- Email: [email protected]
PGE2 exerts dose-dependent opposite effects on net water and chloride absorption from the rat colon
Prostaglandins & other Lipid Mediators 79 (2006) 43–52
PGE2 exerts dose-dependent opposite effects on net water and chloride absorption from the rat colon Sawsan Ibrahim Kreydiyyeh ∗ , Sarine Markossian, Rawad F. Hodeify Department of Biology, Faculty of Arts & Sciences, American University of Beirut, Beirut, Lebanon Received 28 May 2005; received in revised form 22 July 2005; accepted 25 July 2005 Available online 13 February 2006
Abstract This work investigated the effect of different doses of PGE2 on net water and Cl− absorption from the rat colon, using an in situ perfusion technique. PGE2 exerted opposite effects at different concentrations. Net water and Cl− absorption was significantly reduced at low doses with a minimum at 0.4 g/100 g BW, and significantly elevated at high doses with an observed maximal effect at 21 g/100 g BW. At low doses, PGE2 increased in superficial cells, the activity of the Na+ –K+ ATPase and the protein expression of the Na+ K+ 2Cl− cotransporter, but reduced them in crypt cells. Thus, the reduction in net water and Cl− absorption was ascribed to an increase in secretion by surface cells that masked absorptive processes. At high doses, PGE2 increased significantly the activity of the Na+ –K+ ATPase in superficial cells only, and was without any effect on the protein expression of the cotransporter and the pump in both surface and crypt cells. The observed increase in net water and Cl− absorption was attributed in this case to an increase in absorptive processes with no effect on secretion. © 2005 Elsevier Inc. All rights reserved. Keywords: PGE2; Water; Chloride ;Na+ –K+ ATPase; Na+ –K+ –2Cl− ; Colon
PGE2 is an eicosanoid with diverse biological actions on the reproductive, gastrointestinal and neuroendocrine system. Derived from the cyclo-oxygenase (COX) mediated metabolism of arachidonic acid, it has abroad range of physiological and pharmacological effects, and acts via its different receptors to maintain homeostasis [1]. PGE2 is a known modulator of water and electrolyte movements in the colon. When released into the colonic lumen of patients with ulcerative colitis, it reduced net mucosal to serosal fluxes of Na+ and Cl− and contributed to the diarrhea associated with the disease [2]. PGE2 was shown also to inhibit the HCO3 − dependent Cl− absorption in porcine distal colon epithelium [3], and stimulate K+ and Cl− secretion in rabbit distal colon. In rat colonic crypt base cells it increased Cl− secretion by inducing a marked depolarization that reduces the basal K+ conductance [4]. Because most studies showed an inhibition in intestinal fluid and chloride absorption by PGE2, the prostaglandin has always been recognized as a secretagogue in the gastrointestinal tract [5–8]. Net water movement in the colon is directly dependent on the rate of net electrolytes transport [9]. Ions like Na+ , K+ and Cl− are transported across the colonic epithelium from the mucosal to the serosal side or vice versa, and cause the movement of water by osmosis. Net fluid transport is the resultant of absorptive and secretory processes and depends on the activity of major transporters like the Na+ –K+ ATPase, the Na/H+ exchanger and the ∗
Corresponding author. Fax: +961 1 744461. E-mail address: [email protected] (S.I. Kreydiyyeh).
1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2005.07.004
44
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
Na+ K+ 2Cl− symporter [10]. Sodium absorption can be electrogenic via sodium channels or electroneutral via parallel Na+ /H+ and Cl− /HCO3 − exchange, and is geared by the sodium gradient established by the Na+ –K+ pump situated on the basolateral membrane. Bulk sodium transport in the rat colon takes place in both crypts and surface epithelium [11] of proximal and distal colon and is dominated by electroneutral absorption [12]. The Na+ K+ 2Cl− cotransporter, situated on the basolateral side, is responsible for chloride and potassium secretion. Under control conditions, ion transport activities result in net absorption, and secretory properties become apparent only upon stimulation by seretagogues. As a secretagocue, PGE2 may be altering absorptive and secretory processes by targeting electrolyte transporters and changing their activity. This work studies the effect of different doses of PGE2 on net water and chloride absorption from the rat colon and attempts to delineate its mode of action by investigating its effect on the Na+ –K+ pump, a major regulator of transport processes, and on the Na+ K+ 2Cl− symporter, a major player in secretion. The results demonstrate, a dose-dependent dual stimulatory and inhibitory role of PGE2 on net water and Cl− absorption from the rat colon, and shows that the prostaglandin does not act always as a secretagogue but can at some concentrations stimulate fluid and electrolyte absorption. 1. Materials and methods 1.1. Animal treatments Male Sprague-Dawley rats (Rattus norvegicus) weighing 150–500 g were handled all through in accordance with the Guide for Laboratory Animal Facilities and Care, US Department of Health, Education and Welfare. Rats weighing around 250 g were injected i.p. with PGE2 (21; 4; 1; 04; 0.18; 0.05 g/100 g BW) 15 min before the beginning of the experiment. PGE2 was dissolved in ethanol. Control rats were injected with a similar volume of the vehicle. In all other experiments high and low PGE2 doses refer respectively to 21 and 0.4 g/100 g BW). 1.1.1. Measurement of luminal water movements in the rat colon Rats were anesthetized by i.p. injection of pentobarbital (5 mg/100 g body weight). The abdomen was then opened through a midline incision to expose the colon. L-shaped inlet and outlet catheters connected to polyethylene tubing were introduced at each end of the colon keeping the mesenteries and vasculature intact. The end of the inlet catheter was connected to a perfusion pump whose rate was maintained at 0.7 ml/min, while the outlet tube drained into a beaker to allow for the collection of the effluent buffer. The colon was perfused first with oxygenated (95% O2 ; 5% CO2 ) Krebs Improved Ringer buffer pH 7.4 (KIRB) (123.3 mM NaCl; 6.17 mM KCl; 3.29 mM CaCl2 ·7H2 O; 0.78 mM MgSO4 ·7H2 O; 32.14 mM NaHCO3 ; 1.54 mM KH2 PO4 ; 6.4 mM sodium glutamate; 6.4 mM sodium pyruvate; 7 mM sodium fumarate; 11.1 mM glucose) for 15 min to clean it from wastes. Then, it was perfused with the same buffer for another 30 min and the effluent buffer collected. At the end of the perfusion, the volume of the infused and collected buffers was measured and the colon was excised, cut longitudinally, its widih and length measured and its surface area calculated. Water absorption, during the second perfusion step, was calculated as the difference between the volumes of infused and collected buffers divided by the surface area of the perfused colon. 1.1.2. Measurement of net chloride absorption in the rat colon Samples from the inlet and outlet buffers were drawn and assayed for their Cl− content by titration with silver nitrate AgNO3 (0.1 M) according to Mohr’s method [13]. K2 CrO4 (2.5 M) was used as an indicator. Chloride absorption was calculated as the difference in the number of moles of chloride present in the infused and collected buffers. 1.1.3. Isolation of colon superficial and crypt cells Surface and crypt cells were isolated from the colon and characterized as described by Homaidan et al. [14]. Briefly, the colon was removed and washed consecutively with ice cold saline-DTT (0.154 M NaCl; 0.1 mM DTT (dithiothreitol)) and ice cold phosphate buffer saline (PBS) pH 7.3 (0.088 M NaCl; 0.01 M Na2 HPO4 ; 8 mM KH2 PO4 ) to clean it from wastes. Then, it was everted, filled with PBS pH 7.3, clamped at both ends, and incubated at 37 ◦ C first, in citrate buffer (27 mM Na citrate, 96 Mm NaCl, 1.5 mM KCl, 8 mM KH2 PO4 , 5.6 mM Na2 HPO4 , 1 mM DTT, pH 7.5)
