0 1 2 3 4
R u m e n A b o m a s u m J e ju n u m C o lo n
*
*
*
*
*
*
( 1 4 ) ( 1 0 ) ( 1 6 ) ( 1 1 )
( 1 6 ) ( 6 ) ( 8 ) ( 1 1 ) ( 8 )
*
Isc (µeqcm-2 h-1 )
Figure 1: Increase in transepithelial current.
Increase in transepithelial current (Δ Isc) across different epithelia due to an increase in mucosal K concentration from 4 mmol·l-1 to 100 mmol·l-1, Na-free conditions on the mucosal side. (N) = number of animals, * significantly different from 0.
Goat Sheep Cattle G o a t
R u m e n
Figure 2: Isc at mucosal K concentrations of 4, 25, 50 and 100 mmol∙l-1.
Isc measured across goat epithelia. Na-free conditions on the mucosal side. Rumen N = 13, abomasum N = 9, jejunum N = 7, colon N = 8.
G o a t R u m e n
Figure 3: Effect of K and Na on Isc across different epithelia.
+ p<0.05 versus Na-free,
* p<0.05 versus 4 mmol∙l-1 K mucosal, rumen N = 8, abomasum N = 5, jejunum N = 5, colon N = 4.
4 mmol∙l-1 K 50 mmol∙l-1 K
S h e e p r u m e n
- 1 1 2 3 4 5 6
- 1 1 2 3 4 5
K I s c ( µ e qc m- 2h- 1) Rb Isc (µeqcm-2 h-1 )
S h e e p c o lo n
- 1 1 2 3 4 5 6
- 1 1 2 3 4 5 6
K I s c ( µ e qc m- 2h- 1) Rb Isc (µeqcm-2 h-1 )
C a t t l e r u m e n
- 1 1 2 3 4 5 6
- 1 1 2 3 4 5
K I s c ( µ e qc m- 2h- 1) Rb Isc (µeqcm-2 h-1 )
Figure 4: Correlation between K and Rb currents.
Isc at 4 mmol·l-1 K or Rb Isc at 100 mmol·l-1 K or Rb
Δ Isc (Isc at 100 mmol·l-1 – Isc at 4 mmol·l-1)
─ Linear regression for Isc --- Linear regression for Δ Isc
- 0 . 4
Figure 5: Mucosal and serosal changes in K concentration with 4 mmol·l-1 K mucosal.
Mucosal and serosal changes in K concentration (mmol∙l-1) within 30 minutes after incubation with 4 mmol·l-1 K buffer solution on both sides with an exposed area of 3.14 cm², (n) = number of samples, two measuring periods per epithelium, two epithelia per sheep; *significantly different from 0 (p<0.05)
R u m e n A b o m a s u m J e ju n u m C o lo n
0 .0 0 .5 1 .0
*
* *
*
( 5 3 ) ( 4 7 ) ( 5 6 ) ( 4 4 )
change in K concentration (mmoll-1 )
Figure 6: Serosal changes in K concentration after incubation with 100 mmol·l-1 K mucosal.
Serosal changes in K concentration (mmol∙l-1) within 30 minutes after incubation with 100 mmol·l-1 K buffer solution on the mucosal side with an exposed area of 3.14 cm², (n) = number of samples, two measuring periods per epithelium, two epithelia per sheep; *significantly different from 0 (p<0.05)
0 1 2 3 4 5
R u m e n A b o m a s u m J e ju n u m C o lo n
( 1 4 ) ( 6 ) ( 1 4 ) ( 6 )
*
*
b
a b
a
b
µeqcm-2 h-1
Figure 7: K induced currents and increases in K absorption.
K induced currents (Δ Isc) and increases in K absorption across gastrointestinal epithelia from sheep due to a change in mucosal K concentration from 4 to 100 mmol·l-1, Na-free conditions on the mucosal side. Values with different letters differ with p<0.05, * significantly different from respective Δ Isc, (N) = number of animals.
