhibited by various SCFA in cell Ž systems as diverse as cultured MDCKcells Moyer . et al., 1999 , cultured rabbit renal proximaltubules Ž . Ž Tang et al., 1995 and rat cerebral cortex Wyse .    et al., 1998 . Na   K -ATPase is required forboth colonic Na  absorption and Cl  secretionby virtue of the transmembrane electro-chemicalgradients generated by its activity. Consideringthe effects of SCFA to stimulate Na  absorptionand inhibit Cl  secretion, it would be of interest Ž to determine whether this enzyme is inhibited or . stimulated by these agents.In the present study we systematically ex-amined whether the SCFA most plentiful in the Ž mammalian colon acetate, propionate and bu- . tyrate have differing effects on proximal anddistal colonic Na  absorption. We also de-termined whether SCFA metabolism or modula-tion of certain mucosal enzymes involved in in-     testinal absorption and secretion i.e. Na   K - Ž .  ATPase, carbonic anhydrase CA , adenylyl cy-clase, guanylyl cyclase and phosphodiesterase Ž .  PDE mediate their effects. The findings havepractical experimental as well as physiologic im-plications. For various reasons many studies of SCFA examine only a single SCFA, colonic seg-ment or secretagogue. Our results establish abasis for each of these choices. 2. Materials and methods  Approvals of the VA Subcommittee for AnimalStudies and the VA R&D Committee were ob- Ž tained. Male Sprague  Dawley rats  Rattus . Ž .  nor     egicus  250  350 g were maintained on astandard chow diet with free access to water. Ž   1 . Pentobarbital sodium 5 mg 100 g of body wt. was used for anesthesia. The proximal 6 cm ordistal 10 cm of colon was removed, rinsed with0.9% saline and prepared for either Na  or Cl  flux measurements or enzyme assays.Discarded human colon removed during colonsurgery at the VA Medical Center was studied.The specimens were not accepted if patientstested positive for HIV or hepatitis B or C infec-  ( )V. Zaharia et al.  Comparati    e Biochemistry and Physiology Part A 128 2001 335   347   337 tion, or if the tissue was abnormal on histologic Ž exam. Specimens were labeled proximal ascend- . Ž ing and transverse colon or distal descending . and sigmoid colon by the surgeon, kept in icedRinger solution and stripped of serosa within 30min of removal.  2.1. Ion flux measurements The methods used to measure ion flux were Ž previously described Goldfarb et al., 1988; . Dagher et al., 1992 . Briefly, resected, stripped ratand human colonic segments were studied undershort circuit conditions in modified Ussing half-chambers exposing a 1.12-cm 2 surface area. Tis- Ž . sue conductance  G  was calculated from periodicbipolar pulses of 0.5 mV. Tissues were paired forNa  flux studies when their values for  G  differed Ž . by less than 25%. The potential difference PD Ž .  was calculated from the short circuit current  I  sc divided by  G , and was referenced to the mucosalside.Unidirectional fluxes of Na   were measured by 22   Ž   1 adding 2   Ci Na 100 Ci  g specific activity; . New England Nuclear, Boston, MA to the mu-cosal side of one member of each tissue pair andthe serosal side of the other. Mucosal-to-serosal Ž . Ž . Ž  J   and serosal-to-mucosal  J   fluxes ex- ms sm  2   1 . pressed as   eq. cm h were measured for 16min after an initial 30-min equilibration. For eachnew steady state 12 min was allowed. Net flux, Ž .  J   , was calculated as  J     J   . In other tissues, net ms sm Ž . the unidirectional serosal to mucosal flux   J   of  sm   36   Ž Cl was measured by adding 1   Ci Cl 100Ci  g  1 specific activity; New England Nuclear, . Boston, MA to the serosal side of each tissue.Equilibration, steady state and flux periods weresimilar to the Na  flux protocols.Reagent grade chemicals were obtained from Ž . Sigma Chemical Co. St. Louis, MO unless oth-erwise indicated. HCO  -free Hepes Ringer con- 3 tained 99.2 mM Na  , 4 mM K   , 1.2 mM Ca 2  , 1mM Mg 2  , 93 mM Cl  , 2.4 mM HPO 2  , 0.4 mM 4 H PO 1  , 2.2 mM SO 2  , 10 mM glucose, and 5 2 4 4 mM Hepes. This solution was gassed with 100%O and titrated with 2 M H SO to a pH of 7.4. 