4.5.1 Effect of Na on the K induced current
Changes in short circuit current after an increase in mucosal K concentration could be caused by a higher K absorption or by a K dependent increase in Na absorption [30,31]. To investigate the contribution of Na to the observed effects we compared currents in the pre- and absence of Na on the mucosal side. In rumen, the K dependent currents did not differ between the presence or absence of Na, thereby excluding a Na effect on the K dependent currents. In contrast, the presence of Na increased the K dependent currents in proximal colon (Fig. 3). While K absorption in the distal colon is described as Na independent [32], a competition between mucosal K and Na has been described for the proximal colon [33]. This should however result in decreased K currents in the presence of Na, and higher effects at low K concentration, which we did not observe. In jejunum, the presence of Na decreased the K dependent currents, while the currents across abomasal epithelia showed no or only slight effects in the presence of Na. Due to all these slight and different Na effects on the K currents in epithelia from abomasum, jejunum and colon, we worked with Na free buffer solutions in the subsequent experiments.
4.5.2 Measurement of K absorption with Rb
The usual way to investigate the absorption of electrolytes in the Ussing chamber system is by use of radioactive isotopes. For the investigation of K transport processes 42K as well as 86Rb have been used as tracers. Both isotopes have a relatively high radiation. Furthermore, 42K has a very short half-life. For this reason most studies use Rb isotopes to measure K transport.
Calculations of the Na+/K+ATPase-mediated influxes of K based on 86Rb or 42K as tracer yielded very similar values for both isotopes in smooth muscle cell preparations [34,35] and skeletal muscle [18]. However, the effluxes calculated with the aid of 86Rb ranged from 56 to 82 % of the 42K efflux, dependent on the state of stimulation and the added chemicals in that study.
In rat and mouse soleus muscle Rb transport experiments wrongly suggested the existence of a Na-K-2Cl-cotransporter [36]. Using 86Rb as K tracer in smooth muscle cells resulted in a calculation of a reduced efflux rate [37]. For the Kv1.3 K channel, a voltage-gated K channel which is also described in the intestine [38], the required verapamil concentration to inhibit 50 % of the current in patch-clamp experiments current was 3.2 fold higher with intracellular Rb than with intracellular K [39].
Obviously, Rb and K are not transported in the same way in all pathways of K transport. The mechanisms of intestinal K transport in ruminants are mostly unknown. So, we had to investigate, if Rb could be a reliable tracer for K in the gastrointestinal tract of ruminants.
In our own experiments, half to two-thirds of the K current were not represented by Rb currents (Fig. 4). In cattle rumen a direct comparison between the K and Rb currents did not show any correlation. A re-calculation of data from sheep rumen [40],
that had been measured with the aid of 42K and 86Rb in the presence of 2.5 mmol·l-1 K and 2.5 mmol·l-1 Rb on both sides of the tissues, supported the results of the current study. In these former experiments, the absorptive Rb flux from the mucosal to the serosal compartment amounted to 33 % of the respective K Flux (R2 = 0.49), while the Rb flux in the opposite direction from serosal to mucosal amounted to 61%
of the respective K flux (R2 = 0.86). Thus, since the Rb current reflected only a part of the K current (or showed no correlation at all), we used atomic absorption spectroscopy to determine K absorption directly.
4.5.3 Measurement of K absorption with atomic absorption spectroscopy When [K]0 was set to 4 mmol∙l-1 K on both sides an increase of the mucosal K concentration was correlated with a decrease of the serosal K concentration in the rumen and jejunum, pointing to a K secretion under these conditions. In colon and abomasum, the increase in mucosal concentration was only paralleled with a decrease by trend in the same experiments. These discrepancies between mucosal and serosal changes in K concentrations at 4 mmol∙l-1 K mucosal could be due to a liberation of intracellular K because of some cell damages in colon and abomasum.
To achieve the measured changes in K concentration 0.8 cm2 (abomasum) and 1.1 cm2 (colon) of the surface has to be damaged [41]. At mucosal K concentrations of 100 mmol∙l-1 such an increase in mucosal K concentration was no longer observed.
High mucosal K concentrations decreased with all epithelial preparations during the incubation period. The calculated decreases in mucosal K concentration were much higher, however, than the increases in serosal K concentration and had higher deviations. This might be due to technical reasons. To measure the K concentration via atom absorption spectroscopy we had to dilute the samples containing about 100 mmol∙l-1 K in proportion 1:500, which was made in two steps. The samples containing about 4 mmol∙l-1 K where only diluted by 1:50 and obviously included less errors of measurement. The K absorption was therefore calculated from the change in serosal K concentrations, were we did not observe problems like those described above.
In the rumen and colon the calculated K absorption rates correlated significantly with the respective K induced currents. In rumen, the increase in K absorption amounted to 80±22 % (R² = 0.32) of the increase in K current. In colon, the increase in K absorption amounted to 69±17 % (R² = 0.46) of the increase in K current.