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
45
for 10 min and then in PBS-EDTA buffer (140 mM NaCl, 8 mM Na2 HPO4 , 2.8 mM KCl, 1.5 mM KH2 PO4 , 0.5 mM DTT, 1.5 mM Na2 EDTA, pH 7.4) for l5 min in a shaking water bath (75 cycles/min) while gassing with 95% O2 , 5% CO2 . The cells shed in the latter buffer were collected by a 5-min centrifugation at 770 × g and 4 ◦ C and corresponded to villus cells. Subsequently, two sequential fractions were collected after two consecutive 15-min incubation periods in PBS-EDTA buffer. The cells released in the last fraction were collected by centrifugation as before and corresponded to crypt cells. After collection, villus and crypt cells were washed twice with Tris buffer. The collected superficial and crypt cells were resuspended in Tris buffer pH 7.4 (buffer A) (200 mM NaCl; 5 mM MgCl2 ; 2 mM EGTA; 5 mM KCl; 200 mM Trizmabase) (0.4 g/ml) and homogenized for 3 min with a polytron (20,000–22,000 rpm) at 4 ◦ C, then spun at 3300 × g for 10 min at 4 ◦ C to prepare a crude membrane homogenate. A mixture of protease inhibitors was then added to the homogenate which was saved at −20 ◦ C for later use. 1.1.4. Incubation of isolated cells with agonists/antagonists To determine the type of receptors involved in the effect of PGE2 on the pump, specific agonist and antagonists (Cayman Chemical Company, MI, USA) were used. Butaprost and sulprostone were chosen as respective selective agonists for EP2 and, EP3 and EP1 receptors, while SC-19220 was chosen as a selective antagonist of PGE2 at the EP1 receptor, and AH-6809 as an antagonist with equal affinity for EP1, EP2 and EP3 as reported by the providing company. Superficial and crypt cells were isolated as described before, re-suspended (0.07 g/ml) in DMEM culture medium containing 4500 mg/l Glucose, sodium pyruvate, 15% FBS, 1% PS; Penicillin (100 g/ml), and incubated with continuous oxygenation (95% O2 ; 5% CO2 ) for 30 min in presence of the agonists or, with PGE2 and its antagonists. PGE2 was added at 1 nM concentration. Agonists were added at a concentration of 1 M and the antagonists at a concentration of 10 M. The viability of the cells was checked every 10 min and did not drop below 80% by the end of the experiment. Antagonists were added 10 min before PGE2. At the end of the incubation period, the cells were collected by centrifugation and crude membrane homogenates were prepared as described before and used to assay for the Na+ –K+ ATPase activity. 1.1.5. Na+ –K+ ATPase assay Membrane homogenates were diluted in Tris buffer (buffer A) to a final concentration of 2 mg/ml protein and incubated for 30 min with saponin (0.02% final concentration) at room temperature. After a pre-incubation at 37 ◦ C for 10 min in presence or absence of ouabain (4 mM final concentration), the reaction was initiated by addition of ATP to a final concentration of 1.25 mM, and terminated after a one hour incubation at 37 ◦ C by addition of trichloroacetic acid (200 l, 11%). The amount of inorganic phosphate liberated was measured colorimetrically according to the meihod of Taussky and Shorr [15] and the activity of the enzyme was determined by measuring the ouabain-inhibitable inorganic phosphate liberated. The percent inhibition of the enzyme activity was calculated as follows: 1−
Pi (treatment) − Pi (treatment + ouabain) × 100. Pi (control) − Pi (control + ouabain)
1.1.6. Western blot analysis Membrane proteins were quantified using the Bio-Rad reagent and were equally loaded and resolved on 8% polyacrylamide gels, then transferred to a PVDF membrane (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA). Protein expression of  actin was used to check for equal loading. The PVDF membrane was then washed, blocked and incubated overnight at 4 ◦ C with a rabbit Anti-Na+ /K+ ATPase ␣1 IgG (Upstate biotechnology, Lake Placid, NY 12946), or with an anti-rat sodium potassium chloride cotransporter antibody (Alpha Diagnostic International, TX, USA). Detection of the signal was by enhanced chemiluminescence using luminol reagent (Santa Cruiz Biotechnology Inc., CA, USA). The thickness of the bands was determined by densitometry using a gel-pro analyzer (2) software. 1.1.7. Statistical analysis Results are reported as means ± SEM. Statistical significance was tested by a Student t-test or a one-way analysis of variance followed by a Tukey–Kramer multiple comparisons.
46
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
Fig. 1. Effect of different doses of PGE2 on the net absorption of water from the colon. Letters above the bars indicate significant differences. P < 0.05: (a,e); P < 0.01: (a,b); P < 0.001: (b,d), (b,e), (b,f), (b,g).
2. Results 2.1. Dose dependent effect of PGE2 on net water and chloride absorption from the colon The effect of PGE2 on net water and chloride absorption from the colon was dose-dependent (Figs. 1 and 2). Net water and chloride absorption was significantly reduced at low doses, and significantly enhanced at higher ones. Doses lower than 0.18 g/100 g BW exerted no significant effect. The same profile was observed for both water and chloride transport. 2.1.1. Animal treatment with a low dose of PGE2 2.1.1.1. Effect of PGE2 on the Na+ –K+ ATPase activity and protein expression in surface and crypt colonocytes isolated from treated animals. PGE2 injected to animals at a relatively low dose (0.4 g/100 g) exerted opposite effects on the Na+ –K+ ATPase activity in surface and crypt cells (Fig. 3). The activity was significantly enhanced in surface cell and
Fig. 2. Effect of different doses of PGE2 on the net absorption of chloride from the colon. Letters above the bars indicate significant differences. P < 0.05: (a,f); P < 0.01: (a,b); P < 0.001: (b,d), (b,e), (b,f), (b,g).
Fig. 3. Effectof low doses of PGE2 on the Na+ –K+ ATPase activity in superficialand crypt cells. Samples from every experiment were run in triplicates. Results report the mean of three experiments.
