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Department of Physiology, School of Veterinary Medicine, Hannover, Germany
1 Corresponding author: sabine.leonhard-marek{at}tiho-hannover.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: calcium absorption • chloride • rumen • short-chain fatty acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Feeding experiments have shown that the preintestinal localization of Ca absorption gains importance if Ca intake increases (Schröder, 1996; Khorasani et al., 1997). Sheep with a Ca intake of 2.7 g/d showed a preintestinal Ca secretion of 0.8 g/d and an intestinal Ca absorption of 1.6 g/d (Wylie et al., 1985), whereas sheep with a Ca intake of between 4.8 and 13.6 g/d showed a preintestinal Ca absorption of between 0.7 and 4.3 g/d and a Ca secretion in the small intestine of between 1.2 and 2.4 g/d, which was partly compensated for by Ca absorption in the large intestine (Grace et al., 1974). Comparable observations have been made in cows. Lactating cows with a Ca intake of between 115 and 231 g/d showed a preintestinal Ca movement ranging from a small secretion of –0.9 g/d at a low Ca intake to an absorption of 49.8 g/d at a high Ca intake (Khorasani et al., 1997).
The addition of Ca salts to the diets of cows is a frequent strategy at the onset of lactation, because dairy cows lose considerable amounts of calcium to milk production. Compared with an immediately available Ca pool of 3 to 4 g in the circulating blood of a cow (calculated on the basis of 
2.25 mM Ca and 65 mL of blood/kg of BW), the Ca drain with milk can amount to 50 g/d (assuming a content of 1.2 g of Ca/L of milk). To prevent or correct a Ca imbalance in lactating cows, Ca salts are added to their diet (e.g., Ca-chloride, Ca-propionate). After ingesting the diet, these salts dissociate in the rumen fluid; therefore, the respective anions may influence Ca transport rates across the rumen epithelium.
The mechanisms of ruminal Ca absorption have been studied in small ruminants, where different groups have shown the absorption of Ca across the rumen of sheep to be stimulated by short-chain fatty acids (SCFA; Schröder et al., 1997, 1999; Wadhwa and Care, 2000; Uppal et al., 2003) and by feeding concentrate (Uppal et al., 2003). Further, preliminary studies have suggested a stimulatory effect on ruminal Ca absorption by chloride anions (Leonhard-Marek et al., 2000). However, all those experiments were conducted as anion-exchange in vitro studies in which the anions investigated were replaced by gluconate in the control solutions. This may be a disadvantage, because gluconate is capable of binding Ca2+ ions to some extent (Skibsted and Kilde, 1972; Christoffersen and Skibsted, 1975; Kenyon and Gibbons, 1977). This knowledge has not been considered in most measurements of the Ca flux rates.
We therefore studied the distinct effects of chloride, SCFA, gluconate, and sulfate on Ca absorption across isolated epithelia from sheep rumen and measured the effects of these anions on ionized Ca2+ concentrations ([Ca2+]ion) in the bathing solutions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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) at 38°C, where the mucosa was stripped from the underlying muscle layers and the serosa (Leonhard-Marek et al., 1998). The tissue was then cut into smaller pieces of about 2 x 2 cm.
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, buffer A). These SCFA anions were replaced by 60 mM gluconate in the solution bathing the serosal side of the epithelia (buffer C). Both solutions contained 68 mM Cl. In these first experiments, Cl was replaced by gluconate to obtain a low-Cl solution (buffer B).
In the second series of experiments, we (re)evaluated the effects of different anions on Ca flux rates, starting from a control buffer that contained mannitol (and no gluconate, buffer D; Table 1). To obtain different anion solutions, we replaced mannitol by Tris-OH and the respective acid (hydrochloric acid, sulfuric acid, or SCFA, buffers E to G; Table 1).
The compositions of the different solutions are presented in Table 1. All solutions had a pH of 7.4 when gassed with 95% O2 and 5% CO2 at 38°C and an osmolarity of 300 mosmol/L. All chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany) or from Sigma-Aldrich (Deisenhofen, Germany).