Δ Isc
Δ absorption
R u m e n
- 1 1 2 3 4 5 6 7
- 6 - 5 - 4 - 3 - 2 - 1 1 e + 0 0 0 2 3 4 5 6
Is c ( µ e qc m- 1h- 1) K absorption (µeqcm-1 h-1 )
-1 1 2 3 4 5 6 7
-6 -5 -4 -3 -2 -1 1 2 3 4 5 6
Colon
Isc (µeqcm-1h-1) K absorption (µeqcm-1 h-1 )
Figure 8: Linear correlation between K current and K absorption.
Linear correlation between the K currents and the K absorption across the rumen and colon from sheep, Na-free conditions on the mucosal side.
T A P
Figure 9: Effect of different blockers on the K current.
(n) = numbers of epithelia, * significantly different from respective unblocked Isc. The effect of barium in rumen epithelia was tested with cattle, all other epithelia were from sheep.
Isc unblocked (100 mmol·l-1 K) TAP (10 mmol·l-1 mucosal and serosal)
Verapamil (100 µmol·l-1 mucosal) Barium chloride mucosal (3 mmol·l-1) additional Barium chloride serosal (3 mmol·l-1)
5 Manuskript II
Influence of hypokalemia and stress on gastrointestinal potassium transport in ruminant tissues
Nina Katrin Kronshage§1 and Sabine Leonhard-Marek1,2
1Department of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany
²University Library, University of Veterinary Medicine Hannover, Bünteweg 2, 30559 Hannover, Germany
Short title: K transport and stress in ruminants
§Corresponding author
Addresses:
NKK: ninakronshage@gmail.com, Department of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, 30173 Hannover, Germany
SLM: sabine.leonhard-marek@tiho-hannover.de, Department of Physiology and University Library, University of Veterinary Medicine Hannover, Bünteweg 2, 30559 Hannover, Germany
5.1 Abstract
Ruminants are able to absorb potassium (K) in the whole gastrointestinal tract.
Intensive K fertilisation causes a high K intake of dairy cows. Regardless, cows that suffer from abomasal displacement often show a hypokalemia. A reduced gastrointestinal absorption might be a reason. We investigated the regulation of K absorption across epithelia from rumen, abomasum, jejunum and colon of goat, sheep or cattle with mucosal K concentrations of 4, 50 and 100 mmol·l-1, using the Ussing chamber technique. Simulation of a hypokalemia by changing the serosal K concentration from 4 mmol∙l-1 to 2 mmol∙l-1 had no effect on the current of the tested epithelia. To investigate an effect of stress, forskolin, adrenaline and isoproterenole were added to rumen epithelia. Forskolin caused an increase in current at mucosal K concentrations of 100 mmol∙l-1 in sheep and in two of 6 cattle, while it had no effect at mucosal K concentrations of 4 mmol∙l-1. To distinguish between an opening of nonspecific cation channels or K channels, Calcium (Ca2+) and Magnesium (Mg2+) as blockers of the nonselective cation channel were removed, or the K channel blocker verapamil was added. Removal of Mg2+ had no significant effect on the K current and on the forskolin effect. Removal of Ca2+ and Mg2+ increased the K current, but did not change the effect of forskolin. Addition of verapamil abolished the forskolin effect.
Thus, cAMP seems to increase the K current via K channels. Addition of adrenaline and isoproterenole had no effect on the K currents across rumen epithelia, which excludes the involvement of ß-adrenergic receptors in the cAMP effect.
Ruminants, potassium, hypokalemia, stress, abomasal displacement
5.2 Introduction
In lactating cows 74 to 97 % of the ingested K is absorbed in the gastrointestinal tract [1,2]. The location of K absorption seems to depend on K intake. Steers, as well as lambs fed 0.6 % K absorbed K mainly in the small intestine [1,3]. Khorasani et al. [2]
likewise observed a primarily intestinal K absorption with a low K intake in cows. At a high K intake the preintestinal K absorption increased [1,2,4,5]. In a previous in vitro study we observed an increase in K absorption after increasing the luminal K concentration from 4 to 100 mmol∙l-1 by 2.5 µeq∙cm-2∙h 1 in the rumen, by 3.5 µeq∙cm-2∙h-1 in the abomasum, by 4.5 µeq∙cm-2∙h-1 in the jejunum and by 2.9 µeq∙cm-2∙h-1 in the colon. Thus, preintestinal regions can absorb relevant amounts of K, especially at high luminal K concentrations.