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
47
Fig. 4. Effect of low doses of PGE2 on the protein expression of Na+ –K+ ATPase in superficial and crypt cells. Results are representative of an experiment repeated three times.
Fig. 5. Effect of low doses of PGE2 on the protein expression of the Na+ –K+ 2Cl− cotransporter in superficial (a) and crypt cells (b). Results are representative of an experiment repeated three times. Results for superficial (c) and crypt cells (d) were quantified using a gel-pro analyser (2) software.
significantly reduced in crypt cells. No significant difference was observed however in the protein expression of the pump in both types of cells (Fig. 4). 2.1.1.2. Effect pf PGE2 on the protein expression of the Na+ K+ 2Cl− cotransporter. The effect of PGE2 on the protein expression of the cotransporter showed a similar profile to that observed with the pump activity. The Na+ K+ 2Cl− symporter was up-regulated in surface cell and down-regulated in crypt cells (Fig. 5). 2.1.2. Animal treatment with a high dose of PGE2 2.1.2.1. Effect of PGE2 on the Na+ –K+ ATPase activity and protein expression in surface and crypt colonocytes isolated from treated animals. At high doses also (21 g/100 g BW), PGE2 increased the activity of the Na+ –K+ ATPase in surface colonocytes but decreased it in crypt cells (Fig. 6). This effect was not associated with any change in the protein expression of the pump (Fig. 7).
Fig. 6. Effect of high doses of PGE2 on the Na+ –K+ ATPase activity in superficial and crypt cells. Samples from every experiment were run in triplicates. Results report the mean of three experiments.
48
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
Fig. 7. Effect of high doses of PGE2 on the protein expression of Na+ –K+ ATPase in superficial and crypt cells. Results are representative of an experiment repeated three times.
2.1.2.2. Effect on the protein expression of the Na+ K+ 2Cl− cotransporter. At high doses PGE2 did not affect the protein expression of the Na+ K+ 2Cl− cotransporter (Fig. 8). 2.1.3. Effect of different agonists/antagonists on the pump activity in isolated colonocytes 2.1.3.1. Superficial cells. Butaprost, a specific agonist for EP2 receptors, increased significantly the pump activity, as did PGE2 (Fig. 9). When the prostaglandin was added in presence of AH-6809, i.e. when EP1, EP2 and EP3 receptors were blocked, an increase of a similar order of magnitude was observed. Sulprostone, an agonist for EP1 and EP3 receptors, caused however a significant reduction in the ATPase activity, which disappeared in presence of SC-19220, an EP1 antagonist (Fig. 9). 2.1.3.2. Crypt cells. In crypt cells, butaprost did not affect the pump activity, while sulprostone decreased it significantly (Fig. 10). The decrease observed with sulprostone was less pronounced in presence of SC-19220. The inhibitory effect of PGE2 disappeared in presence of AH-6809 (Fig. 10).
Fig. 8. Effect of high doses of PGE2 on the protein expression of Na+ –K+ 2Cl− cotransporter in superficial and crypt cells. Results are representative of an experiment repeated three times.
Fig. 9. Effect of butaprost (buta), sulprostone (sulpr), SC-19220. AH-6809, and PGE2 on the activity of the Na+ –K+ pump in superficial colonocytes (N = 3). Letters above the bars indicate significant differences. Bars not sharing a common superscript are significantly different from each other P < 0.01.
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
49
Fig. 10. Effect of butaprost (buta), sulprostone (sulpr), SC-19220. AH-6809, and PGE2 on the activity of the Na+ –K+ pump in crypt colonocytes. Results are the mean of three experiments. Letters above the bars indicate significant differences. Bars not sharing a common superscript are significantly different from each other P < 0.01.
3. Discussion This work demonstrated opposite effects of PGE2 on water movements across the rat colon and showed for the first time that PGE2 is not always a secretagogue in the intestinal tract as it is commonly known. PGE2 acted dosedependently to increase or decrease net water uptake. This dual effect may be ascribed to the presence of different PGE2 receptors (EP), having different affinities for the prostaglandin, and signaling through different pathways that affect differentially the major transporters involved in electrolyte movements. Water transport in the colon follows the movement of electrolytes and in particular that of Na+ . Net water absorption from the colon is the resultant of absorptive and secretory processes. Thus, water absorption may be affecting either electrolyte absorption or electrolyte secretion. The rat colon is dominated by electroneutral absorption of NaCl by the coupled activity of the Na+ /H+ and Cl− /HCO3 − exchangers present in the luminal brush border membrane of colonic epithelial cells [12]. This transport is geared by the Na+ –K+ pump and requires the hydrolysis of one mole of ATP per 3 moles of NaCl absorbed [16]. Consequently, any change in the Na+ –K+ ATPase activity is expected to alter the activity of the Na+ /H+ exchanger (NHE) and alter the net uptake of sodium chloride and water from the gastrointestinal tract. A family of six NHE isoforms has been described, three of which, have been identified in the intestine NHE1, NHE2, and NHE3 [17]. NHEl is ubiquitous, localized to the basolateral membrane, and involved in the regulation of cell volume and intracellular pH (pH) [18–20]. NHE2 and NHE3 are localized to the apical membrane and are involved in vectorial Na+ transport [20]. The three intestinal NHE isoforms are regulated differentially by external signals and second messengers. NHE1 and NHE2 isoforms are stimulated by growth factors,hormones [21], PKA and PKC, while NHE3 is inhibited by the latter two kinases [22]. NHE2 is expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells [23,24]. The major transporter involved in secretion is the Na+ K+ 2Cl− cotransporter situated on the basolateral membrane. The sodium gradient established by the Na+ –K+ pump provides also the driving force for this symporter, which transports Na, K and Cl− to the inside of the cell. K+ is recycled through basolateral K+ channels, while Cl− that accumulates intracellularly above its electrochemical equilibrium leaves through luminal Cl− channels. The rat colon is set to maximal absorption and the effect of regulatory mechanisms is typically to reduce absorption and induce electrolyte secretion [25]. Thus under basal conditions, the Na+ K+ 2Cl− cotransporter has a relatively low activity but becomes stimulated by secretagogues that induce the release of