Electrical Measurements
Mucosal tissues were mounted between the two halves of Ussing incubation chambers (Department of Physiology, School of Veterinary Medicine, Hannover, Germany) with an exposed area of 1.13 cm2. Damage to the tissue edges was minimized by placing rings of silicon rubber on both sides of the tissues. Incubation chambers were connected to circulation reservoirs (glassblower shop, Hannover Medical School, Hannover, Germany) containing 10 mL of buffer solution on each side. The solutions were maintained at 38°C by a water jacket and were continuously stirred by use of a gas lift system that supplied 95% O2 and 5% CO2. The Ussing chambers were also connected to a computer-controlled voltage clamp device (Mussler Scientific Instruments, Aachen, Germany). Transepithelial potential differences (Vt) were measured through buffer solution agar bridges and calomel electrodes with reference to the mucosal solution. Transepithelial tissue conductances (Gt) were determined from the changes in Vt induced by bipolar current pulses of 100 µA/cm2 of 500 ms duration. The currents were passed through buffer solution agar bridges connected to Ag-AgCl electrodes in 3 M KCl. In each setup, fluid resistances and junction potentials were measured before mounting the mucosal tissues and were corrected for during the experiments. The experiments were performed under short-circuit conditions, unless otherwise stated.
Ca Flux Rates
Unidirectional flux rates of Ca were measured using 45Ca as a tracer, which was added as 45CaCl2 (specific activity >370 GBq/g; PerkinElmer Life Sciences, Rodgau-Jügesheim, Germany) in each chamber to the mucosal or the serosal side of the epithelia. After an equilibration period of 20 min, 10 to 12 buffer samples of 500 µL were taken at 30-min intervals and replaced by aliquots of the same unlabeled solution. Radioactivity was measured in a conventional liquid scintillation counter (Tricarb Packard, Dreieich, Germany). Flux rates were calculated from the rate of tracer appearance on the other side of the epithelia. In the absence of any manipulation, flux rates were stable from the second to the ninth flux period in the epithelia used for the experiments under short-circuit conditions, and from the third to the eleventh flux period in the epithelia used for the voltage clamp experiments. On the basis of steady-state values, a mean flux rate was calculated for each epithelial sheet and each condition. The effects of different anions on Ca flux rates were tested with adjacent pieces of epithelia from the same sheep.
To study the effect of different transepithelial potential differences on Ca flux rates, we incubated 2 epithelia for each buffer solution and each flux direction from each sheep. One of these tissues was subsequently clamped to –25, +25, and 0 mV, and the other tissue was clamped to +25, –25, and 0 mV. To exclude an influence of the sequence of the applied potential differences, the steady-state fluxes measured at the respective Vt were averaged between the 2 tissues. Unidirectional fluxes measured at different potential differences were divided into their electrogenic and electroneutral components according to a mathematical model described by Frizzell and Schultz (1972): J = Jd ·
–0.5 + Jm, and = e(zFV/RT), where J is the total unidirectional flux of an ion, Jd · 0.5 is its diffusible (electrogenic) part, and Jm is its electroneutral (voltage-independent) proportion. V gives the potential difference across the epithelium in flux direction, z is the valence of the ion, F is the Faraday constant, R is the gas constant, and T is the absolute temperature.
Calcium flux rates were measured 1) in the presence of gluconate or chloride (corresponding to the classical ion-exchange experiments) and at different transepithelial potential differences (allowing for the calculation of electrogenic and electroneutral flux components); 2) in the presence of gluconate and chloride (to follow up on the distinct effects of each anion on the same tissues; see below); and 3) in the absence of gluconate (allowing us to monitor the specific effect of an anion in relation to a mannitol control).
For the second purpose, we added gluconate to a high-Cl solution or Cl to a high-gluconate solution. The addition of these salts increased the osmolarity by about 100 mosmol/L on the luminal side. This increase is in the range of changes that can occur after food intake (Warner and Stacy, 1965; von Engelhardt, 1969) and induces changes in transepithelial conductance that can be measured in in vitro studies. These increases in Gt have been attributed to an increase in passive paracellular movements (Schweigel et al., 2005). Apart from these osmolarity-induced increases in Gt, the transepithelial conductance, which was taken as a measure of tissue viability, remained stable for the duration of the experiments.