As a result of high K concentrations in the grass rations for dairy cows their K intake is severalfold higher than the K requirement. Regardless, cows that suffer from abomasal displacement often show a (sometimes severe) hypokalemia [6,7,8], with plasma K concentrations of 3.1 mmol∙l-1 [9] or 3.49 mmol∙l-1 [10], respectively. The reference range is specified as 3.5 to 5 mmol∙l-1 [11] or 4.0 to 5 mmol∙l-1, respectively [12]. This drop in plasma K might be the cause as well as the consequence of
abomasal displacement. Some researchers postulate, that the reduction in plasma K develops secondary to abomasal displacement as a consequence of reduced K absorption from the intestine [13,14]. If the abomasum is displaced, the intestine, and especially the jejunum could be omitted from K absorption because of the blocked passage. As described above, ruminal K absorption, however, should compensate for this deficit, if the ruminal K concentration were sufficient. It is conceivable, that peripartal stress or a beginning decrease in serum K concentration could affect the conductance of rumen epithelial K channels and thereby inhibit K absorption. The developing – primary - hypokalemia could promote an abomasal displacement. Türck and Leonhard-Marek [15] have shown that a reduction in extracellular K concentration decreases the contraction activity of abomasal muscles. An abomasal atony favours an abomasal displacement. Regardless of a primary or secondary concentrations. Low extracellular K causes a hyperpolarisation of the cellular membrane [18]. This could open voltage gated K channels. Voltage gated K channels are preferentially expressed in excitable cells, but they are also found in nonexcitable cells as in the epithelia of the gastrointestinal tract [19,20]. On the other hand, the absence of a K sensing mechanism for K absorption could introduce a hypokalemia, themselves. Rabinowitz [17] calculated an augmented K excretion at high ruminal K concentrations. If an abomasal displacement has any negative effects on the K absorption and, as a result, the high ruminal amounts of K cannot be absorbed in sufficient amounts, this could induce a hypokalemia. Peripartal stress might be another factor that could inhibit the K absorption. In the present study cyclic adenosine monophosphate (cAMP), adrenaline and isoproterenole as a ß-agonist were used to simulate a stress situation.
cAMP is a second messenger for diverse epithelial transport processes [21]. cAMP dependent K channels are described in the human [22] and murine stomach [23], in the human duodenum [22], and in the rat and rabbit colon [24,25]. In the colon of rats [26, 27, 28] and rabbits [25] an effect of cAMP on K channels is described, with both activating and inactivating effects.
In sheep rumen epithelia, an increase in cAMP decreased or increased the short circuit current in previous studies [29,30]. The explanations for the cAMP and forskolin effect differed. The effect of forskolin on the K current, especially in the presence of a high transepithelial K gradient is unknown.
Adrenaline has a regulatory effect on the K transport. It can shift K from extra- to intracellular compartments [31,32]. Furthermore it acts on K transport in the gastrointestinal tract. In the rabbit and guinea pig colon adrenaline affected the K conductance, probably via a Ca2+ dependent effect [33,34,35]. The colon is presumed to be functionally related to the rumen [2]. In the rumen adrenaline inhibits
the rumen motility [22]. In addition, it affects ruminal absorption. Adrenaline stimulates glucose absorption via β2 adrenergic receptors, mediated by cAMP [36]. In a further study Aschenbach et al. [37] observed an increase in Isc after addition of adrenaline, while the β-agonist isoproterenole decreased Isc. The increase in Isc after adrenaline addition was paralleled by an increase in a net Na flux rate. K flux rates were not investigated. Since the Na transport is associated with K transport via the Na/K ATPase it is conceivable that adrenaline might have an effect on K transport as well. The effects of adrenaline are partly mediated via cAMP as second messenger [13,34].