4.5.4 K transport across the rumen
Wylie [1] showed, that the preintestine of sheep secrets K when the K intake is low (4.31 g∙d-1). These findings correspond with those observed in cows, where a preintestinal secretion of 18 g/day on a K intake of about 200 g/day could be observed [5] and in lambs who showed a K secretion of 0.72 g/day at an intake of 5.5 g/day [42]. In contrast, the rumen absorbs K when the ruminal K concentration raises threefold over the plasma K concentration [6]. Khorasani et al. [5] developed the following formula for the net absorption of K before the small intestine (Y):
Y = 104.2 + 0.43X, P < .01, r2 = .41, X = intake, this equation also fits to the K uptake and absorption values reported by Greene et al. [4]. Conversely, Warner & Stacy [43]
calculated positive K absorption rates only with ruminal K concentrations higher than 100 mmol∙l-1.
In our current experiments with low K concentration on both sides of the rumen epithelia an increase of the mucosal K concentration was paralleled with a decrease of the serosal K concentration, pointing to a K secretion under these conditions. This is in line with the above mentioned in vivo data at low K intake and with previous absorb relevant amounts of K at high luminal K concentration. Additionally, we saw a linear increase in short circuit current when raising mucosal K concentrations between 4 and 100 mmol∙l-1 (Fig. 2). A phenomenon that has also been observed by Scott [6]. The observed increase in K absorption of 2.5 µeq∙cm-2∙h-1 when changing from 4 to 100 mmol∙l-1 K (Fig. 9) corresponds to an amount of 0.7 mol per day across the estimated 1.2 m² of ruminal surface in sheep [7]. This equals the amount of K absorption observed in vivo by Scott [6], but is higher than the amounts observed by Warner and Stacy [43] at a K concentration of 100 mmol∙l-1.
The K concentrations that can be obtained in the rumen amount to 35 per cent of the intake according to Scott [6]. Calculating with a K concentration in forage of 15 – 30 g∙kg-1 dry matter and a dry matter intake of 3 - 3.8 % of body mass [45], a ration of 18 kg forage may contain 270 – 540 g or 6.9 - 13.8 mol K. 35 % of this amount [6]
diluted in a total of 100 l rumen liquid amounts to a calculated K concentration of 24 to 48 mmol∙l-1. For bulls K concentrations of 50.3 mmol∙l-1 have been described [46].
Bailey [47] found K concentrations in the rumen fluid of cows between 24 and 85 mmol∙l-1. Under Na-deficient conditions, the stimulation of the salivary glands with aldosterone may even rise the ruminal K concentration up to 100 mmol∙l-1 [48,49].
According to Scott [50] the ruminal K absorption can be estimated by the following formula: K absorption (mmol/day) = 5.1 ∙ (ruminal K concentration) – 54. According to this equation, every ruminal K concentrations above 10.6 mmol∙l-1 should cause a K absorption. Thus, we have to assume, that the rumen absorbs K in a physiological situation.
Since the amounts of K absorption and short circuit currents were in the same order of magnitude, the current appeared to express ruminal K absorption. We therefore conclude that an electrogenic K transport seems to dominate in the rumen. This electrogenic K transport across the rumen is mediated via barium and verapamil sensitive K channels and a TAP sensitive paracellular component. Based on our
blocker experiments, 70 % of the K absorption might be via transcellular and 30 % via paracellular pathways. Stumpff [51] concluded a predominantly cellular K transport as well. Parthasarathy & Phillipson [52] assumed a passive K transport because of the reversibility of the K flow, dependent of the concentration ratio across the rumen wall. The transcellular component of ruminal K transport at low mucosal K concentration seems to respond to the following transport model [40,53]: a basolateral K uptake by the Na-K-pump and a recycling via basolateral K channels as well as a secretion to the rumen fluid via K channels in the luminal membrane. In the absence of a K gradient, addition of barium chloride as a K channel blocker to the mucosal side resulted in a decrease of K secretion, while an addition of barium chloride to the serosal side increased the K secretion [40]. At high K concentrations, when K is absorbed, a K channel block of K channels with barium or verapamil (Fig.
9) decreased the K absorption. This argues for a cellular K transport via barium and verapamil sensitive K channels with changing directions according to the K concentration. The existence of K channels in rumen epithelial cells has also been shown in patch clamp experiments [54,55,56].
The paracellular pathway via solvent drag has not the same relevance as in the small intestine, because of the differences in epithelial resistance, but obviously K can be transported via the paracellular pathway in a substantial amount (Fig. 9 rumen, TAP).
This transport is passive and determined by the electrochemical gradient.