2 2 4 Fluxes were also measured in HCO  -Ringer simi- 3 lar to Hepes Ringer except that NaCl was re-duced to allow for the substitution of 21 mMNaHCO for 5 mM Na  Hepes. This solution 3  was gassed with 5% CO   95% O to obtain a 2 2  P  CO  value of 35 mmHg and a pH value of 7.4. 2 Bathing solutions were maintained at 37  C, andpH and  P  CO  were measured with a Radiometer 2 BMS 3 Mk 2 system with a PHM 73 acid base Ž . analyzer The London Company, Cleveland, OH .In some experiments, Na  salts of various SC- Ž   FAs were added to the mucosal bath Na gluco- . nate to the serosal bath at a final concentrationof 50 mM. The effects of the membrane permeant Ž CA inhibitor methazolamide 0.1 mM added to . both bathing solutions and the relatively im- Ž permeant CA inhibitor benzolamide Presig et al., . Ž . 1987 0.1 mM added to the mucosal bath weretested in other experiments. In a third set of experiments, the effect of bilateral addition of  Ž   . 300   M 2-bromo-octanoate Na salt was ex-amined. A concentration of 60   M has beenshown to inhibit   - oxidation and the conversionof butyrate to   -hydroxybutyrate in perfused rat Ž . liver Bojes and Thurman, 1996 . Concentrationsup to 600   M inhibit   - oxidation and SCFA metabolism but do not alter basal levels of oxygen Ž . consumption Bojes and Thurman, 1996 . Finally,to examine whether SCFA inhibition of Na   K   -ATPase affected intestinal secretion,  J  Clsm  was measured after diutyryl cAMP was added tothe serosal bathing solution at a final concentra-tion of 1 mM. This was followed by the serosaladdition of ouabain at a final concentration of 0.05 mM or 0.1 mM. An ouabain concentration of 0.1 mM has been shown to cause 50% inhibitionof Na   K   -ATPase activity in rat distal colon asdetermined by enzyme assay and measurement of   I   in the presence of mucosal nystatin and serosal sc Ž . ouabain Pacha et al., 1991 .  2.2. Enzyme assays The mucosa of resected proximal and distal ratcolon was harvested by scraping with a glass slide. Ž  After addition of 1  2 ml of 50 mM tris hydroxy- . Ž . methyl aminomethane Tris buffer plus 5 mM Ž . Ž . MgCl pH 7.5 PDE assay or 50 mM Tris 2 buffer plus 0.5 mM 3-isobutyl-1-methylxanthine Ž . Ž . Ž IBMX pH 7.6 guanylyl and adenylyl cyclase . assays or 130 mM NaCl plus 5 mM EDTA plus30 mM imidazole HCl plus 2.4 mM Na  deoxy- Ž . Ž . cholate pH 7.4 ATPase assay the mucosa washomogenized using an iced glass cylinder andTeflon pestle. In some experiments the homo-genate was centrifuged at 100000   g   at 3  5  Cfor 60 min. The supernatant and pellet wereassayed for PDE activity, and guanylyl and adeny-  ( )V. Zaharia et al.  Comparati    e Biochemistry and Physiology Part A 128 2001 335   347  338 lyl cyclase activities, respectively. In other experi-ments the homogenate was centrifuged at 770   g  for 10 min and the resulting supernatant wascentrifuged at 10000   g   at 3  5  C for 10 min.The pellet was then assayed for Na   K   -ATPaseactivity.For all enzymes, linearity with assay time andprotein concentration were demonstrated, and as-says were performed in duplicate with appropri-ate blanks and vehicle controls in the presenceand absence of 50 mM acetate, propionate, bu- Ž   . tyrate or isobutyrate Na salt . Preincubation of SCFA with assay fractions for up to 10 min didnot alter the observed enzyme activities. A SCFA concentration of 50 mM was studied because thisconcentration reliably stimulates colonic Na  ab-  Ž sorption and inhibits Cl secretion Dagher et al., . 