Ionized Ca2+
The concentrations of free Ca2+ ions in the solutions were measured with a blood gas and electrolyte analyzer (Bayer Diagnostics, Fenwald, Germany) equipped with an ion-selective Ca2+ electrode. Samples were analyzed at 37°C and corrected to a pH of 7.4. The ionized Ca2+ concentration was measured 1) in gluconate and Cl solutions at different times, in parallel to the protocol that had been used for the flux experiments; 2) after addition of gluconate or chloride; and 3) in mannitol, sulfate, and SCFA solutions.
Statistics
Results are given as means ± standard error of the means. Unless otherwise stated, n represents the number of tissues. Wherever possible, 2 pieces of tissues were incubated per animal and per experimental condition. Epithelial preparations that showed large unspecific increases in transepithelial conductance were excluded from the experiments. Most experiments were conducted with tissues from a group of 6 animals; in 2 instances, 9 and 4 animals were available. Statistical significance was evaluated using ANOVA (one-way) if 3 or more groups had to be compared. For the comparison of 2 conditions, we used Student’s t-test, paired or unpaired as appropriate. A value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). These SCFA anions were replaced by gluconate on the serosal side of the epithelia (buffer C). Both solutions contained 68 mM Cl. Under this high-Cl condition, we observed a Ca flux rate from the mucosal to the serosal side (JCams) of 20.3 ± 1.4 nmol cm–2 h–1 (n = 12; Figure 1) and a Ca flux rate from the serosal to the mucosal compartment (JCasm) of 2.3 ± 0.4 nmol cm–2 h–1 (n = 6), resulting in a significant net Ca absorption (JCanet = JCams – JCasm) of 18.0 ± 1.1 nmol cm–2 h–1 (n = 6) across the rumen epithelium.
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), but JCams was significantly reduced to 13.4 ± 0.7 nmol cm–2 h–1 (n = 12, P < 0.001 vs. high Cl). The JCanet was reduced to 10.7 ± 1.1 nmol cm–2 h–1 (n = 6).
The results (Figure 1) showed a significant effect of Cl (or gluconate) on JCams under short-circuit conditions (at zero electrochemical potential). Subsequently, we measured JCams and JCasm at +25, 0, and –25 mV of Vt. The analysis of these Ca flux rates at varying Vt (according to Frizzell and Schultz, 1972; see the Materials and Methods section) and linear regressions of the Ca fluxes on the electric driving force (–0.5) resulted in the following equations (n = 27 observations in 9 sheep):
High-Cl
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Low-Cl
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A comparison of the Cl effects on the basis of the number of animals (n = 9) showed that Cl increased both the electrogenic and the electroneutral component of JCams (P < 0.01). Chloride also had a small but significant effect on the electroneutral component of JCasm (P < 0.05), whereas the electrogenic component of JCasm remained unaffected.
Ca Flux Rates in the Presence of Gluconate and Chloride
This first study, corresponding to the classical ion-exchange experiments, did not allow us to discriminate between a stimulatory effect of Cl or a blocking effect of gluconate. We therefore simulated an additional salt intake after the fifth flux period and added 58 mM NaCl to the luminal side of the epithelia bathed in the low-Cl (and high-gluconate) buffer. At the same time, adjacent epithelia that were incubated in a high-Cl (and low-gluconate) buffer received an addition of 58 mM Na-gluconate to the luminal side (Figure 2). Both groups of epithelia thus faced the same composition of buffer solution during the final flux rates.
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Despite the changes in the paracellular pathway, the addition of Cl ions to the low-Cl, high-gluconate solution had no effect on JCams (14.0 ± 1.1 nmol cm–2 h–1 before vs. 13.1 ± 2.4 nmol cm–2 h–1 after addition of chloride, n = 6). In contrast, the addition of gluconate to the high-Cl, low-gluconate solution reduced JCams from 18.9 ± 1.8 nmol cm–2 h–1 (before addition of gluconate) to 11.5 ± 0.5 nmol cm–2 h–1 (n = 6, P < 0.01; Figure 2). These data showed that, in a standard ion-exchange experiment, the higher Ca flux rates in a Cl buffer were not due to a stimulating effect of Cl, but could be attributed to a blocking effect of gluconate in the control solution.
