Divalent cations reduce the electrogenic transport of monovalent cations across rumen epithelium

The rumen epithelium of sheep and goats showed an increase in short circuit current ( Isc) and transepithelial conductance (gt) upon mucosal removal of divalent cations. A divalent-sensitive Isc and gt were present in Na+, K+ or Rb+ buffer, but nearly abolished in mucosal NMDG+ (N-methyl-D-glucamine) buffer. High K buffer, addition of BaCl2 or of ouabain on the serosal side also reduced or abolished the divalent-sensitive Isc. Mucosal Ca2+ was more potent in blocking Isc, but had the same potency as Mg2+ in blocking gt. A prolonged mucosal deprivation of Mg2+ ions increased gt, potential difference and basal as well as the Ca2+-sensitive Isc. Mucosal addition of Mg2+ had a smaller effect on gt after serosal preincubation with Ba. The data suggest that rumen epithelial cells exhibit an apical non-selective cation conductance, which permits the passage of monovalents in the mucosal absence of divalents. The development of a divalent-sensitive Isc in Na buffer requires Na+/K+ pumps and K+ recycling through Ba2+-sensitive K+ conductances on the basolateral side. This Isc is blocked by extracellular Ca2+ and both extracellular and intracellular Mg2+ ions. A prolonged deprivation of mucosal Mg2+ alone seems to affect intracellular Mg2+ in this Mg2+-absorbing tissue.     

Studies on the absorption of sodium and chloride from the rumen of sheep

The net absorption of Na and Cl from the temporarily isolated rumen of sheep was studied using an artificial ruminal fluid with different Na and K and constant Cl concentrations. The net absorption of Na and Cl was linearly correlated. The net absorption of Cl was abolished and a small net secretion was observed when no sodium was in the artificial rumen fluid. The net absorption of Na was significantly reduced under chloride free conditions. It is concluded that the active transport of Na and Cl is coupled. The mechanism of an Na-Cl cotransport can not be deduced from these studies.     

Sodium chloride absorption across the ileal epithelium of the lizardGallotia galloti

Summary Ion transport processes in the ileum of the lizard,Gallotia (=Lacerta) galloti was examined in vitro by measuring Na²² and Cl³⁶ fluxes across short-circuited preparations.In Ringer-bicarbonate solution there was both a net sodium flux ( ) and a net chloride flux ( ) from mucosa to serosa. The inequality between and short-circuit current (I _sc) suggests that part of the net sodium transport is the result of an electrically neutral transport mechanism or that another electrogenic mechanism opposite in sign is contributing to the short-circuit current.In the absence of sodium, the short-circuit current and net chloride flux were abolished. In the absence of chloride, the net sodium was reduced but not abolished and the short-circuit current was unchanged.From an analysis of the effects of the inhibitors furosemide, amiloride, disulfonic stilbene (DIDS) and acetazolamide, a plausible model was developed to explain the characteristics of these transports. It is proposed that the entry of sodium into the cell across the luminal membrane occurs by two pathways. Part occurs by the antiport Na⁺H⁺ and part by an electrogenic pathway. The entry of chloride is by the antiport Cl^–HCO ₃ ^– .Symbols and abbreviations DIDS 4,4 diisothiocyanatostilbene-2,2-disulfonic acid - G _t tissue conductance - I _sc short circuit current - m mucosal - PD potential difference - s serosal     

Modulation of electroneutral Na transport in sheep rumen epithelium by luminal ammonia