different intracellular messengers. An increase in cAMP and a subsequent activation of PKA was reported to lead to up-regulation of the cotransporter [26], while PKC and calcium lead to its down-regulation [26,27]. The observed PGE2-induced changes in net water absorption may be due to changes in absorption, or secretion, or both. Since the activity of the transporters involved in both processes is geared by the Na gradient established by the Na+ –K+ ATPase, PGE2 may be targeting directly or indirectly the pump and altering its activity and/or protein expression. This work showed that at low doses of PGE2, the activity of the pump was increased in surface colonocytes but reduced in crypt cells. Changes in the protein expression of the Na+ K+ 2Cl cotransporter followed the same profile: the cotransporter was up-regulated in surface cells and down-regulated in crypt colonocytes. Stimulation of the pump by PGE2 is expected to increase the sodium gradient needed for NaCl entry from the luminal side by the coupled Na+ /H+
50
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
and Cl− /HCO3 transport, and water would follow by osmosis. However no increase in net water uptake was observed at low doses probably because it was overcome by an increase in secretion. PGE2 is known to bind to four different types of prostanoid receptors known as EP1-4, having different affinities for PGE2. The Ki values reported for these receptors are 0.8, 1.9, 12 and 20 nM for respectively EP3, EP4, EP2 and EP1 [28]. EP receptors are linked to different G proteins, and activate different intracellular signal transduction pathways [29]. The EP1 receptor mediates Gq activation leading to a PLC-dependent calcium release and PKC activation [30]. EP2 and EP4 receptors couple to a Gs -type G protein leading to cAMP production and PKA stimulation, whereas EP3 receptor couples to a Gi -type G protein that inhibits PKA [31,32]. Of the four EP receptors, EP1 receptors have the lowest affinity for PGE2 and are expected to be active only at high concentrations. Lower PGE2 concentrations involve receptors with higher affinities like EP2 and EP4. Thus, at low concentrations, PGE2 is expected to signal through cAMP/PKA. The effect of cAMP on the Na+ –K+ pump was highly studied but is still controversial. It varies from stimulation to activation and is species-dependent and tissue-specific. It can be direct through phosphorylation of the ␣ subunit on Serine residues 35 or indirect through a change in the activity of other Na+ transporters, leading to changes in intracellular Na+ concentrantion [33,34]. It can be speculated that the observed increase in the activity of the pump at low doses of PGE2, may be caused by an elevation in cAMP levels which are expected to increase the gradient needed for NaCl uptake through the Na+ /H+ exchanger and thus increase electrolyte and water absorption. However no increase in net water absorption was observed at low PGE2 concentrations, probably because absorption was masked by a greater increase in secretion or because the NHE was already inhibited. The most abundant NHE isoform on the apical side of the surface intestinal epithelial cells is NHE3. cAMP was previously shown to inhibit NHE3 [22], activate apical chloride channels [10], and up-regulate the NaKCl cotransproter [26]. As a consequence, although the stimulation of the pump caused an increase in sodium gradient, no increase in Na+ uptake resulted because the exchanger was inhibited by cAMP. On the other hand, cAMP is expected to stimulate indirectly the Na+ –K+ 2Cl− cotransporter through an activation of Cl− channels leading to a decrease in intracellular Cl− levels, an increase in the Cl− gradient across the membrane and a stimulation of the cotransporter [35]. The stimulation of the pump is also another factor contributing to the activation of the cotransporter because it increases the Na gradient. Activation of the pump could be also a consequence of the activation of the cotransporter. Activation of Cl− channels and the Na+ –K+ 2Cl− symporter leads to an increase in Cl− secretion and water retention in the lumen of the colon resulting in a decrease in net water absorption. It can be concluded that at low concentrations, PGE2 activates in surface cells, receptors that increase cAMP and cause a decrease in absorption and an increase in secretion. In crypt cells low doses of PGE2 decreased the activity of the pump and down-regulated the cotransproter. This may be ascribed to the presence of other types of PGE2 receptors that activate different signaling pathways. At high concentrations the net water and Cl− absorption was significantly increased. The activity of the pump was enhanced in surface cells but unchanged in crypt colonocytes. The protein expression of the cotransporter was also not affected. At high doses, PGE2 binds to receptors with high and low affinity and thus would signal through both PKA and PKC/Ca2+ . PKC can activate or inhibit the Na+ –K+ pump depending on the tissue and animal species [36] PKC is known also to inhibit NHE3 by translocating the exchanger from the membrane to sub-apical cytoplasmic compartments [37]. The outcome on the pump, of a simultaneous activation of both PKA and PKC, has not been reported. In this work, high doses of PGE2, which signal via both kinases, stimulated the pump in surface cells, but were without any effect on crypt colonocytes. This may be due to a different distribution of EP receptors and NHE transporter isoforms in the two types of cells. Similarly no change in the protein expression of the cotransporter was observed. To determine the type of receptors involved in the effect of PGE2 on the pump, isolated superficial and crypt colonocytes were incubated with different PGE2 agonists and antagonists. In superficial cells (Fig. 9), an increase in the pump activity was observed with the EP2 agonist butaprost, inferring that EP2 receptors are present in these cells, and their activation, which causes an increase in cAMP levels, leads to a stimulation of the Na+ –K+ ATPase. On the other hand, sulprostone, a PGE2 agonist at EP1 and EP3 receptors, reduced the activity of the pump and its effect disappeared in presence of SC-19220, a specific antagonist for EP1 receptors, suggesting that the effect of sulprostone is mediated via EP1 receptors. The increase in the pump activity observed with PGE2 was not affected by the simultaneous presence of AH-6809, a PGE2 antagonist with equal affinity for EP1, EP2 and EP3 receptors, inferring that the effect of the prostaglandin is not mediated via any of these receptors, but rather via EP4 receptors.