Ionized Ca2+ Concentration in Gluconate and Cl Solutions
The extent of JCams reduction attributable to gluconate was rather astonishing. We therefore expanded the study by measuring the concentration of free Ca2+ ions in all buffer solutions (Figure 3). All buffer solutions had a total Ca concentration of 1.2 mM. Of these we found 0.8 ± 0.02 mM [Ca2+]ion in the high-Cl (SCFA) solution (buffer A; n = 4 freshly prepared solutions), but only 0.2 ± 0.0 mM [Ca2+]ion in the low-Cl solution (high-gluconate and SCFA, buffer B; n = 3 solutions). The buffer solution on the serosal side (high-Cl, high-gluconate, no SCFA, buffer C) showed a [Ca2+]ion concentration of 0.3 ± 0.03 mM (n = 4 solutions). These concentrations of [Ca2+]ion did not change between measurements carried out in the buffer reservoir in the Ussing chambers 5 min before the placement of the epithelia and 300 min after incubation of the epithelia (Figure 3), indicating that the tissues themselves had no effect on the concentration of [Ca2+]ion. We then repeated the Ca flux protocol and added Na-gluconate or NaCl (58 mM each) to the buffer solutions on the mucosal side. Gluconate immediately decreased the concentration of free Ca2+ ions to 0.3 ± 0.01 mM (P < 0.001, n = 5; Figure 3), in parallel with the reduction of the Ca flux rates shown above (JCams; Figure 2). Chloride, which had no effect on JCams (Figure 2), had also almost no effect on the concentration of free Ca2+ ions (0.27 ± 0.01 mM before and 0.29 ± 0.01 mM after addition of 58 mM NaCl, P = 0.07, n = 5; Figure 3).
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Effect of Anions on Ca Fluxes (Without Gluconate)
In a second series, we (re)evaluated the effects of different anions on Ca flux rates, starting from a control buffer that contained mannitol (and no gluconate; buffer D in Table 1). To obtain different anion solutions, we replaced mannitol by Tris-OH and the respective acid (hydrochloric acid, sulfuric acid, or SCFA; buffers E to G in Table 1 and Figure 4).
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Ionized Ca2+ Concentration in Cl, SCFA, and Sulfate Solutions
To compare the observed Ca flux rates to the concentration of free Ca2+ ions, we again measured the concentration of ionized Ca2+ in these solutions. As in the first series, the total Ca concentration amounted to 1.2 mM in all buffer solutions. These measurements showed that not only gluconate, but also sulfate and SCFA could reduce the concentration of ionized Ca2+ (Table 2), which was not always correlated with a reduction in the Ca flux rate.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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) and found that Cl is indeed able to stimulate JCams and net Ca absorption from the rumen (Figure 4).
Calcium absorption across the sheep rumen epithelium is at least partly active (Höller et al., 1988; Schröder et al., 1997) and involves electrogenic and electroneutral components (Höller et al., 1988; Wadhwa and Care, 2000), which was confirmed here. The electrogenic component of JCams cannot be explained by mere paracellular transport, because it clearly differed from the respective component of JCasm. The majority of potential-dependent JCams has therefore to be considered as an electrogenic transport through the cells (Figure 5). Both components of JCams, electrogenic and electro-neutral, were stimulated by mucosal Cl in the present study.