Ammonia is an abundant fermentation product in the forestomachs of ruminants and the intestine of other species. Uptake as NH3 or NH4+ should modulate cytosolic pH and sodium-proton exchange via Na+/H+ exchanger (NHE). Transport rates of Na+, NH4+, and NH3 across the isolated rumen epithelium were studied at various luminal ammonia concentrations and pH values using the Ussing chamber method. The patch-clamp technique was used to identify an uptake route for NH4+. The data show that luminal ammonia inhibits electroneutral Na transport at pH 7.4 and abolishes it at 30 mM (P < 0.05). In contrast, at pH 6.4, ammonia stimulates Na transport (P < 0.05). Flux data reveal that at pH 6.4, approximately 70% of ammonia is absorbed in the form of NH4+, whereas at pH 7.4, uptake of NH3 exceeds that of NH4+ by a factor of approximately four. The patch-clamp data show a quinidine-sensitive permeability for NH4+ and K+ but not Na+. Conductance was 135 +/- 12 pS in symmetrical NH(4)Cl solution (130 mM). Permeability was modulated by the concentration of permeant ions, with P(K) > P(NH4) at high and P(NH4) > P(K) at lower external concentrations. Joint application of both ions led to anomalous mole fraction effects. In conclusion, the luminal pH determines the predominant form of ammonia absorption from the rumen and the effect of ammonia on electroneutral Na transport. Protons that enter the cytosol through potassium channels in the form of NH4+ stimulate and nonionic diffusion of NH3 blocks NHE, thus contributing to sodium transport and regulation of pH.     

Luminal hyperosmolarity decreases Na transport and impairs barrier function of sheep rumen epithelium

The effects of luminal hyperosmolarity on Na and Cl transport were studied in rumen epithelium of sheep. An increase of luminal osmotic pressure with mannitol (350 and 450 mosm/l) caused a significant increase of tissue conductance, G _T, which is linearly correlated with flux rates of ⁵¹Cr-EDTA and indicates an increase of passive permeability. Studies with microelectrodes revealed, that an increase of the osmotic pressure caused a significant increase of the conductance of the shunt pathway from 1.23±0.10 (control) to 1.92±0.14 mS cm⁻² (450 mosm/l) without a change of fractional resistance. Hyperosmolarity significantly increased J _sm and reduced J _net Na. The effect of hyperosmolarity on J _ms Na is explained by two independent and opposed effects: increase of passive permeability and inhibition of the Na⁺/H⁺ exchanger. Hypertonic buffer solution induced a decrease of the intracellular pH (pH_i) of isolated ruminal cells, which is consistent with an inhibition of Na⁺/H⁺ exchange, probably isoform NHE-3, because NHE-3-mRNA was detectable in rumen epithelium. These data are in contrast to previous reports and reveal a disturbed Na transport and an impaired barrier function of the rumen epithelium, which predisposes translocation of rumen endotoxins and penetration of bacteria.     

K+-conductance and electrogenic Na+/K+ transport of cultured bovine pigmented ciliary epithelium

Summary Using intracellular microelectrode technique, we investigated the changes in membrane voltage (V) of cultured bovine pigmented ciliary epithelial cells induced by different extracellular solutions. (1)V in 213 cells under steady-state conditions averaged –46.1±0.6 mV (sem). (2) Increasing extracellular K⁺ concentration ([K⁺] _o ) depolarizedV. Addition of Ba²⁺ could diminish this response. (3) Depolarization on doubling [K⁺] _o was increased at higher [K⁺] _o (or low voltage). (4) Removing extracellular Ca²⁺ decreasedV and reduced theV amplitude on increasing [K⁺] _o . (5)V was pH sensitive. Extra-and intracellular acidification depolarizedV; alkalinization induced a hyperpolarization.V responses to high [K⁺] _o were reduced at acidic extracellular pH. (6) Removing K _o ⁺ depolarized, K _o ⁺ readdition after K⁺ depletion transiently hyperpolarizedV. These responses were insensitive to Ba²⁺ but were abolished in the presence of ouabain or in Na⁺-free medium. (7) Na⁺ readdition after Na⁺ depletion transiently hyperpolarizedV. This reaction was markedly reduced in the presence of ouabain or in K⁺-free solution but unchanged by Ba²⁺. It is concluded that in cultured bovine pigmented ciliary epithelial cells K⁺ conductance depends on Ca²⁺, pH and [K⁺] _o (or voltage). An electrogenic Na⁺/K⁺-transport is present, which is stimulated during recovery from K⁺ or Na⁺ depletion. This transport is inhibited by ouabain and in K⁺-or Na⁺-free medium.     

