Ketogenic regulation by certain metabolites in rumen epithelium

In experiments with rumen epithelium incubated in vitro in the presence of butyrate, the ketogenic effect of glucose was shared by epimeric monosaccharides but not by non-metabolizable analogues. 14C from glucose was not incorporated into ketone bodies. Malate increased ketogenesis from butyrate and decreased its oxidation, pyruvate and NH4+ had the opposite effect, and malonate inhibited both processes. The ketogenic effect of glucose was also effective with isovalerate maintaining the high proportion of acetoacetate which is characteristic of this substrate. Rumen epithelium transformed added acetoacetate into 3-hydroxybutyrate. It is concluded that reducing equivalents produced from glucose and other metabolizable substrates are responsible regulators of ketogenesis from butyrate. The results are discussed in view of the functional role of ruminal ketogenesis.     

3-Hydroxybutyrate dehydrogenase in tissues from normal and ketonaemic sheep

1. In liver, rumen epithelium and kidney cortex of the sheep, a dehydrogenase active against dl-3-hydroxybutyrate occurred in both the cytosol and particulate fractions of the tissues. In brain, heart, skeletal and smooth muscles, the enzyme occurred only in the particulate fraction. 2. Enzyme activity in the cytoplasmic fraction of liver and rumen epithelium was similar with either d(-)-3-hydroxybutyrate or dl-3-hydroxbutyrate, but was less with acetoacetate as the substrate. The cytosol fraction of kidney cortex showed very little activity with d(-)-3-hydroxybutyrate, confirming that most of the activity with dl-3-hydroxybutyrate was with the l(+) isomer in this tissue. 3. 3-Hydroxybutyrate dehydrogenase activities in the cytosol and particulate fractions of liver, rumen epithelium and kidney cortex and in the particulate fraction of brain tissue were not stimulated by phosphatidylcholine, unlike the enzyme in sheep muscle and in tissues of other species. 4. The activity of 3-hydroxybutyrate dehydrogenase was not increased significantly in any of the tissues of ketonaemic sheep. 5. Comparison of rates of 3-hydroxybutyrate production in vivo with the enzyme activity in ketogenic tissue suggested that in sheep the maximum rate of production might be limited by this activity.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

Activation of volatile fatty acids in bovine liver and rumen epithelium. Evidence for control by autoregulation

1. The total capacities of homogenates of bovine liver and rumen epithelium to activate acetate, propionate and butyrate were determined. 2. Activating capacities were assayed by measuring the rate of formation of the corresponding CoA esters. The methods used for determining the concentrations of the CoA esters allowed the CoA esters of acetate, propionate and butyrate to be distinguished. It was thus possible to investigate the effect of the presence of a second volatile fatty acid on the rate at which a given volatile fatty acid was activated. 3. The propionate-activating capacity in rumen epithelium was decreased by about 87% in the presence of butyrate, the acetate-activating capacity in liver was decreased by about 55% in the presence of either propionate or butyrate, and the butyrate-activating capacity in liver was decreased by about 40-50% in the presence of propionate. 4. All three activating capacities in liver appeared to be located in the mitochondrial matrix and membrane. The three activating capacities had similar locations to each other in rumen epithelium as well, although in this case activity was more evenly divided between the mitochondria and the cytoplasm. 5. The relative activating capacities towards the volatile fatty acids in the two tissues, together with the ability of one volatile fatty acid to inhibit the activation of another volatile fatty acid, appear to ensure that butyrate is mainly metabolized in the rumen epithelium and that propionate is metabolized in the liver.     

Competition of glycerol with other oxidizable substrates in rat brain.

The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.     

Effect of starter diet supplementation on rumen epithelial morphology and expression of genes involved in cell proliferation and metabolism in pre-weaned lambs