The aim of the present study was to investigate a potential regulation of electrogenic K absorption via serosal K concentration and an effect of stress. To investigate a potential effect of hypokalemia on the electrogenic K absorption, the serosal K concentration was degraded under a physiological degree. Stress was simulated by an increase of cAMP, adrenaline or a ß2-agonist. Blocker studies were used to further clarify the involved Ion channels, where effects were observable.
5.3 Methods
The study was performed with the Ussing chamber technique. The Ussing chamber simulates a physiological system to measure ion transports across epithelial tissues [38].
5.3.1 Tissues
Pieces of the ventral rumen wall, the greater abomasal curvature, middle part of jejunum and of the flexura centralis of proximal colon were taken from slaughtered sheep and goats, furthermore pieces of the ventral rumen wall were taken from slaughtered cattle. The pieces were immediately immersed in buffer solution. The mucosa was stripped from the underlying muscle layers and the serosa. The tissues were by-products from slaughters for the food production.
5.3.2 Incubations and electrical measurements
Mucosal tissues were mounted between the two halves of Ussing incubation chambers with an exposed area of 1 cm2 or 3.14 cm2. Incubation chambers were connected to reservoirs containing 10 or 13 ml buffer solution on each side. The solutions were kept at 38°C and were continuously stirred by the use of a gas lift system that supplied 95 % O2 and 5 % CO2.
We minimized edge damage by placing rings of silicone rubber on both sides of the tissues. The chambers were connected to a computer controlled voltage clamp device (Epithelial Voltage Clamp Model EC 825, Warner Instrument Corp., Hamden, USA). Transepithelial potential differences (Vt) were measured through buffer solution agar bridges and calomel electrodes with reference to the mucosal solution.
Transepithelial conductances (Gt) were determined from the changes in Vt caused by bipolar current pulses of 100 µA·cm-² of 500 ms duration. The currents were passed through buffer solution agar bridges connected to Ag/AgCl electrodes placed in each half of the Ussing chamber. In each setup, fluid resistances and junction potentials
were measured before mounting the mucosal tissues and corrected for during the experiments. The experiments were performed under short circuit conditions.
5.3.3 Experimental set-up
5.3.3.1 Effect of serosal K concentration on transepithelial current
These experiments were performed with goat tissues. Isolated epithelia from rumen, abomasum, jejunum and colon were incubated in standard buffer solution containing Na on the serosal side and in Na-free K buffer solution on the mucosal side. The mucosal K concentration was 100 mmol l-1. To investigate a potential effect of hypokalemia the serosal K concentration was changed between 4 and 2 mmol∙l-1 and back to 4 mmol∙l-1.
5.3.3.2 Effect of stress on the K+ current
Stress was investigated as an increase in adrenaline and isoproterenole or as an increase of the second messenger cAMP.
5.3.3.3 Effect of forskolin on the K+ current
Isolated epithelia from the rumen of sheep and cattle were incubated in standard buffer solution. The mucosal K concentration was changed between 4 and 100 mmol·l-1 in sheep and between 4, 50 and 100 mmol·l-1 in cattle. The serosal buffer solution remained unchanged. 10 µmol∙l-1 forskolin as a stimulator of the adenylate cyclase [39] was added to the mucosal and serosal side of rumen epithelia to increase the intracellular cAMP level. cAMP activates the protein kinase A, which is able to modify the activity of transport proteins. To diminish the cAMP degradation via phosphodiesterase 10 µmol∙l-1 isobutyl-methyl-xanthine (IBMX) as a blocker of the phosphodiesterase [40,41] was added parallel to the forskolin addition. All buffer solutions contained 1 µmol∙l-1 indomethacine to inhibit the endogenous production of prostaglandins, which would on their part affect the intracellular level of cAMP [37].
5.3.3.4 Effect of Ca2+ and Mg2+ on the K current
To investigate a potential involvement of the unspecific cation channel in the forskolin effect [29,43], the mucosal side of sheep rumen epithelia was incubated in Mg2+-free buffer solution as well as Ca2+- and Mg2+-free buffer solution in a parallel set-up.