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
51
It can be concluded that in superficial cells EP2 and EP4 receptors are present, and their activation stimulates the pump through a phosphorylation by PKA, since both receptors signal via cAMP. EP1 receptors are also present but cause a reduction in the pump activity. In crypt colonocytes (Fig. 10), butaprost did not exert any effect on the pump activity suggesting that either EP2 receptors are not present, or if present, they do not mediate the effect of PGE2 on the pump. PGE2 and sulprostone exerted an inhibitory effect on the pump, which was of a similar order of magnitude. In presence of SC-19220, sulprostone which activates only EP3 receptors, caused a smaller inhibitory effect on the pump than the one observed with sulprostone alone. The results suggest that both EP1 and EP3 decrease the activity of the pump, and when activated together, they exert additive effects. On the other hand the inhibitory effect of PGE2 disappeared in presence of AH6809, i.e. when EP1, EP2 and EP3 are blocked. Since EP2 receptors were shown not to be involved in the effect of PGE2 on the pump, then the prostaglandin is acting on the pump via EP1 and EP3 receptors. It can be concluded that at high concentrations, the increase in water and chloride absorption is due to stimulation of the pump in surface cells leading to an increase in Na+ absorption. Water secretion is not affected and crypt cells appear not to be involved at high doses. Thus, the reduction in net water and Cl− transport demonstrated in this work at low PGE2 concentrations is due to an increase in secretion, while the net increase in water and Cl− absorption observed at high doses resulted from an increase in absorption. In both cases crypt cells seemed not to be involved in these regulatory processes. Acknowledgement This work was supported by a grant from the University Research Board. References [1] Hatae N, Sugimoto Y, Ichikawa A. Prostaglandin receptors: Advances in the study of EP3 receptor signalling. J Biochem (Tokyo) 2002;131:781–4. [2] Sernka TJ, Rood RP, Mah MY, Tseng CH. Antiabsorptive effects of 16,16 dimethyl prostaglandin E2 in isolated rat colon. Prostaglandins 1982;23:411–26. [3] Traynor TR, Brown DR, O’Grady SM. Effects of inflammatory mediators on electrolyte transport across the porcine distal colon epithelium. J Pharmacol Exp Ther 1993;264:61–6. [4] Greger R, Bleich M, Riedemann N, van Driessche W, Ecke D, Warth R. The role of K+ channels in colonic Cl− secretion. Comp Biochem Physiol A Physiol 1997;118:271–5. [5] Frieling T, Dobreva G, Weber E, et al. Different tachykinin receptors mediate chloride secretion in the distal colon through activation of submucosal neurons. Naunyn Schmiedebergs Arch Pharmacol 1999;359:71–9. [6] Frieling T, Rupprecht C, Dobreva G, Schemann M. Prostaglandin E2 (PGE2)-evoked chloride secretion in guinea-pig colon is mediated by nerve- dependent and nerve-independent mechanisms. Neurogastroenterol Motil 1994;6:95–102. [7] Peterson JW, Ochoa LG. Role of prostaglandins and cAMP in the secretory effects of cholera toxin. Science 1989;245:857–9. [8] Schmitz H, Fromm M, Bode H, Scholz P, Riecken EO, Schulzke JD. Tumor necrosis factor-a induces Cl− and K+ secretion in human distal colon driven byprostaglandin E2. Am J Physiol Gastrointest Liver Physiol 1996;271:G669–74. [9] Curran PF, Schwartz GF. Na, Cl and water transport by rat colon. J Gen Physiol 1960;43:555–71. [10] Kunzelmann K, Mall M. Electrolyte transport in the Mammalian colon: mechanisms and implications for disease. Physiol Rev 2002;82:245–89. [11] Singh SK, Binder HJ, Boron WF, Geibel JP. Fluid absorption in isolated perfused colonic crypts. J Clin Invest 1995;96:2373–9. [12] Binder HJ, Foster ES, Budinger ME, Hayslett JP. Mehcanism of electroneutral sodium chloride absorption in distal colon of the rat. Gastroenterology 1987;93:449–55. [13] Hargis L. Analytical chemistry, principles and techniques. Prentice Hall; 1988. p. 314. [14] Homaidan FR, Zhao L, Donovan V, Shinowara NL, Burakoff R. Separation of pure populations of epithelial cells from rabbit distal colon. Anal Biochem 1995;224:134–9. [15] Taussky HH, Shorr E. Microcolorimetric Method for Determination of Inorganic Phosphorous. J Biol Chem 1953;202:675–85. [16] Jorgensen PL. Sodium and potassium ion pump in kidney tubules. Physiol Rev 1980;60:864–917. [17] Dudeja PK, Rao DD, Syed I, et al. Intestinal distribution of human Na+ /H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA. Am J Physiol Gastrointest Liver Physiol 1996;271:G483–93. [18] Sardet C, Franchi A, Pouyssegur J. Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+ /H+ antiporter. Cell 1989;56:271–80. [19] Tse CM, Ma AI, Yang VW, et al. Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+ /H+ exchanger. EMBO J 1991;10:1957–67. [20] Tyagi S, Joshi V, Alrefai WA, Gill RA, Ramaswamy K, Dudeja PK. Evidence for a Na+ -H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons. Dig Dis Sci 2000;45:2282–9.
52
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
[21] Tse M, Levine S, Yun C, et al. Structure/function studies of the epithelial isoforms of the mammalian Na+ /H+ exchanger gene family. J Membr Biol 1993;135:93–108. [22] Kandasamy RA, Yu FH, Harris R, Boucher A, Hanrahan JW, Orlowski J. Plasma membrane Na+ /H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. JBC 1995;270:29209–16. [23] Janecki AJ, Montrose MH, Zimniak P, et al. Subcellular redistribution is involved in acute regulation of the brush border Na+ /H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 1998;273:8790–8. [24] Bachmann O, Riederer B, Rossmann H, et al. The Na+ /H+ exchanger isoform 2 is the predominant NHE isoform in murine coIonic crypts and its lack causes NHE3 upregulation. Am J Physiol Gastrointest Liver Physiol 2004;287:G125–33. [25] Andres H, Rock R, Bridges RJ, Rummel W, Schneider J. Sub-mucosal plexus and electrolyte transport across rat colonic mucosa. J Physiol (London) 1985;364:301–12. [26] D’Andrea L, Lytle C, Matthews JB, Hofman P, Forbush B, Madar JL. Na-K-2Cl cotransporter (NKCC) of intestinal epithelial cells. Surface expression in response to cAMP. J Biol Chem 1996;271:28969–76. [27] Layne J, Yip S, Crook RB. Down-regulation of Na-K-Cl cotransport by protein kinase C is mediated by protein phosphatase 1 in pigmented ciliary epithelial cells. Exp Eye Res 2001;72:371–9. [28] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999;79:1193–226. [29] Hata AN, Breyer RM. Pharmacology and signaling of prostaglandin receptors: Multiple roles in inflammation and immune modulation. Pharmacol Therapeut 2004;103:147–66. [30] Hebert RL, Jacobson HR, Breyer MD. PGE2 inhibits AVP-induced water flow in cortical collecting ducts by protein kinase C activation. Am J Physiol 1990;259:F318–25. [31] Bos CL, Richel DJ, Ritsema T, Peppelenbosch MP, Versteeg HH. Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 2004;36:1187–205. [32] Abramovitz M, Adam M, Boie Y, et al. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta 2000;1483:285–93. [33] Fisone G, Cheng SX, Nairn AC, et al. Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+ , K(+)-ATPase and effects of site-directed mutagenesis. J Biol Chem 1994;269:9368–73. [34] Stewart WC, Pekala PH, Lieberman EM. Acute and chronic regulation of Na+ /K+ ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production. Glia 1998;23:349–60. [35] Russel JM. Sodium-potassium-chloride cotransport. Physiol Rev 2000;80:211–76. [36] Therien AG, Rhoda B. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 2000;279:C541–66. [37] Janecki AJ, Montrose MH, Zimniak P, et al. Subcellular redistribution is involved in acute regulation of the brush border Na+ /H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 1998;173:8790–8.