Starter feeding is usually used in lamb production to improve rumen development and to facilitate the weaning process, but molecular mechanism of which is not well understood. Therefore, the objective of this study is to investigate the effect of starter feeding on the expression of ruminal epithelial genes involved in cell proliferation, apoptosis and metabolism in pre-weaned lambs. We selected eight pairs of 10-day-old lamb twins. One twin was fed ewe milk (M, n=8), while the other was fed ewe milk plus starter (M+S, n=8). The lambs were sacrificed at 56 days age. Results showed that the lambs fed M+S had lower pH in the rumen and a higher concentration of acetate, propionate, butyrate and total volatile fatty acid (VFA). Compared with the M group, the concentration of β-hydroxybutyric acid in plasma had an increased trend, and the concentration of IGF-1 in plasma had an decreased trend in the M+S group. The length, width and surface of rumen papillae increased in the M+S group compared with the M group; this was associated with increased cell layers in the stratum corneum, stratum granulosum and total epithelia. Messenger RNA (mRNA) expression of proliferative genes of cyclin A, cyclin D1 and cyclin-dependent kinase 2 in the ruminal epithelia of M+S lambs was increased compared with M only lambs. The mRNA expression of apoptosis genes of caspase-3, caspase-8, B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) in the M+S group was decreased compared with M group, but the ratio of Bcl-2 to Bax were not changed between the two groups. Expression of IGF-1 mRNA was decreased, but the mRNA expression of IGF-1 receptor was higher in ruminal epithelia in the M+S group. Furthermore, the mRNA expression of VFA absorption and metabolism genes of β-hydroxybutyrate dehydrogenase isoforms 1 and 3-hydroxy-3-methylglutaryl-CoA lyase had an increased trend in the M+S group than in the M group, but the mRNA expression of 3-hydroxy-3-methylglutaryl-CoA synthase isoform 1, monocarboxylate transporter isoform 1 and putative anion transporter isoform 1 had a decreased trend in the M+S group than in the M group. These results suggest that starter feeding increased proliferation and inhibited apoptosis of ruminal epithelial cells, and may promote the VFA metabolism in ruminal epithelium in pre-weaned lambs. These findings provide new insights into improving rumen development by nutritional intervention strategies in pre-weaned lambs.     

Glucose alleviates ammonia-induced inhibition of short-chain fatty acid metabolism in rat colonic epithelial cells

Ammonia decreased metabolism by rat colonic epithelial cells of butyrate and acetate to CO2 and ketones but increased oxidation of glucose and glutamine. Ammonia decreased cellular concentrations of oxaloacetate for all substrates evaluated. The extent to which butyrate carbon was oxidized to CO2 after entering the tricarboxylic acid (TCA) cycle was not significantly influenced by ammonia, suggesting there was no major shift toward efflux of carbon from the TCA cycle. Ammonia reduced entry of butyrate carbon into the TCA cycle, and the proportion of CoA esterified with acetate and butyrate correlated positively with the production of CO2 and ketone bodies. Also, ammonia reduced oxidation of propionate but had no effect on oxidation of 3-hydroxybutyrate. Inclusion of glucose, lactate, or glutamine with butyrate and acetate counteracted the ability of ammonia to decrease their oxidation. In rat colonocytes, it appears that ammonia suppresses short-chain fatty acid (SCFA) oxidation by inhibiting a step before or during their activation. This inhibition is alleviated by glucose and other energy-generating compounds. These results suggest that ammonia may only affect SCFA metabolism in vivo when glucose availability is compromised.     

Competition among oxidizable substrates in brains of young and adult rats. Dissociated cells.

The rates of conversion of D-(-)-3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate, [6-14C]glucose and [U-14C]glutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to [6-14C]glucose or unlabelled glucose was added to [U-14C]glutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy[3-14C]butyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to [U-14C]glutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to [6-14C]glucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of [6-14C]glucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of [3-14C]acetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy[3-14C]butyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.     

Calcium propionate supplementation improves development of rumen epithelium in calves via stimulating G protein-coupled receptors

In the present study, calcium propionate (CaP) was used as feed additive in the diet of calves to investigate their effects on rumen fermentation and the development of rumen epithelium in calves. To elucidate the mechanism in which CaP improves development of calf rumen epithelium via stimulating the messenger RNA (mRNA) expression of G protein-coupled receptors, a total of 54 male Jersey calves (age=7±1 days, BW=23.1±1.2 kg) were randomly divided into three treatment groups: control without CaP supplementation (Con), 5% CaP supplementation (5% CaP) and 10% CaP supplementation (10% CaP). The experiment lasted 160 days and was divided into three feeding stages: Stage 1 (days 0 to 30), Stage 2 (days 31 to 90) and Stage 3 (days 91 to 160). Calcium propionate supplementation percentages were calculated on a dry matter basis. In total, six calves from each group were randomly selected and slaughtered on days 30, 90 and 160 at the conclusion of each experimental feeding stage. Rumen fermentation was improved with increasing concentration of CaP supplementation in calves through the first 30 days (Stage 1). No effects of CaP supplementation were observed on rumen fermentation in calves during Stage 2 (days 31 to 90). Supplementation with 5% CaP increased propionate concentration, but not acetate and butyrate in calves during Stage 3 (days 91 to 160). The rumen papillae length of calves in the 5% CaP supplementation group was greater than that of Con groups in calves after 160 days feeding. The mRNA expression of G protein-coupled receptor 41 (GPR41) and GPR43 supplemented with 5% CaP were greater than the control group and 10% CaP group in feeding 160 days calves. 5% CaP supplementation increased the mRNA expression of cyclin D1, whereas did not increase the mRNA expression of cyclin-dependent kinase 4 compared with the control group in feeding 160-day calves. These results indicate that propionate may act as a signaling molecule to improve rumen epithelium development through stimulating mRNA expression of GPR41 and GPR43.     

Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells

Tumors of peripheral tissues contain low levels of succinyl CoA-acetoacetate CoA transferase activity which is not induced in vitro by prolonged cultivation in 2.5 mM DL-3-hydroxybutyrate. Although this enzyme is considered to be the main agent controlling the extent to which ketone bodies serve as metabolic substrates such tumors metabolize D(-)-3-hydroxy[3(14)C]butyrate to 14CO2. Also addition of 3-hydroxybutyrate and/or acetoacetate reduces the amount of 14CO2 produced from D-[U-14C] glucose suggesting a common metabolic intermediate. These observations can be accounted for by the presence of acetoacetyl-CoA synthetase, an enzyme which is able to synthesize acetoacetyl-CoA directly from acetoacetate, ATP and coenzyme A. This is the first demonstration of this enzyme in tumor tissue. The rate of metabolism of acetoacetate by this enzyme is sufficient to account for the production of CO2 from 3-hydroxybutyrate.     

Effects of nutritional conditions around weaning on carbonic anhydrase activity in the parotid gland and ruminal and abomasal epithelia of Holstein calves

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.     

Transport of butyrate across the isolated bovine rumen epithelium--interaction with sodium, chloride and bicarbonate

The Ussing chamber technique was used for studying unidirectional fluxes of 14C-butyrate across the bovine rumen epithelium in vitro. Significant amounts of butyrate were absorbed across the bovine rumen epithelium in vitro, without any external driving force. The paracellular pathway was quantitatively insignificant. The transcellular pathway was predominately voltage-insensitive. The serosal to mucosal (SM) pathway was regulated by mass action, whereas the mucosal to serosal (MS) pathway further includes a saturable process, which accounted for 30 to 55% of the MS flux. The studied transport process for 14C-butyrate across the epithelium could include metabolic processes and transport of 14C-labelled butyrate metabolites. The transport of butyrate interacted with Na+, Cl- and HCO3-, and there was a linear relationship between butyrate and sodium net transport. Lowering the sodium concentration from 140 to 10 mmol l-1 decreased the butyrate MS flux significantly. Amiloride (1 mmol l-1) did, however, not reduce the butyrate flux significantly. Chloride concentration in itself did not seem to influence the transport of butyrate, but chloride-free conditions tended to increase the MS and SM flux of butyrate by a DIDS-sensitive pathway. DIDS (bilateral 0.5 mmol l-1) did further decrease the butyrate SM flux significantly at all chloride concentrations. Removing bicarbonate from the experimental solutions decreased the MS and increased the SM flux of butyrate significantly, and abolished net butyrate flux. There were no significant effects of the carbonic anhydrase inhibitor Acetazolamide (bilateral 1.0 mmol l-1). The results can be explained by a model where butyrate and butyrate metabolites are transported both by passive diffusion and by an electroneutral anion-exchange with bicarbonate. The model couples sodium and butyrate via CO2 from metabolism of butyrate, and intracellular pH.     

Ruminal transport and metabolism of short-chain fatty acids (SCFA) in vitro: effect of SCFA chain length and pH

The unidirectional transport and metabolism of 14C-labeled acetate, propionate and butyrate across the isolated bovine rumen epithelium was measured in vitro by the Ussing chamber technique. There was a significant, but relatively small, net secretion of acetate and propionate, and a large and significant net absorption of butyrate. The results demonstrate that the mucosal-serosal (MS) pathway for short-chain fatty acids (SCFA) is different from the serosal-mucosal (SM) pathway, and that butyrate is treated differently from acetate and propionate by the epithelium. The results support that the main route for epithelial SCFA transport is transcellular. The correlation between SCFA lipophility and the flux rate was positive but weak at both pH 7.3 and 6.0. Decreasing pH increased all SCFA fluxes significantly, but not proportionally to the increase of protonized SCFA in the bathing solution. There was a significant and apparently non-competitive interaction between the transport of acetate, propionate and butyrate. It seems that mediated transport mechanisms must be involved in epithelial SCFA transport in the bovine rumen, but the data do not exclude that passive diffusion could account for a significant part of the flux. The metabolism of SCFA in the Ussing chamber system was considerable, and there was a clear preference for excretion of CO2 from this metabolism to the mucosal side, while side preference for non-CO2 metabolite excretion was not studied. Of the propionate and butyrate transported in the MS direction, 78 and 95% was metabolised, while only 37 and 38% was metabolised in the SM direction (acetate metabolism could not be measured). There was, however, no simple relation between the degree of metabolism and the transport rate or the transport asymmetry of the SCFA.     