1996a,b; Charney et al., 1999 . Protein concentra-tion in all samples was determined by the Lowry Ž . method Lowry et al., 1951 . Pure  E. coli  ST a enterotoxin was a gift of Dr Ralph Giannella, U.of Cincinnati.  2.2.1. Cyclic GMP and cAMP PDE Mucosal samples obtained from proximal anddistal colon were used for the cGMP PDE andcAMP PDE assays, respectively. The 100-  l reac- Ž tion mixture contained tissue sample 5  32   g . Ž supernatant protein , substrate supplemented  3   .  with 0.1   Ci H -cGMP or cAMP , 50 mM Tris Ž . buffer pH 7.5 and 5 mM MgCl . Substrate con- 2 centration was chosen as 1 or 50   M cGMP to Ž . Ž . measure low  K   2.5   M or high  K   17   M m m cGMP PDE or as 1 or 50   M cAMP to measure Ž . Ž . low  K   5   M or high  K   52   M cAMP PDE m m Ž . Craven et al., 1983 . The reaction was started bythe addition of substrate, carried out at 37  C for10 min and terminated by immersion in boiling water. Substrate and product were separated bypolyacrylamide-boronate affinity gel chromato-graphy. The results were expressed as pmol 5  GMP or AMP mg  1 protein min  1 .  2.2.2. Adenylyl cyclase The 100000   g   pellet obtained from rat distalcolon was assayed for adenylyl cyclase activity.The 100-  l reaction mixture contained tissue Ž . sample 70  85   g pellet protein , 1 mM ATP, 50 Ž . mM Tris buffer pH 7.6 , 0.5 mM IBMX, 5 mM Ž MgCl and 0 or 1   M forskolin dissolved in 2 . dimethyl sulfoxide, final concentration 0.1% . Thereaction was started by the addition of sample,carried out at 37  C for 8 min and terminated byimmersion in boiling water. Following centrifuga-tion at 15000   g   for 3 min, cAMP in the super- Ž natant was measured by radioimmunoassay Bio-trak, Amersham Life Science, Arlington Heights, . IL . The results were expressed as pmol cAMPmg  1 protein min  1 .  2.2.3. Guanylyl cyclase The 100000   g   pellet obtained from rat proxi-mal colon was assayed for guanylyl cyclase activ-ity. The 100-  l reaction mixture contained tissue Ž . sample 70  100   g pellet protein , 1 mM GTP, Ž . 50 mM Tris buffer pH 7.6 , 0.5 mM IBMX, 5mM MgCl and 0 or 0.25   M ST . The reaction 2 a  was started by the addition of sample, carried outat 37  C for 5 min and terminated by addition of  Ž . 400   l of 50 mM sodium acetate pH 4 andimmersion in boiling water. Following centrifuga-tion at 15000   g   for 3 min, cGMP in the super- Ž natant was measured by radioimmunoassay Bio-trak, Amersham Life Science, Arlington Heights, . IL . The results were expressed as pmol cGMPmg  1 protein min  1 .  2.2.4. Na    K   -ATPase The 10000   g   pellet obtained from rat distalcolon was assayed for ATPase activity. The 1-ml Ž reaction mixture contained sample 7  30   g pel- . let protein , 5.4 mM ATP, 10 mM imidazole buf- Ž . fer pH 6.8 , 120 mM NaCl, 6 mM MgCl , and 0 2 Ž . or 20 mM KCl substituted for NaCl . The reac-tion was started by the addition of sample, carriedout at 37  C for 15 min and terminated by additionof 500   l of 27% trichloroacetic acid. Inorganicphosphate was measured by the method of Fiske Ž . and Subbarow Fiske and Subbarow, 1925 byspectrometry. The difference between the activityin the presence and absence of K    was con-sidered Na   K   -ATPase activity. The results were expressed as   mol Pi mg  1 protein h  1 .  2.3. Mucosal cAMP content The accumulation of cAMP was measured inmucosal scrapings of stripped rat distal colon.Colonic segments were incubated at 37  C for 60min in HCO  -free Hepes Ringer containing 149.2 3 mM Na  , 4 mM K   , 1.2 mM Ca 2  , 1 mM Mg 2  ,93 mM Cl  , 2.4 mM HPO 2  , 0.4 mM H PO 1  , 4 2 4 2.2 mM SO 2  , 10 mM glucose, 5 mM Hepes and 4 either 50 mM gluconate or a SCFA. This solution  ( )V. Zaharia et al.  