Mechanisms of Na and Cl absorption across the distal colon epithelium of the pig

Summary Porcine distal colon epithelium was mounted in Ussing chambers and bathed in plasma-like Ringer solution. Tissue conductances ranged from 10 to 15 mS and the short-circuit current (I_sc) ranged from-15 to 220 A·cm^-2. Variations in basal I_sc resulted from differences in the amount of amiloride (10M mucosal addition)-sensitive Na⁺ absorption. Ion substitution and transepithelial flux experiments showed that 10 M amiloride produced a decrease in the mucosal-to-serosal (M-S) and net Na flux, and that this effect on I_sc was independent of Cl^- and HCO ₃ ^- replacement. When the concentration of mucosal amiloride was increased from 10 to 100 M, little change in I_sc was observed. However, increasing the concentration to 1 mM produced a further inhibition, which often reversed the polarity of the I_sc. The decrease in I_sc due to 1 mM amiloride was dependent on both Cl^- and HCO ₃ ^- , and was attributed to reductions in the M-S and net Na⁺ fluxes as well as the M-S unidirectional Cl^- flux. Ion replacement experiments demonstrated that Cl^- substitution reduced the M-S and net Na fluxes, while replacement of HCO ₃ ^- with HEPES abolished net Cl^- absorption by reducing the M-S unidirectional Cl^- flux. From these data it can be concluded that: (1) Na⁺ absorption is mediated by two distinct amiloride-sensitive transport pathways, and (2) Cl^- absorption is completely HCO ₃ ^- -dependent (presumably mediated by Cl^-/HCO ₃ ^- exchange) and occurs independently of Na⁺ absorption.Abbreviations G_t tissue conductance - HEPES tris (hydroxymethyl) aminomethane - (tris) N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - I_sc short-circuit current - J_r residual flux - M-S mucosal-to-scrosal - S-M serosal-to-mucosal - TTX tetrodotoxin     

Basolateral Mg2+/Na+ exchange regulates apical nonselective cation channel in sheep rumen epithelium via cytosolic Mg2+

High potassium diets lead to an inverse regulation of sodium and magnesium absorption in ruminants, suggesting some form of cross talk. Previous Ussing chamber experiments have demonstrated a divalent sensitive Na(+) conductance in the apical membrane of ruminal epithelium. Using patch-clamped ruminal epithelial cells, we could observe a divalent sensitive, nonselective cation conductance (NSCC) with K(+) permeability > Cs(+) permeability > Na(+) permeability. Conductance increased and rectification decreased when either Mg(2+) or both Ca(2+) and Mg(2+) were removed from the internal or external solution or both. The conductance could be blocked by Ba(2+), but not by tetraethylammonium (TEA). Subsequently, we studied this conductance measured as short-circuit current (I(sc)) in Ussing chambers. Forskolin, IBMX, and theophylline are known to block both I(sc) and Na transport across ruminal epithelium in the presence of divalent cations. When the NSCC was stimulated by removing mucosal calcium, an initial decrease in I(sc) was followed by a subsequent increase. The cAMP-mediated increase in I(sc) was reduced by low serosal Na(+) and serosal addition of imipramine or serosal amiloride and depended on the availability of mucosal magnesium. Luminal amiloride had no effect. Flux studies showed that low serosal Na(+) reduced (28)Mg fluxes from mucosal to serosal. The data suggest that cAMP stimulates basolateral Na(+)/Mg(2+) exchange, reducing cytosolic Mg. This increases sodium uptake through a magnesium-sensitive NSCC in the apical membrane. Likewise, the reduction in magnesium uptake that follows ingestion of high potassium fodder may facilitate sodium absorption, as observed in studies of ruminal osmoregulation. Possibly, grass tetany (hypomagnesemia) is a side effect of this useful mechanism.     

Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium

Intracellular microelectrode techniques and intracellular pH (pHi) measurements using the fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were employed to characterize an electrogenic bicarbonate transport mechanism at the apical membrane of the frog retinal pigment epithelium (RPE). Reductions in apical concentrations of both [HCO3]o (at constant Pco2 or pHo) or [Na]o caused rapid depolarization of the apical membrane potential (Vap). Both of these voltage responses were inhibited when the concentration of the other ion was reduced or when 1 mM diisothiocyano-2-2 disulfonic acid stilbene (DIDS) was present in the apical bath. Reductions in apical [HCO3]o or [Na]o also produced a rapid acidification of the cell interior that was inhibited by apical DIDS. Elevating pHi at constant Pco2 (and consequently [HCO3]i) by the addition of apical NH4 (20 mM) produced an immediate depolarization of Vap. This response was much smaller when either apical [HCO3]o or [Na]o was reduced or when DIDS was added apically. These results strongly suggest the presence of an electrogenic NaHCO3 cotransporter at the apical membrane. Apical DIDS rapidly depolarized Vap by 2-3 mV and decreased pHi (and [HCO3]i), indicating that the transporter moves NaHCO3 and net negative charge into the cell. The voltage dependence of the transporter was assessed by altering Vap with transepithelial current and then measuring the DIDS-induced change in Vap. Depolarization of Vap increased the magnitude of the DIDS-induced depolarization, whereas hyperpolarization decreased it. Hyperpolarizing Vap beyond -114 mV caused the DIDS-induced voltage change to reverse direction. Based on this reversal potential, we calculate that the stoichiometry of the transporter is 1.6-2.4 (HCO3/Na).     

Intracellular Na+ activity as a function of Na+ transport rate across a tight epithelium.

Liquid Na+ resin microelectrodes were used to measure intracellular Na+ activities (alpha iNa+) in the mammalian tight epithelium, rabbit urinary bladder. alpha iNa+ averaged 7 +/- 1 mM and was independent of Na+ transport rate over the range of 2 to 8 muA/muF. (1 mF congruent to 1 cm2 apical membrane area). After Na+ loading the cells the Na+ pump in the basolateral membrane was measurably electrogenic. A method for shielding the Na+-sensitive microelectrodes is described which increases the response time and eliminates an electrical shunting artifact.     

Progesterone transport across the wall of the rumen of sheep

Data on content of fiber, lignin and progesterone in reticulo-ruminal chyme and progesterone in blood serum and saliva of sheep throughout the reproductive cycle and at intramuscular injection of progesterone, are presented. A direct correlation between concentration of progesterone in blood and chyme was revealed. 1.5% of progesterone entered the reticulo-rumen with saliva. Transport of hormone through the wall of forestomach is the main way of progesterone entering the reticulo-rumen. The transport is carried out against the gradient of hormone concentration. Possible participation of progesterone adsorption on lignin in explanation of this transport, is discussed.     

Effects of pulsed electromagnetic fields on Na+ fluxes across stripped rabbit colon epithelium

The effect of pulsed electromagnetic fields on the electrical potential and two-way flux of Na+ across the epithelium of the rabbit colon in vitro was investigated. In control experiments the transepithelial mucosal-to-serosal and serosal-to-mucosal fluxes (Jm----s and Js----m) were constant over the experimental period. When the epithelium was at right angles to the applied electromagnetic field, the Jm----s flux of Na+ was reduced, whereas Js----m was enhanced. When the epithelium was rotated 180 degrees, Jm----s was enhanced, whereas Js----m was reduced. When the epithelium was mounted parallel to the magnetic field, both Jm----s and Js----m of Na+ were increased, the latter continuing to increase after the field was turned off. When the tissue was rotated 180 degrees, the same enhanced flux was observed, but now the Jm----s flux showed the greatest increase, which again occurred in the period after the field was turned off. The rate of decrease of transepithelial potential difference in all orientations was less than the control. Also, the conductance increased in orientations 2-4 and decreased in orientation 1 after the field had been applied. This suggests that pulsed electromagnetic fields can have a direct effect on the movement of Na+ across tissue and transepithelial potentials. The mechanism may depend on several factors, such as induced changes in certain ion pumps, the membrane potential, and the surface charge of cell wall proteins.     