丁酸调控幼龄反刍动物瘤胃上皮发育研究进展

瘤胃是反刍动物营养物质消化吸收和代谢的重要器官,其发育状态直接影响反刍动物生产性能和健康。初生犊牛和羔羊,瘤胃功能尚未发育完全,不能够充分消化和吸收固体饲料。因此,在幼龄时期,通过营养调控手段促进反刍动物的瘤胃发育对维持动物健康及提高生产性能具有重要意义。丁酸是瘤胃微生物降解植物性饲料的主要产物,也是瘤胃上皮及宿主的重要能量来源。丁酸调控幼龄反刍动物瘤胃上皮发育是一个历久弥新的话题。主要介绍了幼龄反刍动物瘤胃上皮形态及功能的发育以及丁酸调控幼龄反刍动物瘤胃上皮发育的研究进展。     

Effects of L-Alanine on ketogenesis in vitro

Alanine (5 mM) increased ¹⁴CO₂ production from [1-¹⁴C]oleate by 130% and from [1-¹⁴C]butyrate by 101%. Alanine inhibited ketone-body production by 37.5% in the presence of butyrate but did not affect ketogenesis in the presence of oleate. Alanine decreased the [3-hydroxybutyrate]/[acetoacetate] ratio when either butyrate or oleate was present. The results are discussed with reference to the hypoketonaemic action of alanine in vivo.     

Glucose, glutamine and ketone body utilisation by resting and concanavalin A activated rat splenic lymphocytes

The utilisation of glucose, glutamine, acetoacetate and D-3-hydroxybutyrate were investigated over 72 h of incubation of rat splenic lymphocytes, with and without concanavalin A. Lymphocytes consumed both ketone bodies; acetoacetate was consumed preferentially. The ketone bodies reduced glucose consumption by 30-50%, but had little effect on lactate production. Glutamine uptake was concentration dependent up to 4 mM, and consumption was increased in the presence of concanavalin. Glutamine stimulated glucose consumption and lactate production in both resting and activated cells. Complete oxidation contributed 65% of glucose-derived ATP, but less than 40% of glutamine-derived ATP. Glutamine metabolism makes only a minor contribution to lymphocyte ATP generation.     

Impaired sensitivity to insulin of rat livers perfused with blood of diminished haematocrit.

1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.     

Induction of Encystment and Poly-β-Hydroxybutyric Acid Production by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201, when grown under nitrogen-free conditions with excess glucose, accumulated poly-β-hydroxybutyric acid amounting to 75% of cell dry weight at the late exponential phase. This led to induction of encystment, which increased steadily with concomitant intracellular degradation of the polymer. Increase in encystment and PHB production were parallel up to 0.5% (wt/vol) glucose. Further increase in glucose reduced cyst formation but enhanced PHB accumulation. Replacement of glucose by n-butyl alcohol and metabolically related compounds identified crotonate as the best encystment inducer followed by β-hydroxybutyrate and butyrate, but PHB production was inhibited in general. Supplementation of medium with these compounds enhanced the onset of encystment, and only β-hydroxybutyrate increased PHB accumulation significantly. Received: 23 April 1997 / Accepted: 31 May 1997     

Effect of chloride on pH microclimate and electrogenic Na+ absorption across the rumen epithelium of goat and sheep

Active Na+ absorption across rumen epithelium comprises Na+/H+ exchange and a nonselective cation conductance (NSCC). Luminal chloride is able to stimulate Na+ absorption, which has been attributed to an interaction between Cl-/HCO3- and Na+/H+ exchangers. However, isolated rumen epithelial cells also express a Cl- conductance. We investigated whether Cl- has an additional effect on electrogenic Na+ absorption via NSCC. NSCC was estimated from short-circuit current (Isc) across epithelia of goat and sheep rumen in Ussing chambers. Epithelial surface pH (pHs) was measured with 5-N-hexadecanoyl-aminofluorescence. Membrane potentials were measured with microelelectrodes. Luminal, but not serosal, Cl- stimulated the Ca2+ and Mg2+ sensitive Isc. This effect was independent of the replacing anion (gluconate or acetate) and of the presence of bicarbonate. The mean pHs of rumen epithelium amounted to 7.47 +/- 0.03 in a low-Cl- solution. It was increased by 0.21 pH units when luminal Cl- was increased from 10 to 68 mM. Increasing mucosal pH from 7.5 to 8.0 also increased the Ca2+ and Mg2+ sensitive Isc and transepithelial conductance and reduced the fractional resistance of the apical membrane. Luminal Cl- depolarized the apical membrane of rumen epithelium. 5-Nitro-2-(3-phenylpropylamino)-benzoate reduced the divalent cation sensitive Isc, but only in low-Cl- solutions. The results show that luminal Cl- can increase the microclimate pH via apical Cl-/HCO3- or Cl-/OH- exchangers. Electrogenic Na+ absorption via NSCC increases with pH, explaining part of the Cl- effects on Na+ absorption. The data further show that the Cl- conductance of rumen epithelium must be located at the basolateral membrane.