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Luminal Cl is, however, able to increase the pH at the epithelial surface via a stimulation of apical localized Cl–/HCO3– or Cl–/OH– transporters (Figure 5; Leonhard-Marek et al., 2006). In vivo studies with the temporarily isolated rumen have shown that an increase in luminal pH can in turn increase ruminal Ca absorption (Wagner, 1998). The structures of the proteins involved in ruminal Ca transport are not yet known, but we have recently shown that the apical membrane of rumen epithelia from sheep and goats exhibits a nonselective cation conductance, which allows for a current of monovalent cations in the absence of divalent cations and is regulated by extracellular Ca2+ and Mg2+ as well as by intracellular Mg2+ ions (Leonhard-Marek, 2002; Leonhard-Marek et al., 2005). This phenomenon, the passage of monovalents in the absence of divalent cations, is a feature common to many Ca2+ channels (Sather and McCleskey, 2003; Hoenderop et al., 2005). In the rumen, the Ca2+ channel agonist Bay K 8644 (50 nM) was able to increase JCams (Wadhwa and Care, 2000), whereas verapamil, a channel blocker, decreased net Ca absorption (Schröder et al., 1997; Wadhwa and Care, 2000), which could indicate the presence of L-type Ca2+ channels in the rumen epithelium. Although many epithelia use transient receptor potential vanilloid-type Ca2+ channels for Ca absorption (Hoenderop et al., 2005), L-type Ca2+ channels have been suggested to contribute additionally to intestinal Ca absorption (Morgan et al., 2003). Both the epithelial transient receptor potential vanilloid-type Ca2+ channels and L-type Ca2+ channels increase their conductance with alkaline pH (Pietrobon et al., 1989; Vennekens et al., 2001). An increase in mucosal Cl concentration in the rumen could thus stimulate the electrogenic part of ruminal Ca absorption via an increase in surface pH followed by a pH-dependent increase in luminal Ca2+ conductance (Figure 5).
For the electroneutral part of ruminal Ca absorption, a Ca2+/2H+ exchange mechanisms has been suggested (Schröder et al., 1997; Wadhwa and Care, 2000). An increase in mucosal Cl concentration increases the activity of luminal Cl–/HCO3–(OH–) exchange. This has been shown by an increase in bicarbonate secretion (Gäbel et al., 1991), an increase in surface pH (Leonhard-Marek et al., 2006), and an increased recovery of isolated cells from an alkaline load in the presence of extracellular Cl (Bilk et al., 2005). Therefore, the Cl-dependent stimulation of electroneutral Ca absorption in the present study may be the consequence of an interaction between Cl–/HCO3–(OH–) and Ca2+/2H+ exchange via intracellular pH or surface pH (Figure 5).
Effects of SCFA on Ca Absorption
A stimulatory effect of SCFA on Ca absorption across the sheep rumen epithelium has been reported by different groups (Schröder et al., 1997, 1999; Wadhwa and Care, 2000; Uppal et al., 2003) and has recently also been shown with rumen epithelia from slaughtered cattle (Ricken 2005). However, all these studies used buffer solutions in which 25 to 100 mM SCFA were replaced by gluconate in the control solution. The current experiment demonstrated that the addition of 58 mM gluconate to a standard rumen buffer decreased the concentration of free Ca2+ ions from 0.8 to 0.3 mM (Figure 3). This implies that the change from a control (gluconate) solution to an experimental (gluconate-free) solution may more than double the concentration of free Ca2+ ions. We therefore hypothesized that the stimulatory effect of SCFA might alternatively have been a blocking effect of gluconate on Ca flux rates under "control" conditions. When JCams was measured in SCFA solutions without gluconate (19.2 ± 3.0 nmol cm–2 h–1, n = 7; buffer G; SCFA in Figure 4) or with gluconate (13.4 ± 0.7 nmol cm–2 h–1, n = 12; buffer B; low Cl in Figure 1), we found that gluconate could reduce Ca flux rates in the presence of SCFA (P < 0.05). In the same buffers, we also observed a huge difference in [Ca2+]ion, ranging from 0.77 ± 0.01 mM in the presence of SCFA alone (n = 4, buffer G) to 0.2 ± 0.0 mM in the presence of SCFA and gluconate (n = 3, buffer B). However, when we exchanged mannitol for a combination of Tris-OH and SCFA without increasing the free Ca2+ concentration (Table 1), we could confirm that SCFA were indeed able to stimulate net Ca absorption across the rumen epithelium because of an increase in JCams (Figure 4). This means that the effects attributed to SCFA in the stimulation of Ca flux rates have been overestimated in previous studies, because gluconate should have decreased the control Ca flux rates in those experiments (Schröder et al., 1997, 1999; Wadhwa and Care, 2000; Uppal et al., 2003; Ricken 2005). However, the general conclusion of those studies was correct, that SCFA stimulate ruminal Ca absorption.