Forskolin was added to the mucosal and serosal side at mucosal K concentrations of 100 mmol∙l-1. 2 mmol∙l-1 Ca2+ was added to the mucosal side after forskolin addition to investigate, if the effect was reversible.
5.3.3.5 Effect of verapamil on the forskolin effect
Verapamil as a putative K channel blocker in rumen [44] was added in a concentration of 100 µmol·l-1 to the mucosal side of the epithelia before and after addition of 10 µmol∙l-1 forskolin. For this study we used epithelia from the rumen of sheep. The mucosal K concentration was 100 mmol∙l-1.
5.3.3.6 Effect of adrenaline and isoproterenole on the K current
Isolated epithelia from the rumen of cattle were incubated in standard buffer solution.
The mucosal K concentration was 50 mmol·l-1. Adrenaline and isoproterenole were added at the serosal side of rumen epithelia. Adrenaline was added in steps of 1, 10 and 100 µmol∙l-1 or directly with the maximal concentration of 100 µmol∙l-1. Isoproterenole was added in concentrations of 1 or 100 µmol∙l-1.
5.3.3.7 Time dependent effects
To exclude effects due to the duration of the experiments or due to an irritation of the epithelia after changing the buffer solutions, the order of the different K side. In high K (Na free) buffer solutions Na+ was replaced by N-methyl-D-glucamine (NMDG), which can hardly be absorbed in the gastrointestinal tract. The Na-free buffer contained (in mmol∙l-1) 4, 50 or 100 K+, with 141.4, 95.4 or 45.4 NMDG, respectively. This buffer was used for incubation on the mucosal side. In Mg2+- free or in Ca2+- and Mg2+ free solutions Mg2+ or Ca2+ and Mg2+ were replaced by NMDG.
The buffer contained (in mmol∙l-1) 46.6 or 47.8 NMDG, respectively.
The pH of all solutions was 7.4, when gassed with 95% O2/5% CO2. Osmolarity was adjusted to 300 mosmol·l-1 with mannitol.
5.3.5 Chemicals
CaCl2, MgCl2 and HCl were from Merck (Darmstadt, Germany). All other chemicals were obtained from Sigma (Steinheim, Germany).
Forskolin was dissolved in 2 and 2.6 µl Dimethyl sulfoxide (DMSO) and added to the incubation volume of 10 and 13 ml, respectively. This DMSO volume produced no electrophysiological effects in control tissues incubated in parallel. IBMX was dissolved in ethanol. The ethanol concentration never exceeded 0.1 % which had no effect (46). Adrenaline and isoproterenole were dissolved in water. Substances, which were added during the measuring period, were diluted in stock solutions with high concentrations in order to achieve minimal change of the volume of the buffer solution.
5.3.6 Statistics
Data are presented as means ± SEM; n indicates the number of epithelia, N indicates the number of animals. Statistical significance was evaluated with analysis of variance and Student’s t-test or Wilcoxon signed rank test. Statistical analyses and graphs were performed with Graph Pad Prism 4. Differences were assumed significant when P values were lower than 0.05, highly significant when P values
were lower than 0.01 and very highly significant when P values were lower than 0.001.
5.4 Results
5.4.1 Effect of the serosal K concentration on the transepithelial current
Decreasing the serosal K concentration from 4 to 2 mmol∙l-1 and increasing it again to 4 mmol∙l-1 had no effect on the current of rumen, abomasum and jejunum at mucosal K concentrations of 100 mmol∙l-1. In colon, the current increased with time (Fig. 1).
5.4.2 Effect of forskolin on the K current
Serosal addition of forskolin increased the K current across rumen epithelia of sheep at mucosal K concentrations of 100 mmol∙l-1, but not at mucosal K concentrations of 4 mmol∙l-1 (Fig. 2). In cattle, addition of forskolin showed considerable effects like in sheep across 2 of 6 epithelia (Fig. 3). To investigate a potential effect at lower K concentrations in cattle, we added forskolin at mucosal K concentrations of 50 mmol∙l-1. No forskolin effect was observable.
