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A higher expression of the sodium glucose-linked transporter, SGLT-1, has been previously demonstrated in the intestine of adult fallow deer compared to sheep. The functional presence of SGLT-1 in the rumen of both species was investigated in the present study. Isolated ruminal epithelia were incubated in Ussing chambers. D-Glucose or 3-O-methyl-alpha-D-glucose (3-OMG) were applied at 10 mmol x l-1 to the mucosal side of tissues and the changes in short-circuit current (Delta I(sc)) were recorded. Alternatively, apical uptake of [14C]-D-glucose (200 micro mol x l-1) was determined in the presence or absence of phlorizin (100 micro mol x l-1) or Na+ (115 mmol x l-1). In both species, mucosal D-glucose addition induced a larger ( P<0.05) Delta I(sc) than the mucosal addition of 3-OMG. When comparing the effects of D-glucose or 3-OMG between species, the Delta I(sc) was two-fold larger ( P<0.01) in sheep compared to fallow deer. Accordingly, phlorizin-sensitive D-glucose uptake was approximately 50% smaller ( P<0.05) in fallow deer. It is concluded that D-glucose can be absorbed via SGLT-1 from the forestomachs of both species. However, the intermediate-mixed feeding habit of fallow deer does not seem to coincide with a higher activity of the ruminal SGLT-1.

Luminal histamine concentrations can be very high in the intestine without eliciting clinical histaminosis. The intestinal mechanisms that possibly prevent systemic intoxication by luminal histamine were investigated in the present study. Porcine colonic epithelia were mounted in Ussing chambers. After unilateral application of partially (3)H-labelled histamine, mucosal-to-serosal (MS) and serosal-to-mucosal (SM) flux rates were calculated from the contralateral appearance of histamine and the radioactive histamine label (hist-rad; representing histamine plus catabolites). A discrepancy between histamine and hist-rad fluxes was observed in both flux directions, indicating efficient histamine catabolism at all histamine concentrations tested (5, 50 and 100 microM). Catabolism exceeded 65% at 100 microM histamine and reached approximately 100% at 5 microM histamine. Blockade of histamine N-methyltransferase by amodiaquin (100 microM) abolished catabolism completely, whereas blockade of diamine oxidase by aminoguanidine (100 microM) was less effective. MS fluxes of histamine and hist-rad correlated with mannitol fluxes. Mast cell stimulation with the calcium ionophore A23187 (1 microM) induced a twofold larger histamine appearance on the serosal side than the mucosal application of 100 microM histamine. It is concluded that histamine permeation across the porcine proximal colon is restricted by low permeability and sequential catabolism by histamine N-methyltransferase and diamine oxidase.

The motility patterns of the reticulorumen evoke mainly mixing of the ingesta. So far unknown, intrinsic neural circuits of the enteric nervous system are involved in the control of these motility patterns. The aim of the study was to characterize neurochemically sheep ruminal myenteric neurones, in particular the neural pathways innervating the ruminal muscle layers. Cell bodies within the myenteric plexus projecting to the longitudinal or circular muscle layer were retrogradely labelled by direct application of the fluorescent tracer 1,1'-didodecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) onto the circular or longitudinal muscle. The neurochemical code of myenteric neurones was identified by their immunoreactivity for choline acetyltransferase (ChAT), nitric oxide synthase (NOS), substance P (SP) and vasoactive intestinal peptide (VIP). According to their neurochemical code, ruminal myenteric neurones were divided into three populations: ChAT/SP (68% of all myenteric neurones), NOS/VIP (26% of all myenteric neurones) and ChAT/- (5% of all myenteric neurones). Application of DiI onto the circular or longitudinal muscle revealed on average 64 or 44 labelled cell bodies in the myenteric plexus, respectively. DiI-labelled neurones expressed the code ChAT/SP or NOS/VIP. In the pathways to circular or longitudinal muscle, ChAT/SP-positive neurones outnumbered NOS/VIP-immunoreactive neurones by 5:1 and 2:1. Pathways to the circular or longitudinal muscle did not exhibit any pronounced polarized innervation patterns. This study demonstrated specific projections of myenteric neurones to the ruminal muscle. Neurones expressing the code ChAT/SP might function as excitatory muscle motor neurones, whereas NOS/VIP neurones are likely to act as inhibitory muscle motor neurones.