Comparati    e Biochemistry and Physiology Part A 128 2001 335   347   339  was gassed with 100% O and titrated with 2 M 2 H SO to a pH of 7.4. In some tissues, after 40 2 4 min 10   M forskolin was added. After an additio-nal 20 min the segments were opened along themesenteric border, blotted with filter paper andthe mucosa scraped with a glass slide. The mu-cosa was then homogenized in twice its weight of  Ž . 50 mM Tris  HCl pH 7.5 containing 4 mMEDTA in a Teflon-glass homogenizer. After analiquot was removed for protein assay, the homo-genate was placed in a boiling water bath for 1min, and centrifuged at 1650   g   for 10 min. Ž .  Aliquots 50   l of supernatant were assayed for Ž cAMP by a competitive binding assay Biotrak, .  Amersham Life Science, Arlington Heights, IL .The results were expressed as pmol mg  1 protein.  2.4. Statistics Data were expressed as means  S.E. and ana-lyzed by paired or unpaired Student’s  t- test or Ž . analysis of variance ANOVA . Two-tailed  P   val-ues   0.05 were considered significant. 3. Results  3.1. Na   flux measurements 3.1.1. Proximal colon  As shown in Table 1, in rat proximal colon alow level of net Na  absorption was present inHepes Ringer associated with a serosa positivePD. These flux levels were similar to previous Ž studies of this tissue under these conditions Fos- . ter et al., 1986 . Acetate, propionate, butyrate andisobutyrate at 50 mM all stimulated  J   ,  J   ,  J   , ms sm net and  G , and reduced  I   and PD. The increments sc in  J   were approximately threefold. There were net no differences among the SCFA with regard totheir effects on these transport parameters by Ž .  ANOVA   F   0.63, NS . Ž The role of CA in colonic transport Charney . et al., 1986 suggested that this enzyme may berequired for the effects of SCFA. We tested forthis possibility by adding the CA inhibitor metha-zolamide to both bathing solutions. As shown inTable 2, CA inhibition did not affect basal colonicNa  absorption in Hepes Ringer in proximalcolon although  I   and PD were reduced. In sc addition, methazolamide did not affect the stimu-lation of Na  transport by 50 mM propionate orbutyrate. The effects of 50 mM propionate also were not inhibited by 0.1 mM benzolamide, arelatively membrane impermeant CA inhibitor Ž . data not shown .Table 3 shows that net Na  absorption and aserosa positive PD were present in human proxi-mal colon under basal conditions. Propionate andacetate at 50 mM each stimulated  J   and  J  ms sm and reduced PD. Increases in net Na  absorptionand  G , and decreases in  I   did not reach statisti- sc Table 1 a Effect of SCFA on electrolyte transport in rat proximal colon   I   PD  G  Na flux  sc  2   1   2 Ž . Ž . Ž .  eq. cm h MV mS cm  J J J  ms sm net  2   1   2   1   2   1 Ž . Ž . Ž .  eq. cm h   eq. cm h   eq. cm hControl 2.3  0.2 5.5  0.4 10.7  0.5 7.4  0.6 5.7  0.7 1.7  0.7   Ž .  Acetate 50 mM 1.7  0.1 3.1  0.5 16.6  2.4 13.5  0.5  n  7.5  0.5  n  6.0  0.5  Control 1.3  0.2 3.6  0.5 10.0  0.5 6.9  0.7 5.3  0.4 1.5  0.6   Ž . Propionate 50 mM 0.8  0.1 1.3  0.3   18.8  2.0  n  12.2  0.5  n  7.7  0.8   4.5  1.0Control 1.8  0.2 4.7  0.4 10.4  0.5 6.8  0.5 4.8  0.4 2.1  0.5 Ž . Butyrate 50 mM 1.0  0.1  n  1.4  0.1  n  20.0  1.3  n  15.6  0.9  n  8.3  1.0  n  7.4  0.7  n Control 1.9  0.1 5.5  0.4 9.4  0.7 5.3  0.6 4.4  0.8 0.9  1.3 Ž . Isobutyrate 50 mM 1.2  0.1   1.9  0.2   17.5  1.3  n  12.0  0.6  n  7.2  1.0   4.9  1.4  a Values are means  S.E. All tissues were studied in Hepes Ringer containing 5 mM Hepes at pH 7.4. Control  acetate,  n  5;control  propionate,  n  8; control  butyrate,  n  9; control  isobutyrate,  n  5.    P   0.05,    P   0.01,  nP   0.001 when compared tocontrol by paired Student’s  t- test.

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May 17, 2018
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