PGE2 exerts dose-dependent opposite effects on net water and chloride absorption from the rat colon Sawsan Ibrahim Kreydiyyeh ∗ , Sarine Markossian, Rawad F. Hodeify Department of Biology, Faculty of Arts & Sciences, American University of Beirut, Beirut, Lebanon Received 28 May 2005; received in revised form 22 July 2005; accepted 25 July 2005 Available online 13 February 2006
Abstract This work investigated the effect of different doses of PGE2 on net water and Cl− absorption from the rat colon, using an in situ perfusion technique. PGE2 exerted opposite effects at different concentrations. Net water and Cl− absorption was significantly reduced at low doses with a minimum at 0.4 g/100 g BW, and significantly elevated at high doses with an observed maximal effect at 21 g/100 g BW. At low doses, PGE2 increased in superficial cells, the activity of the Na+ –K+ ATPase and the protein expression of the Na+ K+ 2Cl− cotransporter, but reduced them in crypt cells. Thus, the reduction in net water and Cl− absorption was ascribed to an increase in secretion by surface cells that masked absorptive processes. At high doses, PGE2 increased significantly the activity of the Na+ –K+ ATPase in superficial cells only, and was without any effect on the protein expression of the cotransporter and the pump in both surface and crypt cells. The observed increase in net water and Cl− absorption was attributed in this case to an increase in absorptive processes with no effect on secretion. © 2005 Elsevier Inc. All rights reserved. Keywords: PGE2; Water; Chloride ;Na+ –K+ ATPase; Na+ –K+ –2Cl− ; Colon
PGE2 is an eicosanoid with diverse biological actions on the reproductive, gastrointestinal and neuroendocrine system. Derived from the cyclo-oxygenase (COX) mediated metabolism of arachidonic acid, it has abroad range of physiological and pharmacological effects, and acts via its different receptors to maintain homeostasis [1]. PGE2 is a known modulator of water and electrolyte movements in the colon. When released into the colonic lumen of patients with ulcerative colitis, it reduced net mucosal to serosal fluxes of Na+ and Cl− and contributed to the diarrhea associated with the disease [2]. PGE2 was shown also to inhibit the HCO3 − dependent Cl− absorption in porcine distal colon epithelium [3], and stimulate K+ and Cl− secretion in rabbit distal colon. In rat colonic crypt base cells it increased Cl− secretion by inducing a marked depolarization that reduces the basal K+ conductance [4]. Because most studies showed an inhibition in intestinal fluid and chloride absorption by PGE2, the prostaglandin has always been recognized as a secretagogue in the gastrointestinal tract [5–8]. Net water movement in the colon is directly dependent on the rate of net electrolytes transport [9]. Ions like Na+ , K+ and Cl− are transported across the colonic epithelium from the mucosal to the serosal side or vice versa, and cause the movement of water by osmosis. Net fluid transport is the resultant of absorptive and secretory processes and depends on the activity of major transporters like the Na+ –K+ ATPase, the Na/H+ exchanger and the ∗
Corresponding author. Fax: +961 1 744461. E-mail address: [email protected] (S.I. Kreydiyyeh).
1098-8823/$ – see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2005.07.004
44
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
Na+ K+ 2Cl− symporter [10]. Sodium absorption can be electrogenic via sodium channels or electroneutral via parallel Na+ /H+ and Cl− /HCO3 − exchange, and is geared by the sodium gradient established by the Na+ –K+ pump situated on the basolateral membrane. Bulk sodium transport in the rat colon takes place in both crypts and surface epithelium [11] of proximal and distal colon and is dominated by electroneutral absorption [12]. The Na+ K+ 2Cl− cotransporter, situated on the basolateral side, is responsible for chloride and potassium secretion. Under control conditions, ion transport activities result in net absorption, and secretory properties become apparent only upon stimulation by seretagogues. As a secretagocue, PGE2 may be altering absorptive and secretory processes by targeting electrolyte transporters and changing their activity. This work studies the effect of different doses of PGE2 on net water and chloride absorption from the rat colon and attempts to delineate its mode of action by investigating its effect on the Na+ –K+ pump, a major regulator of transport processes, and on the Na+ K+ 2Cl− symporter, a major player in secretion. The results demonstrate, a dose-dependent dual stimulatory and inhibitory role of PGE2 on net water and Cl− absorption from the rat colon, and shows that the prostaglandin does not act always as a secretagogue but can at some concentrations stimulate fluid and electrolyte absorption. 1. Materials and methods 1.1. Animal treatments Male Sprague-Dawley rats (Rattus norvegicus) weighing 150–500 g were handled all through in accordance with the Guide for Laboratory Animal Facilities and Care, US Department of Health, Education and Welfare. Rats weighing around 250 g were injected i.p. with PGE2 (21; 4; 1; 04; 0.18; 0.05 g/100 g BW) 15 min before the beginning of the experiment. PGE2 was dissolved in ethanol. Control rats were injected with a similar volume of the vehicle. In all other experiments high and low PGE2 doses refer respectively to 21 and 0.4 g/100 g BW). 1.1.1. Measurement of luminal water movements in the rat colon Rats were anesthetized by i.p. injection of pentobarbital (5 mg/100 g body weight). The abdomen was then opened through a midline incision to expose the colon. L-shaped inlet and outlet catheters connected to polyethylene tubing were introduced at each end of the colon keeping the mesenteries and vasculature intact. The end of the inlet catheter was connected to a perfusion pump whose rate was maintained at 0.7 ml/min, while the outlet tube drained into a beaker to allow for the collection of the effluent buffer. The colon was perfused first with oxygenated (95% O2 ; 5% CO2 ) Krebs Improved Ringer buffer pH 7.4 (KIRB) (123.3 mM NaCl; 6.17 mM KCl; 3.29 mM CaCl2 ·7H2 O; 0.78 mM MgSO4 ·7H2 O; 32.14 mM NaHCO3 ; 1.54 mM KH2 PO4 ; 6.4 mM sodium glutamate; 6.4 mM sodium pyruvate; 7 mM sodium fumarate; 11.1 mM glucose) for 15 min to clean it from wastes. Then, it was perfused with the same buffer for another 30 min and the effluent buffer collected. At the end of the perfusion, the volume of the infused and collected buffers was measured and the colon was excised, cut longitudinally, its widih and length measured and its surface area calculated. Water absorption, during the second perfusion step, was calculated as the difference between the volumes of infused and collected buffers divided by the surface area of the perfused colon. 1.1.2. Measurement of net chloride absorption in the rat colon Samples from the inlet and outlet buffers were drawn and assayed for their Cl− content by titration with silver nitrate AgNO3 (0.1 M) according to Mohr’s method [13]. K2 CrO4 (2.5 M) was used as an indicator. Chloride absorption was calculated as the difference in the number of moles of chloride present in the infused and collected buffers. 1.1.3. Isolation of colon superficial and crypt cells Surface and crypt cells were isolated from the colon and characterized as described by Homaidan et al. [14]. Briefly, the colon was removed and washed consecutively with ice cold saline-DTT (0.154 M NaCl; 0.1 mM DTT (dithiothreitol)) and ice cold phosphate buffer saline (PBS) pH 7.3 (0.088 M NaCl; 0.01 M Na2 HPO4 ; 8 mM KH2 PO4 ) to clean it from wastes. Then, it was everted, filled with PBS pH 7.3, clamped at both ends, and incubated at 37 ◦ C first, in citrate buffer (27 mM Na citrate, 96 Mm NaCl, 1.5 mM KCl, 8 mM KH2 PO4 , 5.6 mM Na2 HPO4 , 1 mM DTT, pH 7.5)