Glucose absorption via the sodium glucose-linked transporter (SGLT)-1, decreases the glucose concentration in the ruminant forestomach and may ameliorate or prevent ruminal lactic acidosis. Because acidotic ruminants show increased sympathetic activity, the possibility of adrenergic modulation of SGLT-1 was investigated. Glucose uptake into ovine ruminal epithelia was measured in Ussing chambers after the addition of 200 micromol/L (14)C-labeled glucose to the mucosal solution. Glucose uptake decreased (P < 0.05) by >50% in comparison with control after mucosal addition of the SGLT-1 inhibitor, phlorizin (100 micromol/L). Serosal preincubation with 100 micromol/L epinephrine increased (P < 0.05) the phlorizin-sensitive glucose uptake in the absence and presence of indomethacin (10 micromol/L). The effect of epinephrine was simulated by beta- (100 micromol/L isoproterenol) and beta(2)-receptor agonists (10 micromol/L terbutaline), as well as by direct stimulation of adenylyl cyclase (10 micromol/L forskolin). The serosal addition of methoxamine, clonidine, dobutamine or BRL 37344 had no effect. Inhibition of protein kinase A with 2 micromol/L H 89 completely abolished the stimulation of glucose uptake by epinephrine. We conclude that ruminal SGLT-1 can be stimulated via beta(2)-dependent generation of cyclic adenosine monophosphate.

Short-chain fatty acids are absorbed in considerable amounts from the rumen. During transit through the epithelial layer, they are intensively metabolised. Interaction between intraepithelial metabolism and absorption, however, is hardly understood. The present study therefore compared the transepithelial transport of the easily metabolised n-butyrate with that of the more metabolism-resistant iso-butyrate both under in vivo conditions (isolated and washed reticulorumen) and in vitro conditions (Ussing chamber). Under in vivo conditions, net absorption of n-butyrate was significantly higher than that of iso-butyrate. The in vitro experiments showed that the higher net flux of n-butyrate was solely due to a higher mucosal-to-serosal flux, whereas the serosal-to-mucosal flux of butyrate was independent from the isoform. Blocking intraepithelial ATP delivery by 2,4-dinitrophenol abolished the net flux of n-butyrate. The study indicates that metabolism and/ or ATP availability stimulates n-butyrate net absorption. By this, the metabolic activity of the epithelium may have a regulatory influence on absorption of n-butyrate.

Because of the effective catabolism of D-glucose to short-chain fatty acids by intraruminal microorganisms, the absorption of D-glucose from the rumen was thought to be of minor importance. However, clinical studies suggested that significant quantities of D-glucose are transported from the ruminal contents to the blood. We therefore tested the ruminal epithelium of sheep for the presence of Na(+)-glucose cotransporter 1 (SGLT-1) on both the functional and mRNA levels. In the absence of an electrochemical gradient, 3-O-methylglucose (3-OMG) was net absorbed across isolated ruminal epithelia mounted in Ussing chambers. The net transport of 3-OMG followed Michaelis-Menten kinetics and was sensitive to phlorizin or decreasing Na(+) concentrations. The mucosal addition of 10 mM D-glucose induced an immediate, phlorizin-sensitive increase in short-circuit current (I(sc)). I(sc) could also be increased by serosal addition of D-glucose or D-mannose, but electrogenic uptake of D-glucose or 3-OMG added on the mucosal side was still detectable after serosal stimulation of I(sc). RT-PCR using primers specific for the ovine intestinal SGLT-1 with subsequent TA cloning and sequencing revealed 100% identity between the cloned cDNA and mRNA fragment 187-621 of ovine intestinal SGLT-1. In conclusion, the ruminal epithelium has a high-affinity SGLT-1, which indicates that it maintains the capacity for D-glucose absorption.

This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pHi) and mechanisms that mediate pHi recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pHi was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2',7'-bis (carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pHi in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 +/- 0.03. In HEPES-buffered solution, a NH4+/NH3-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA: 10 microM) or HOE-694 (200 microM) inhibited pHi recovery from an NH4+/NH3-induced acid load by 58% and 70%, respectively. pHi recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 microM) and HOE-694 (200 microM), respectively. Changing from HEPES-(20 mM) to CO2/HCO3(-)-buffered (5%/20 mM) solution caused a rapid decrease of pHi (P < 0.01), followed by an effective counter-regulation. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 100 microM) blocked the pHi recovery by 88%. The results indicate that intracellular acidification by butyrate and CO2 is effectively counter-regulated by an Na+/H+ exchanger and by DIDS-sensitive, HCO3(-)-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pHi homeostasis of ruminal epithelial cells.