5.4.3 Effect of Ca2+ and Mg2+ on the K current and the forskolin effect
In the absence of Mg2+, the K current across rumen epithelia did not differ from the K current in the presence of Mg2+ at mucosal K concentrations of 100 mmol∙l-1. This was due to 2 epithelia that decreased in current (Fig. 4a). In the absence of Ca2+ and Mg2+ the K current increased (Fig. 4b). Addition of forskolin increased the K current by 0.17 ± 0.05 μeq∙cm-²∙h-1 (6.58±1.83 %) in the absence of Mg2+ and by 0.23±0.03 μeq∙cm-²∙h-1 (7.09±1.32 %) in the absence of Ca2+ and Mg2+, compared to 0.31 ± 0.04 μeq∙cm-²∙h-1 (11.00±1.23 %) in the presence of Ca2+ and Mg2+ (Fig. 2). Based on the increase of current from 4 to 100 mmol∙l-1∙h-1 (Δ Isc) the forskolin effect did not differ from each other in the pre- and the absence of Mg2+ and Ca2+ and Mg2+. Addition of 2 mmol∙l-1 Ca2+ to the mucosal side of the epithelia in the presence of forskolin decreased the K current both in the absence of Mg2+ and in the absence of Ca2+ and Mg2+to basal values.
5.4.4 Effect of forskolin in the absence or presence of verapamil
Mucosal addition of verapamil decreased the K current in the rumen of sheep at mucosal K concentrations of 100 mmol∙l-1 after addition of forskolin. A preincubation with verapamil abolished the forskolin effect. The K current after addition of verapamil did not differ from each other in both experimental set-ups.
5.4.5 Effect of adrenaline and isoproterenole on the K current
Serosal addition of adrenaline and isoproterenole (both between 1 and 100 µmol∙l-1) had no effect on the K current across rumen epithelia from cattle.
5.5 Discussion
5.5.1 Effect of serosal K concentration on transepithelial current
Low extracellular K causes a hyperpolarisation of the cellular membrane. This could open voltage gated ion channels like the HCN proton channel in the heart, which transports preferentially K [46]. In gastric parietal cells of guinea pigs a voltage-gated K channel is activated by hyperpolarisation [19]. In the colon the voltage-gated K channel Kv1.3 has been identified in rabbits [20]. Decreasing the serosal K concentration to investigate a potential effect of hypokalemia had no effect on any of the investigated epithelia in our study. In colon, Isc showed a time dependent increase. Thus, hypokalemia had no influence on electrogenic K absorption under the present conditions. Rabinowitz [17] postulated a K sensing mechanism in the rumen, which could be involved in the regulation of renal K excretion. They calculated an augmented K excretion at high ruminal K concentrations. If an abomasal displacement has any negative effects on the K absorption and, as a result, the high ruminal amounts of K cannot be absorbed in sufficient amounts, this could induce a hypokalemia. This would, in a second step consolidate the abomasal displacement by inhibiting the motility [15,47]. In our study we did not observe a mechanism, which could register and therefore re-elevate a hypokalemia due to an increased K absorption across gastrointestinal epithelia. This missing effect of hypokalemia on gastrointestinal K current and persisting high K concentration in the rumen in vivo could result in a vicious circle via prolonged renal K losses.
5.5.2 Effect of stress on the K+ current
Stress, simulated by cAMP, adrenaline and the ß-agonist isoproterenole, might affect the conductance of rumen epithelial K channels and thereby inhibit K absorption.
5.5.2.1 Effect of cAMP on the K+ current
To increase the intracellular cAMP level forskolin was added to the incubation solutions. This addition of forskolin increased the K current across the rumen of sheep at mucosal K concentrations of 100 mmol∙l-1. This increase was also
To increase the intracellular cAMP level forskolin was added to the incubation solutions. This addition of forskolin increased the K current across the rumen of sheep at mucosal K concentrations of 100 mmol∙l-1. This increase was also