The current understanding of apical SCFA uptake in rumen epithelial cells involves the permeation of protonated SCFA, which are lipid soluble, and the uptake of SCFA– anions in exchange for HCO3– (Gäbel et al., 2002). A stimulation of these pathways by an increase in luminal SCFA concentration will provide protons to the cell interior and bicarbonate to the epithelial surface. This should stimulate a pH-dependent Ca2+ conductance as well as an apical localized Ca2+/2H+ exchanger, as discussed above (Figure 5). The SCFA could thus use the same mechanisms to stimulate Ca absorption, as discussed above for the stimulation of Ca absorption by luminal Cl– ions. This conclusion is supported by a comparison of JCams at a high-SCFA and low-Cl luminal concentration (19.2 ± 3.0 nmol cm–2 h–1, n = 7; Figure 4) with the corresponding flux rate at a high SCFA and high Cl– luminal concentration (20.3 ± 1.4 nmol cm–2 h–1, n = 6; Figure 1). Considering these data, Cl seems to have no further effect on Ca absorption in the presence of SCFA.
Given the electrochemical gradients across the baso-lateral membrane (negative cell interior and low intra-cellular Ca2+ concentration), Ca extrusion to the blood side is an energy-dependent process. This Ca extrusion could be driven by Ca2+-ATPase (Schröder et al., 1997) or by a Ca2+/Na+ exchange energized by the activity of the Na+/K+-ATPase (Höller et al., 1988; Schröder et al., 1999). Intraepithelial metabolism of SCFA, especially of n-butyrate, is the predominant energy source in the rumen epithelium (Baldwin and Jesse, 1992). The SCFA therefore might additionally stimulate ruminal Ca absorption by the provision of ATP. Comparison of the Ca fluxes (JCams) in the presence of Cl (14.5 ± 1.4 nmol cm–2 h–1, n = 8; Figure 4) with those in the presence of Cl and SCFA (20.3 ± 1.4 nmol cm–2 h–1, n = 6; Figure 1) indicated an additional SCFA effect on JCams in the presence of Cl (P < 0.01 between both groups), which would agree with the hypothesized metabolic stimulation (Figure 5).
Concentration of Free Ca2+ Ions in Physiological Buffer Solutions
In the freshly prepared solutions in the first experiment, [Ca2+]ion amounted to 0.75 mM in the high-Cl solution (1.2 mM gluconate) and to 0.22 mM in the low-Cl solution (58.2 mM gluconate). Because the addition of gluconate reduced [Ca2+]ion, whereas the addition of Cl had no effect (Figure 3), the values cited above can be interpreted as a reduction in [Ca2+]ion by 0.53 mM because of the 57 mM gluconate.
The association of Ca2+ ions with gluconate– can be calculated as Kass = [Ca-gluconate+]/(Ca2+ activity · [gluconate–]). For the measurements given above, Kass = 42.66 and log Kass = 1.63. This value is in agreement with the log Kass of 1.81 calculated by Christoffersen and Skibsted (1975). To estimate Kass for sulfate and SCFA, we used the Ca2+ concentrations given in Table 2. The addition of 30 mM sulfate reduced [Ca2+]ion from 1.11 to 0.36 mM, resulting in a Kass of 71.23; the addition of 60 mM SCFA reduced [Ca2+]ion from 1.11 to 0.77 mM, resulting in a Kass of 7.4. The same equation can be used to calculate the concentration of ionized Ca2+ that can be expected in solutions with increasing concentrations of gluconate, sulfate, or SCFA (Figure 6).
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These data shown in Figure 6 can be used as an estimate of [Ca2+]ion reduction in comparable solutions. However, differences in ionic composition between solutions will affect [Ca2+]ion (Günzel et al., 2005). In experiments in which [Ca2+]ion is crucial, it should be measured instead of calculated.