Whereas intraruminal histamine does not affect healthy ruminants, histaminosis is apparent during ruminal acidosis. We therefore investigated the factors that, under physiological circumstances, prevent intoxication by intraruminal histamine and the disturbances occurring during acidotic or hypoxic epithelial damage. After mucosal (m) or serosal (s) application of 80 microM histamine, its flux across the isolated epithelia of the sheep rumen was determined radioactively (hist-rad flux) in Ussing chambers. The non-catabolized component of the hist-rad fluxes was determined by high-pressure liquid chromatography (HPLC) (histamine flux). The difference between hist-rad and histamine fluxes indicated efficient intraepithelial catabolism of histamine at pH 7.4 (m-s direction, 98.7%; s-m direction, 93.3%). Both 0.1 mM 2,4-dinitrophenol (DNP) and mucosal acidification to pH 5.1 increased hist-rad fluxes and decreased catabolic efficiency. pH-dependent secretion of histamine was indicated by differences between m-s and s-m fluxes of histamine and/or hist-rad. Epithelial permeability to hist-rad and mannitol was similar and their fluxes correlated partly. Epithelial release of endogenous histamine was 1.5 pmol x cm(-2) x h(-1) and was not increased by the mast cell stimulator, compound 48/80 (10 ng x ml(-1)). We conclude that histamine absorption across the intact epithelium is efficiently restricted by a low permeability to histamine in combination with catabolic and secretory processes. Especially increases in paracellular permeability and/or inhibition of catabolism enhance histamine absorption.

Sodium is absorbed in considerable amounts across the ruminal epithelium, whilst its transport is strongly interrelated with the permeation of chloride and short-chain fatty acids (SCFAs). However, regulation of ruminal Na+, Cl-, and SCFA absorption is hardly understood. The present study was therefore performed to characterize the influence of cAMP on sodium and sodium-coupled transport mechanisms in short-circuited, stripped ruminal epithelia of sheep. Elevation of intracellular cAMP concentrations by theophylline (10 mM) or theophylline in combination with forskolin (0.1 mM) significantly reduced mucosal-to-serosal sodium transport, leading to a reduction of net transport. The theophylline- or theophylline-forskolin-induced reduction of sodium transport was accompanied by a decrease in chloride net transport but revealed no effect on propionate flux. Short-chain fatty acids stimulated Na+ transport but their stimulatory effect was almost completely blocked by theophylline-forskolin. In solutions with and without SCFAs, the inhibitory effect of 1 mM amiloride on sodium transport was strongly reduced after theophylline-forskolin pretreatment of the tissues. Blocking the production of endogenous prostaglandins by addition of indomethacin (10 microM) led to a theophylline-sensitive stimulation of unidirectional and net fluxes of sodium. The findings indicate that apical, amiloride-sensitive Na+-H+ exchange and/or basolateral Na+-K+-ATPase can effectively be blocked by cAMP, leading to a decrease in sodium and chloride transport. In the ruminal epithelium, cAMP is a second messenger of prostaglandins, which are released spontaneously under in vitro conditions.

Ten, 100 and 1000 microM histamine were applied to ruminal epithelial cells in primary culture. Each of these concentrations diminished cell count as well as the protein and DNA contents of cultures by approximately 20%. On Pappenheim staining, reduced cell count was predominantly attributable to a decrease in relatively chromophobic cells. It is concluded that histamine either induces apoptosis, or increases cell shedding, or interferes with mitosis and cell maturation. Each of these three possibilities implies that histamine may disturb epithelial regeneration after ruminal lactic acidosis.

Short chain fatty acids (SCFAs) and CO2 have been shown to stimulate net Mg2+ efflux from the isolated reticulorumen in vivo. To investigate the underlying mechanisms of Mg2+ transport we performed Ussing chamber and microelectrode experiments and measured 28Mg2+ fluxes across sheep rumen epithelium in vitro. In the presence of SCFAs mucosal-to-serosal Mg2+ flux (Jm-sMg) amounted to 82.3 +/- 7.8 nmol cm-2 h-1 and serosal-to-mucosal Mg2+ flux (Js-mMg) to 3.2 +/- 0.7 nmol cm-2 h-1. Replacing SCFAs with gluconate caused a 50% reduction of Jm-sMg, whereas Js-mMg was not affected. Among the SCFAs, n-butyrate was more effective in stimulating Jm-sMg than acetate, propionate or iso-butyrate. Eliminating HCO3(-)-CO2 from SCFA-containing solutions did not affect Mg2+ fluxes, whereas the same replacement in SCFA-free solutions led to a further reduction in Jm-sMg. Jm-sMg decreased after the addition of ethoxyzolamide to SCFA-free, bicarbonate buffered solutions. Decreasing mucosal pH from 6.4 to 5.4 increased Jm-sMg in SCFA-free, bicarbonate buffered solutions. SCFAs had no effect on the apical membrane potential of rumen epithelial cells. The experiments show that both SCFAs and CO2 stimulate Mg2+ transport through an increase in Jm-sMg, most probably via stimulation of a Mg(2+)-2H+ exchange mechanism. SCFAs may have additional metabolic effects on Mg2+ transport.