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
45
for 10 min and then in PBS-EDTA buffer (140 mM NaCl, 8 mM Na2 HPO4 , 2.8 mM KCl, 1.5 mM KH2 PO4 , 0.5 mM DTT, 1.5 mM Na2 EDTA, pH 7.4) for l5 min in a shaking water bath (75 cycles/min) while gassing with 95% O2 , 5% CO2 . The cells shed in the latter buffer were collected by a 5-min centrifugation at 770 × g and 4 ◦ C and corresponded to villus cells. Subsequently, two sequential fractions were collected after two consecutive 15-min incubation periods in PBS-EDTA buffer. The cells released in the last fraction were collected by centrifugation as before and corresponded to crypt cells. After collection, villus and crypt cells were washed twice with Tris buffer. The collected superficial and crypt cells were resuspended in Tris buffer pH 7.4 (buffer A) (200 mM NaCl; 5 mM MgCl2 ; 2 mM EGTA; 5 mM KCl; 200 mM Trizmabase) (0.4 g/ml) and homogenized for 3 min with a polytron (20,000–22,000 rpm) at 4 ◦ C, then spun at 3300 × g for 10 min at 4 ◦ C to prepare a crude membrane homogenate. A mixture of protease inhibitors was then added to the homogenate which was saved at −20 ◦ C for later use. 1.1.4. Incubation of isolated cells with agonists/antagonists To determine the type of receptors involved in the effect of PGE2 on the pump, specific agonist and antagonists (Cayman Chemical Company, MI, USA) were used. Butaprost and sulprostone were chosen as respective selective agonists for EP2 and, EP3 and EP1 receptors, while SC-19220 was chosen as a selective antagonist of PGE2 at the EP1 receptor, and AH-6809 as an antagonist with equal affinity for EP1, EP2 and EP3 as reported by the providing company. Superficial and crypt cells were isolated as described before, re-suspended (0.07 g/ml) in DMEM culture medium containing 4500 mg/l Glucose, sodium pyruvate, 15% FBS, 1% PS; Penicillin (100 g/ml), and incubated with continuous oxygenation (95% O2 ; 5% CO2 ) for 30 min in presence of the agonists or, with PGE2 and its antagonists. PGE2 was added at 1 nM concentration. Agonists were added at a concentration of 1 M and the antagonists at a concentration of 10 M. The viability of the cells was checked every 10 min and did not drop below 80% by the end of the experiment. Antagonists were added 10 min before PGE2. At the end of the incubation period, the cells were collected by centrifugation and crude membrane homogenates were prepared as described before and used to assay for the Na+ –K+ ATPase activity. 1.1.5. Na+ –K+ ATPase assay Membrane homogenates were diluted in Tris buffer (buffer A) to a final concentration of 2 mg/ml protein and incubated for 30 min with saponin (0.02% final concentration) at room temperature. After a pre-incubation at 37 ◦ C for 10 min in presence or absence of ouabain (4 mM final concentration), the reaction was initiated by addition of ATP to a final concentration of 1.25 mM, and terminated after a one hour incubation at 37 ◦ C by addition of trichloroacetic acid (200 l, 11%). The amount of inorganic phosphate liberated was measured colorimetrically according to the meihod of Taussky and Shorr [15] and the activity of the enzyme was determined by measuring the ouabain-inhibitable inorganic phosphate liberated. The percent inhibition of the enzyme activity was calculated as follows: 1−
Pi (treatment) − Pi (treatment + ouabain) × 100. Pi (control) − Pi (control + ouabain)
1.1.6. Western blot analysis Membrane proteins were quantified using the Bio-Rad reagent and were equally loaded and resolved on 8% polyacrylamide gels, then transferred to a PVDF membrane (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA). Protein expression of  actin was used to check for equal loading. The PVDF membrane was then washed, blocked and incubated overnight at 4 ◦ C with a rabbit Anti-Na+ /K+ ATPase ␣1 IgG (Upstate biotechnology, Lake Placid, NY 12946), or with an anti-rat sodium potassium chloride cotransporter antibody (Alpha Diagnostic International, TX, USA). Detection of the signal was by enhanced chemiluminescence using luminol reagent (Santa Cruiz Biotechnology Inc., CA, USA). The thickness of the bands was determined by densitometry using a gel-pro analyzer (2) software. 1.1.7. Statistical analysis Results are reported as means ± SEM. Statistical significance was tested by a Student t-test or a one-way analysis of variance followed by a Tukey–Kramer multiple comparisons.
46
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
Fig. 1. Effect of different doses of PGE2 on the net absorption of water from the colon. Letters above the bars indicate significant differences. P < 0.05: (a,e); P < 0.01: (a,b); P < 0.001: (b,d), (b,e), (b,f), (b,g).
2. Results 2.1. Dose dependent effect of PGE2 on net water and chloride absorption from the colon The effect of PGE2 on net water and chloride absorption from the colon was dose-dependent (Figs. 1 and 2). Net water and chloride absorption was significantly reduced at low doses, and significantly enhanced at higher ones. Doses lower than 0.18 g/100 g BW exerted no significant effect. The same profile was observed for both water and chloride transport. 2.1.1. Animal treatment with a low dose of PGE2 2.1.1.1. Effect of PGE2 on the Na+ –K+ ATPase activity and protein expression in surface and crypt colonocytes isolated from treated animals. PGE2 injected to animals at a relatively low dose (0.4 g/100 g) exerted opposite effects on the Na+ –K+ ATPase activity in surface and crypt cells (Fig. 3). The activity was significantly enhanced in surface cell and
Fig. 2. Effect of different doses of PGE2 on the net absorption of chloride from the colon. Letters above the bars indicate significant differences. P < 0.05: (a,f); P < 0.01: (a,b); P < 0.001: (b,d), (b,e), (b,f), (b,g).
Fig. 3. Effectof low doses of PGE2 on the Na+ –K+ ATPase activity in superficialand crypt cells. Samples from every experiment were run in triplicates. Results report the mean of three experiments.
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
47
Fig. 4. Effect of low doses of PGE2 on the protein expression of Na+ –K+ ATPase in superficial and crypt cells. Results are representative of an experiment repeated three times.
Fig. 5. Effect of low doses of PGE2 on the protein expression of the Na+ –K+ 2Cl− cotransporter in superficial (a) and crypt cells (b). Results are representative of an experiment repeated three times. Results for superficial (c) and crypt cells (d) were quantified using a gel-pro analyser (2) software.
significantly reduced in crypt cells. No significant difference was observed however in the protein expression of the pump in both types of cells (Fig. 4). 2.1.1.2. Effect pf PGE2 on the protein expression of the Na+ K+ 2Cl− cotransporter. The effect of PGE2 on the protein expression of the cotransporter showed a similar profile to that observed with the pump activity. The Na+ K+ 2Cl− symporter was up-regulated in surface cell and down-regulated in crypt cells (Fig. 5). 2.1.2. Animal treatment with a high dose of PGE2 2.1.2.1. Effect of PGE2 on the Na+ –K+ ATPase activity and protein expression in surface and crypt colonocytes isolated from treated animals. At high doses also (21 g/100 g BW), PGE2 increased the activity of the Na+ –K+ ATPase in surface colonocytes but decreased it in crypt cells (Fig. 6). This effect was not associated with any change in the protein expression of the pump (Fig. 7).