Misestimation of Flux Rates and Possible Effects on Paracellular Permeability
Studies of nutrient and electrolyte transport mechanisms across the forestomach or hindgut epithelia often use an SCFA gradient to mimic what happens in vivo. On the blood side, this amount of SCFA is frequently replaced by gluconate in the respective solutions, because gluconate gradients are considered to have only negligible effects on transport rates. However, in light of the reduced free Ca2+ concentration in a gluconate buffer, as shown above, such a strategy would lead to a misestimation of flux rates from the serosal to the mucosal side, if the flux rate is at all dependent on the available concentration of free Ca2+ ions. For Ca flux rates, this dependence is a direct one.
When the experiments in the present study were compared, there was a significant difference in JCasm between the 2 control conditions (gluconate and mannitol), which was correlated with a difference in free Ca2+ ions. All epithelia bathed in a gluconate solution ([Ca2+]ion = 0.31 mM) on the serosal side showed a mean JCasm of 2.75 ± 0.31 nmol cm–2 h–1 (n = 18), whereas epithelia bathed in the mannitol control solution ([Ca2+]ion = 1.11 mM) showed a higher JCasm of 5.40 ± 0.42 nmol cm–2 h–1 (n = 20, P < 0.001 vs. gluconate solution). This higher transport rate was not due to a higher transepithelial conductance in the second group of tissues. Tissues bathed in the mannitol solution even showed a lower Gt (1.13 ± 0.05 mS cm–2) than those bathed in the gluconate solution (Gt = 2.21 ± 0.13 mS cm–2, P < 0.001), which might point to a higher paracellular permeability in a gluconate solution because of the reduction in [Ca2+]ion.
Besides the increased [Ca2+]ion, an additional explanation for the higher JCasm in the mannitol control solution might be the lower Na concentration in these solutions. (To study the isolated effect of anion addition and end up with the same anion concentrations as in the first experimental series, we had accepted a difference in Na concentration between the 2 series as the smaller deviation.) Up to now, no structural or functional studies have been conducted on the mechanisms mediating basolateral Ca efflux from the rumen epithelium, but the observation that ouabain, a blocker of Na+/K+-ATPase, reduced JCams and increased JCasm (Höller et al., 1988) would support the involvement of Na+/Ca2+-exchangers at the basolateral membrane. The existence of a significant electroneutral component of JCasm (Höller et al., 1988; present study) could further point to a transcellular Ca secretion via Ca exchangers operating in reverse mode. Thus, a reduction in serosal Na concentration together with the increase in [Ca2+]ion might entail basolateral Ca uptake and an increase in JCasm. However, we did not observe a decrease in JCams under the conditions of a reduced Na concentration (cf. SCFA in Figure 4 vs. high Cl and SCFA in Figure 2), which argues against a major contribution of Na+/Ca2+-exchange for transcellular Ca absorption or against its operation in reverse mode despite the reduced Na concentration.
Although we did not find differences between JCasm measured with different solutions on the mucosal side, an altered [Ca2+]ion on the mucosal side might likewise alter the chemical gradient and thereby have an effect on JCasm, if the contribution of other variances is small. This might be one cause for the decreased electroneutral component of JCasm measured in a high-Cl buffer mucosal ([Ca2+]ion, mucosal = 0.8 mM) compared with the value measured in a low-Cl (high-gluconate) solution on the mucosal side ([Ca2+]ion, mucosal = 0.2 mM). The possible misestimation of Jsm flux rates attributable to gluconate solutions discussed here will, as a consequence, lead to a misestimation of net absorption rates, because Jnet = Jms – Jsm.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Based on the effects of Cl and SCFA on ovine ruminal Ca absorption in the present study, and the effects of SCFA on bovine ruminal Ca absorption (Ricken, 2005), Ca salts included in the diets of dairy cows postpartum should be supplied as chlorides (in concentrations or formulations that are not caustic) or as salts of SCFA, preferably as propionate because of its additional glucogenic and energetic benefits.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication June 9, 2006. Accepted for publication October 2, 2006.
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