Short-chain fatty acids are the main end-products of microbial metabolism in the forestomach of ruminants. SCFA produced by the microorganisms are rapidly absorbed across forestomach epithelia and can cover up to 80% of the energy requirement of the animal. Although there is a great concentration gradient for SCFA between the forestomach content and the blood favoring passive transport, (secondary) active transport mechanisms are likely involved in SCFA permeation across the epithelia. (Secondary) active SCFA transport seems to be mediated by an anionic exchange system. The system interacts with other anions like chloride and bicarbonate. Similar to the large intestine of various species, SCFA can stimulate sodium transport probably by activating a Na+/H+ exchange located in the apical membrane. However, in contrast to the large intestine, SCFA transport itself seems to be independent from sodium. Part of the absorbed SCFA does not reach the blood side in the original form because it is metabolized in the epithelial cell. Metabolism, in turn, influences SCFA transport.

The study aimed at testing whether Na+/H+ exchange activity is maintained during isolation and cultivation of rumen epithelial cells (REC) and whether Na+/H+ exchange is altered during proliferation and differentiation. REC were isolated from sheep rumen epithelium by fractional trypsinization and further cultivated for up to 21 days. Characteristics of Na+ uptake into cultured REC were the same as previously described for the Na+/H+ exchange in the intact rumen epithelium. Na+ uptake was increased by intracellular acidification and was inhibited by high doses of amiloride. Amiloride inhibited 3H-thymidine incorporation into REC at concentrations similar to those found for the inhibition of Na+ uptake. Fetal calf serum, a growth factor, stimulated amiloride-sensitive Na+ uptake. We therefore conclude that cultured rumen epithelial cells express Na+/H+ exchange and that the activity of the exchange is at least correlated to the intensity of proliferation.

Investigations on the absorption of shortchain fatty acids across ruminal epithelium of sheep were performed both in vitro (Ussing chamber technique, using propionic acid representatively for short-chain fatty acids) and in vivo (washed, isolated reticulorumen). A pH-induced, nearly tenfold increase in the concentration of undissociated propionate led to an only twofold increase in mucosal-to-serosal flux of propionate (in vitro). Neither amiloride (1 mmol.l-1, in vitro) nor theophylline (10 mmol.l-1, in vivo), inhibitors of the ruminal Na+/H+ exchanger, exerted any significant influence on propionate fluxes or short-chain fatty acids absorption, respectively. Total replacement of luminal Na+ (by choline) did not alter short-chain fatty acids absorption (in vivo). Mucosal 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (0.1 mmol.l-1) or mucosal nitrate (40 mmol.l-1) markedly reduced propionate net flux (in vitro). Increasing mucosal Cl- concentration brought about a significant drop in mucosal-to-serosal flux of propionate (in vitro) and in short-chain fatty acids net absorption (in vivo), respectively. The results obtained suggest that short-chain fatty acids are absorbed both as anions and as undissociated acids across ruminal epithelium of sheep. It is concluded that short-chain fatty acids anions either compete with Cl- for binding sites at a common anion-exchange mechanism or that they are absorbed by an short-chain fatty acids anion/ HCO3- exchanger indirectly coupled to a Cl-/HCO3- exchanger via intracellular bicarbonate.

Net absorption rates for SCFA, sodium, chloride, magnesium, and water were measured in the temporarily isolated and washed reticulorumen of sheep. The animals were either fed hay ad lib. plus concentrates once daily or were deprived of food for two days. Food deprivation led to decreased net absorption of all the substances tested. Comparing the solutes, the relative decreases varied between 43% (butyrate) and 56% (acetate). The decrease in solute absorption was accompanied by diminished water absorption. It is concluded that the reduction in absorption may be beneficial for the animal in that energy consumption is diminished and the reservoir function of the forestomach will be maintained longer. However, the reduced absorptive capacity may lead to a deficiency of magnesium, which is thought to be absorbed mainly by the forestomachs.