Effect of strophanthin on the absorption of sodium and short chain fatty acids from the sheep rumen

In vivo, intraruminal strophantin administration suppressed the sodium absorption by up to 50% in the sheep reticulo-rumen. Absorption of the shortchain fatty acids was not affected. The findings suggest absence of a combined transport of sodium and short-chain fatty acids' anions in basic and lateral membranes of the reticulo-rumen apithelium in sheep.     

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.     

Different modes of electrogenic Na+ absorption in the coprodeum of the chicken embryo: role of extracellular Ca2+

Summary Transepithelial electrogenic Na⁺ transport (I_Na) was investigated in the coprodeum of 20-days-old chicken embryos in Ussing chambers. Short circuit current (I_sc) and transepithelial resistance (R_t) were 14.7±4.8 A · cm^-2 (n=12) and 0.53±0.09 k · cm^-2 (n=12), respectively. I_Na was calculated from changes in I_sc by substitution of mucosal Na⁺ by (N-methyl-d-glucamine) (NMDG). I_sc inversed during Na⁺ removal, and I_Na was found to be 27.8±4.7 A · cm^-2 (n=12). Amiloride (100 mol · l^-1) inhibited only about 60% of I_Na. Analysis of I_sc fluctuations revealed a Lorentzian component in the power density spectrum with a corner frequency of about 57 Hz. This component was not correlated to I_Na, and its origin is still unclear. Removal of mucosal Ca²⁺ increased I_Na about 2.5-fold due to an increase of the amiloride-insensitive component of I_Na in additionally investigated adult tissues. The results clearly show that this is due to a non-selective cation channel with an apparent order of selectivity Cs⁺>Na⁺=K⁺>Rb⁺>Li⁺. The Ca²⁺ concentration required to block 50% of the I_sc was about 18 mol · l^-1. The I _sc ^Ca could also be supressed by other divalent cations such as Mg²⁺ and Ba²⁺. Additionally, an I_Na-linked Lorentzian component occurred which dominated the control spectrum with a significantly higher corner frequency (about 88 Hz). The results indicate that Na⁺ absorption in the coprodeum of the chicken embryo is more complex than in adult hens. However, the Ca²⁺ sensitivity of I_Na is similar to comparable effects described for other epithelia. This possibly reflects the existence of two types of amiloride-insensitive apical cation channels as pathways for Na⁺ absorption, which may be involved to differing degrees in ontogenetic developments of nonselective channels to Na⁺-specific ion channels.Abbreviations DPL direct-linear-plot method - slope of the back-ground noise component - EGTA ethylene glycol-bi(2-amino-ethylether)-N,N,N,N-tetraacetic acid - f frequency - f _c corner frequency of the Lorentzian noise component - G _t transepithelial conductance - HEPES N-hydroxyethylpiperazine-N-ethanesulfonic acid - I _sc short-circuit current - I _Na transepithelial sodium current - I _sc ^Ca Ca²⁺-sensitive short-circuit current - K _m ^Ca Michaelis-Menten constant for Ca²⁺ - K _B power density of the background noise component at f=1Hz - m mucosal - NMDG N-methyl-D-glucamine - R _t transepithelial resistance - s serosal - SEM standard error of mean - S(f) power density of the Lorentzian noise component - S _o plateau value of the Lorentzian noise component