4.5.5 K transport across abomasum
The abomasum is anatomically and histologically comparable to the simple stomach of other mammals [57]. The continuous flow of ingesta from the reticulo-rumen to the abomasum is responsible for its continuous secretory activity, abomasal glands are not able to secrete spontaneously [54]. In the stomach of monogastrians K plays an essential role for the secretion of hydrochloric acid. The H+/K+ATPase secrets H+ against the concentration gradient [59]. The coupling between H+ export and K uptake requires a luminal presence of K for H+ secretion. Apical K channels then allow a K recycling. Diverse apical K channels are described in the parietal cells of mice. The KCNQ1 channel, a voltage-activated K channel, is obviously responsible for the K recycling [60,61], while the function of other K channels in the apical membrane is still open [62]. Basolateral K channels are described in the frog [63] and in rabbit [64] and guinea pigs [65]. Reenstra et al. [66] showed a gastric K absorption in frog of 0.07 µeq∙cm-2∙h-1 in vitro. An in vivo study in dogs showed a gastric K absorption of 0.11 % of the infused K [66]. Both studies were arranged in the absence of a K gradient. In abomasal pouches of sheep, no K absorption was observable [10]. In our own studies the abomasal epithelia showed short circuit currents of 4 to 5 µeq∙cm-2∙h-1, comparable with those in the frog (4 µeq∙cm-2∙h-1) [65].
This short circuit current did however neither respond to an increase in the mucosal K concentration in the absence of Na (Fig. 1 to 3), nor did it change with Na in the presence of low K (Fig. 3). In the presence of 50 mmol·l-1 K, Na had a slight effect on the current.
The K absorption, in contrast, increased by 3.47 µeq∙cm-2∙h-1 when increasing the mucosal K concentration from 4 mmol∙l-1 to 100 mmol∙l-1. This increase in K
absorption was found to be in the same order of magnitude as in the other epithelia.
Neither Δ K absorption in rumen and colon nor in the jejunum did significantly differ from Δ K absorption in the abomasum. The abomasum is obviously able to absorb K in the same magnitude as the jejunum. The absence of an increase in short circuit current suggests an electroneutral pathway of K absorption.
A K transport through the H+/K+APTase is conceivable. Reenstra et al. [66] observed a change from absorption to secretion after blocking the H+/K+ATPase and an increased absorption after blocking the Na+/K+ATPase. The finding, that K was secreted after blocking the H+/K+ATPase is in line with the existence of suggested luminal K channels. In the current study the addition of barium decreased the current by 0.43 µeq∙cm-2∙h-1, pointing to a contribution of apical K channels to K absorption
4.5.6 K transport across jejunum
The small intestine is assumed to be the main site of gastrointestinal potassium absorption [1-3]. In ruminants the absorption of K in the small intestine appears to be qualitatively similar to that shown by non-ruminants [68]. Depending on the K intake up to 100 % of the K is absorbed in the jejunum [1]. An in vivo study in the ileum of sheep observed a net absorption of potassium of 0.64 meq∙h-1 with a mucosal K concentration of 15.7 meq∙l-1 [10].
Earlier studies postulated a passive pathway for K absorption in the jejunum [69,70].
The K absorption is stated as paracellular via solvent drag, due to the high porosity of the zonulae occludentes. [71]. On the other hand, Inagaki et al. [24] supposed a transcellular component of K absorption in the small intestine of mouse and Woodard et al.[72] showed an active K absorption in the piglet jejunum. Cermak et al. [73]
described K secretion via a cellular pathway in rat distal jejunum as well as in lambs [74]. Binder & Murer [75] described a K/Hydrogen exchange in brush border membrane vesicles of the rat ileum. Measurements in the mouse small intestine at a mucosal K concentration of 5.4 mmol∙l-1 K showed an Isc of 0.7 µeq∙cm-2∙h 1 [24], while the Isc at mucosal K concentrations of 5.2 mmol∙l-1 in the jejunum of piglet was 2.24 µeq∙cm-2∙h-1 [72]. Both studies were arranged in the absence of a K gradient and in the presence of Na. In our own study the Isc in goat jejunum amounted to 1.28±0.20 µeq∙cm-2∙h-1 at 4 mmol∙l-1 K mucosal and in the presence of Na. In the absence of Na the current decreased. Increasing the mucosal K concentration from 4 to 100 mmol·l-1 resulted in an increase of current across jejunum of goat and sheep.
This increase in the K current was linearly correlated with the mucosal K concentration (R² = 0.87) and was only seen in the absence of luminal Na. Compared to the K-dependent increases in current across rumen and colon, the increases in K current across sheep jejunum were however marginal. The increases in K absorption
in contrast were significantly higher in jejunum than in rumen (1.83 fold) and colon (1.56 fold), but did not differ from those observed in the abomasum.
In rat jejunum, the K absorption was also largely independent from changes in Isc.