1. 22Na+ and 36Cl- fluxes across isolated reticular epithelium of sheep were measured by using the Ussing-chamber technique. 2. Net NaCl absorption driven by Na(+)-K(+)-ATPase was observed under short-circuit conditions. 3. Evaluation of fluxes measured under voltage-clamp conditions indicated that Na+ absorption is mainly electroneutral. 4. Mucosal application of bumetanide, hydrochlorothiazide, or low dose amiloride (10(-4) M) produced no changes in Na+ transport whereas addition of higher doses of amiloride (> or = 10(-3) M) led to a reduction in net Na+ transport. Short chain fatty acids (SCFA) enhanced the amiloride-sensitive Na+ transport. 5. Alterations of JmsNa induced by inhibitors or by SCFA were always accompanied by qualitatively similar changes of JsmNa. Amiloride-sensitive JsmNa was also decreased at low mucosal Na+ concentration. 6. DIDS, SITS, and nitrate reduced both JmsCl and JsmCl. SCFA did not influence chloride transport. 7. It is concluded that Na+ transport is mediated by Na(+)-H+ exchange and by transport processes operating as Na+ self-exchange. Mucosal-to-serosal chloride transport seems partly to depend on anion exchange systems.

Primary cultures of ovine ruminal epithelial cells were made to study the influence of collagen types I and IV out of medium supplementation with various hormones and Na-n-butyrate on cell morphology and growth characteristics. Both collagen type I and type IV led to increased cell proliferation with the stimulatory effect being more pronounced in collagen IV. In cultures grown on collagen I, both non-stratified and stratified colonies were found, whereas cultures grown on collagen IV showed predominantly stratified growth. Cells in both stratified and non-stratified colonies were positive for cytokeratin antibody. In non-stratified colonies, positive staining with fibronectin antibodies (FN-15) was found in a network over and around the cells. It is suggested that the non-stratified ruminal epithelial cells are in some respects similar to a 'non-differentiating keratinocyte' strain, derived from newborn foreskin epidermis. Cells in stratified colonies bound Ulex europaeus (UEAI) lectin which has been shown to be specific for differentiated epithelial cells in ruminal mucosa. Supplementation of culture medium with glucagon and insulin increased the total cell-overgrown area of collagen I cultures, whereas this effect was absent in cultures grown on collagen IV. In both cultures grown on collagen I or IV, hydrocortisone led to an increase in total cell-overgrown area, whereas Na-n-butyrate inhibited proliferation.

Influences of short-chain fatty acids (SCFA), chloride or lactate on net bicarbonate movement were determined in the temporarily isolated and washed reticulo-rumen of sheep used to 100% hay or 81% concentrate diets. Replacement of SCFA by gluconate and mannitol reversed net bicarbonate secretion into net absorption. The reversal of net bicarbonate movement could not be prevented by presence of D/L-lactate. Replacement of chloride in addition to replacement of SCFA enhanced net absorption of bicarbonate. Feeding concentrate diets resulted in an elevation of net bicarbonate secretion and of net SCFA absorption. It is concluded that under the conditions tested net bicarbonate secretion mainly depends on the luminal presence of SCFA. Furthermore, bicarbonate secretion into the reticulo-rumen may be an important factor for alkalizing the rumen contents, especially in sheep adapted to concentrate diets.

Unidirectional 22Na+ and 36Cl- fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO2/HCO3(-)-containing and CO2/HCO3(-)-free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39mM) significantly enhanced mucosal-to-serosal Na+ absorption without affecting the Cl- transport in the same direction. Short-chain fatty acid stimulation of Na+ transport was at least partly independent of Cl- and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na+ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO2 in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na+ flux. Along with its effect on sodium transport, raising the PCO2 also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO2 permeate the cell membrane and produce a raise in intracellular H+ concentration. This stimulates an apical Na+/H+ exchange, leading to increased Na+ transport. The stimulatory effect of CO2 on Cl- transport is probably mediated by a Cl-/HCO3- exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.

This study was designed to study the mechanism of electroneutral Na and Cl transport across the isolated rumen epithelium of sheep. Net sodium transport (5.75 +/- 0.35 microequiv cm-2 h-1) was significantly higher than the short-circuit current (0.95 +/- 0.08 microequiv cm-2 h-1). Both, net sodium and net chloride transport were markedly reduced by replacement of chloride, bicarbonate and sodium, respectively, but were not changed by the absence of mucosal potassium. Net sodium and net chloride absorption was significantly decreased by 1.0 mM-amiloride. Mucosal addition of bumetanide, furosemide, hydrochlorothiazide or low concentrations of amiloride (less than 0.1 mM) did not change sodium fluxes. These results provide compelling evidence consistent with the presence of Na-H exchange as the predominant electroneutral mechanism for transepithelial sodium movement. The ion replacement studies and data from literature suggest that the Na-H exchange is working in parallel with a Cl-HCO3 exchange although luminal addition of DIDS (4,4'diisothiocyanatostilbene-2,2'-disulphonate, 1 mM) did not significantly influence Cl transport.

The transport of Mg across an isolated preparation of rumen epithelium of sheep was studied using different Mg salts: MgCl2. Mg aspartate, mg pidolate, and Mg-EDTA. The measurements were made under short circuit conditions in Ussing chambers. The obtained results showed that Mg transport rates did not differ significantly between MgCl2, Mg aspartate, and Mg pidolate. However, Mg-EDTA reduced Mg transport significantly and increased the short circuit current (Isc). It was shown that this increased Isc can be abolished by mucosal addition of Mg or Ca. It is concluded that Mg is transported if it is free and in the ionized form.

Experiments were done in vivo and in vitro to study the flux of inorganic phosphate (P1) across the rumen wall in sheep. The technique of the temporarily isolated and washed reticulo-rumen was applied to measure P1 net flux at nine different P1 concentrations in the buffer solutions introduced into the reticulo-rumen for 1 h. Six sheep were either P replete (4.25 g P d-1) or in a P-depleted state (1.05 g P d-1). Under both dietary treatments P1 net secretion into the reticulorumen was found at P1 concentrations between 0.1 and 2.2 mmol l-1 whereas P1 net absorption was observed at P1 concentrations between 4.1 and 15.8 mmol l-1. There were positive linear relationships between P1 concentration and P1 net flux for the whole concentration range studied in repleted and in depleted sheep. There were no indications of active transport components or saturation phenomena. Unidirectional P1 fluxes were then measured in Ussing chambers with 32P using a total of 100 rumen wall epithelia obtained from eighteen sheep of different ages. No P1 net flux was found under short-circuit conditions. Experiments with changed potential differences (+25 or -25 mV at serosal side) showed that P1 flux was influenced by the electrical gradient indicating electrogenic phosphate ion diffusion without involvement of active transport processes. Critical evaluation of the obtained unidirectional flux ratios suggests that part of the P1 flux was electroneutral, e.g. by ion exchange or cotransport. The results from these studies show that the reticulo-rumen substantially contributes to phosphate absorption from the sheep gastrointestinal tract.

Rumen: 1. It is generally accepted that sodium and chloride is transported across the rumen epithelium of ruminants in the mucosal-serosal direction by an active transport mechanism. In all in vitro studies the short circuit current, Isc, was significantly lower than the net transport of sodium, JNanet. It was concluded that most of the sodium is transported by an electrically silent mechanism. A Na, Cl cotransport and/or a double exchange system Na/H and Cl/HCO3 was proposed. 2. Recent in vitro studies in our laboratory revealed that the Na/H exchange inhibitor amiloride (1 mmol/l) reduced JNanet by 70%. The Na/K/2Cl-cotransport inhibitor bumetanide or furosemide (1 mmol/l) had no effect. 3. Replacement of permeable anions led to an inhibition of JNanet by 87%. However, under these experimental conditions Isc and JNanet were not significantly different. The remaining small electrogen transport of sodium was not influenced by mucosal amiloride. 4. It is concluded that two transport systems are operating in the ruminal epithelium: a Na/H exchange system which accounts for 80-90% of JNanet and an electrogen Na transport which is not sensitive against amiloride. Omasum: 1. The data concerning the transport of sodium and chloride in the omasum are very limited. In vivo measurements of flow rates demonstrated that sodium is absorbed by the omasum (40-60% of inflow in bull calves, 10-20% in sheep) and chloride is secreted. 2. In vitro studies with isolated preparation of sheep omasum epithelium demonstrated a net transport of Na and Cl in the mucosal-serosal direction. JNanet is completely abolished by ouabain (0.1 mmol/1) and JClnet by replacement of Na.