Large parts of the jejunal K transport seemed to be electroneutral [73]. An apical K+/H+ATPase that may be a putative pathway for electroneutral K absorption was, however excluded by these authors to participate in K absorption across rat distal jejunum. In contrast, an apical K+/H+ATPase is described in the rat ileum [75] as well as in the Amphiuma jejunum [76].
The commonly postulated passive and paracellular K transport [77,78] should be electrogenic. In line with this assumption, the addition of TAP, a blocker of the paracellular pathway, decreased the K current across the jejunum by 29±7 % in sheep.
The K channel blocker barium decreased the K current by 44±12 % pointing again to a significant contribution of luminal K channels to K absorption. Barium effects on jejunal epithelia have also been described for the rat [73]. But they considered that these effects were due to basolateral K channels. Heitzmann and Warth [62] in contrast, supposed the existence of diverse luminal K channels, but doubted a relevant role of them for K transport. According to Warth [79] these K channels may play an important role in restoring the driving force for Na coupled transport systems.
In the absence of positive Na, and therefore an electrogenic gradient, K might then flow through these K channels along the K gradient, as assumed for ruminal K absorption. This could explain the absence of a K current in the presence of sodium.
Thus, the increases in the K current in the absence of Na could be an artificial phenomenon due to the experimental set-up.
In summary, the jejunum appears to be the main side of K absorption in ruminants.
The ability for K absorption at high mucosal K concentrations in rumen, abomasum and colon, however, has obviously the same order of magnitude per unit of area. The K absorption across sheep jejunum seems to be predominantly electroneutral. The slight electrogenic part of the jejunal K absorption seems to have a TAP sensitive paracellular component and a barium sensitive cellular component.
4.5.7 K transport across colon
Colonic epithelia possess mechanisms for active K absorption and K secretion that increased linear with the intake of K. Pfeffer, et al. [2] observed K absorption rates of 1.51 g/d or 7.9 % of the dietary intake in sheep. Argenzio et al. [83] also demonstrated K absorption from the large intestine, measured in goats.
Measurements of the K concentrations in the large intestine of sheep varied between 17 mmol∙l-1/kg water [84] and 43.5 mmol∙l-1 in the digesta fluid [85]. Hecker & Grovum [82] observed increasing K concentrations from 50 meq∙kg-1 in the proximal colon to 80 meq∙kg-1 at the apex of the spiral colon, which decreased to 20 meq∙kg-1 in the rectum. In our own study the region of the flexura centralis was selected for taking the samples. In this region the physiological K concentration is obviously high and the K concentrations chosen for the experiment reflect the physiological situation.
While we did not observe a K absorption or secretion at mucosal K concentrations of 4 mmol∙l-1, K was absorbed across the colon at mucosal K concentrations of 100 mmol∙l-1. The amounts of K absorption and electric charges transported as short circuit current were in the same order of magnitude. The current thus appeared to represent K absorption. In a linear correlation between Δ Isc and Δ K absorption 69±17 % of the increase in K absorption was explained by an increase in K current.
Electrogenic K transport therefore seems to play an important role in the colon of sheep. However, neither TAP nor the mucosal addition of barium showed any effect on the currents across the colon. Thus, the electrogenic K transport across the colon seems to have neither TAP sensitive paracellular components, nor barium sensitive cellular components. In rat colon barium sensitive K channels, as well as tetraethylammonium chloride, quinidine and Ca2+ sensitive K channels are described [86,87], but most of these K channels are described as responsible for electrogenic K secretion [86-88]. In sheep, barium sensitive K channels have been described in parotid secretory cells [89], while blocker studies of K channels in the colon of ruminants obviously do not exist.
Studies in dogs suggested an active potassium transport in the colon, since the K transport across the epithelia did not follow the concentration gradients [77]. In the colon of rat, rabbit and guinea pig an apical K+/H+ ATPase is described [90,91]. Since our studies suggest a predominantly electrogenic K transport, we assume that an apical K+/H+ ATPase do not play an important role in the colon of ruminants. Sweiry and Binder [33] as well as Del Castillo et al. [92] presumed an apical K+-ATPase with properties that are unlike the gastric K+/H+ ATPase but similar, in part, to Na+/K+ATPase in rats. This transport is mostly unknown, but an involvement in the K transport in sheep might be possible.
Schultz & Dubinsky [31] have described, that an increase in the rate of transcellular Na absorption is accompanied by an increase in the K conductance of the basolateral membrane. Basolateral K channels are characterized as pathways for recirculation of the K needed for the Na+/K+ATPase. With adequate mucosal K concentrations these K channels could be involved in K absorption.
In summary the colon is obviously able to absorb considerable amounts of K. The K absorption appears to be mainly electrogenic. Putative pathways could be barium insensitive K channels and an electrogenic K-ATPase. The colonic K absorption has obviously no paracellular component.