Fig. 6. Effect of high doses of PGE2 on the Na+ –K+ ATPase activity in superficial and crypt cells. Samples from every experiment were run in triplicates. Results report the mean of three experiments.
48
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
Fig. 7. Effect of high doses of PGE2 on the protein expression of Na+ –K+ ATPase in superficial and crypt cells. Results are representative of an experiment repeated three times.
2.1.2.2. Effect on the protein expression of the Na+ K+ 2Cl− cotransporter. At high doses PGE2 did not affect the protein expression of the Na+ K+ 2Cl− cotransporter (Fig. 8). 2.1.3. Effect of different agonists/antagonists on the pump activity in isolated colonocytes 2.1.3.1. Superficial cells. Butaprost, a specific agonist for EP2 receptors, increased significantly the pump activity, as did PGE2 (Fig. 9). When the prostaglandin was added in presence of AH-6809, i.e. when EP1, EP2 and EP3 receptors were blocked, an increase of a similar order of magnitude was observed. Sulprostone, an agonist for EP1 and EP3 receptors, caused however a significant reduction in the ATPase activity, which disappeared in presence of SC-19220, an EP1 antagonist (Fig. 9). 2.1.3.2. Crypt cells. In crypt cells, butaprost did not affect the pump activity, while sulprostone decreased it significantly (Fig. 10). The decrease observed with sulprostone was less pronounced in presence of SC-19220. The inhibitory effect of PGE2 disappeared in presence of AH-6809 (Fig. 10).
Fig. 8. Effect of high doses of PGE2 on the protein expression of Na+ –K+ 2Cl− cotransporter in superficial and crypt cells. Results are representative of an experiment repeated three times.
Fig. 9. Effect of butaprost (buta), sulprostone (sulpr), SC-19220. AH-6809, and PGE2 on the activity of the Na+ –K+ pump in superficial colonocytes (N = 3). Letters above the bars indicate significant differences. Bars not sharing a common superscript are significantly different from each other P < 0.01.
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
49
Fig. 10. Effect of butaprost (buta), sulprostone (sulpr), SC-19220. AH-6809, and PGE2 on the activity of the Na+ –K+ pump in crypt colonocytes. Results are the mean of three experiments. Letters above the bars indicate significant differences. Bars not sharing a common superscript are significantly different from each other P < 0.01.
3. Discussion This work demonstrated opposite effects of PGE2 on water movements across the rat colon and showed for the first time that PGE2 is not always a secretagogue in the intestinal tract as it is commonly known. PGE2 acted dosedependently to increase or decrease net water uptake. This dual effect may be ascribed to the presence of different PGE2 receptors (EP), having different affinities for the prostaglandin, and signaling through different pathways that affect differentially the major transporters involved in electrolyte movements. Water transport in the colon follows the movement of electrolytes and in particular that of Na+ . Net water absorption from the colon is the resultant of absorptive and secretory processes. Thus, water absorption may be affecting either electrolyte absorption or electrolyte secretion. The rat colon is dominated by electroneutral absorption of NaCl by the coupled activity of the Na+ /H+ and Cl− /HCO3 − exchangers present in the luminal brush border membrane of colonic epithelial cells [12]. This transport is geared by the Na+ –K+ pump and requires the hydrolysis of one mole of ATP per 3 moles of NaCl absorbed [16]. Consequently, any change in the Na+ –K+ ATPase activity is expected to alter the activity of the Na+ /H+ exchanger (NHE) and alter the net uptake of sodium chloride and water from the gastrointestinal tract. A family of six NHE isoforms has been described, three of which, have been identified in the intestine NHE1, NHE2, and NHE3 [17]. NHEl is ubiquitous, localized to the basolateral membrane, and involved in the regulation of cell volume and intracellular pH (pH) [18–20]. NHE2 and NHE3 are localized to the apical membrane and are involved in vectorial Na+ transport [20]. The three intestinal NHE isoforms are regulated differentially by external signals and second messengers. NHE1 and NHE2 isoforms are stimulated by growth factors,hormones [21], PKA and PKC, while NHE3 is inhibited by the latter two kinases [22]. NHE2 is expressed most abundantly in crypts, NHE1 equally in crypts and surface cells, and NHE3 much stronger in surface cells [23,24]. The major transporter involved in secretion is the Na+ K+ 2Cl− cotransporter situated on the basolateral membrane. The sodium gradient established by the Na+ –K+ pump provides also the driving force for this symporter, which transports Na, K and Cl− to the inside of the cell. K+ is recycled through basolateral K+ channels, while Cl− that accumulates intracellularly above its electrochemical equilibrium leaves through luminal Cl− channels. The rat colon is set to maximal absorption and the effect of regulatory mechanisms is typically to reduce absorption and induce electrolyte secretion [25]. Thus under basal conditions, the Na+ K+ 2Cl− cotransporter has a relatively low activity but becomes stimulated by secretagogues that induce the release of different intracellular messengers. An increase in cAMP and a subsequent activation of PKA was reported to lead to up-regulation of the cotransporter [26], while PKC and calcium lead to its down-regulation [26,27]. The observed PGE2-induced changes in net water absorption may be due to changes in absorption, or secretion, or both. Since the activity of the transporters involved in both processes is geared by the Na gradient established by the Na+ –K+ ATPase, PGE2 may be targeting directly or indirectly the pump and altering its activity and/or protein expression. This work showed that at low doses of PGE2, the activity of the pump was increased in surface colonocytes but reduced in crypt cells. Changes in the protein expression of the Na+ K+ 2Cl cotransporter followed the same profile: the cotransporter was up-regulated in surface cells and down-regulated in crypt colonocytes. Stimulation of the pump by PGE2 is expected to increase the sodium gradient needed for NaCl entry from the luminal side by the coupled Na+ /H+
50
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
and Cl− /HCO3 transport, and water would follow by osmosis. However no increase in net water uptake was observed at low doses probably because it was overcome by an increase in secretion. PGE2 is known to bind to four different types of prostanoid receptors known as EP1-4, having different affinities for PGE2. The Ki values reported for these receptors are 0.8, 1.9, 12 and 20 nM for respectively EP3, EP4, EP2 and EP1 [28]. EP receptors are linked to different G proteins, and activate different intracellular signal transduction pathways [29]. The EP1 receptor mediates Gq activation leading to a PLC-dependent calcium release and PKC activation [30]. EP2 and EP4 receptors couple to a Gs -type G protein leading to cAMP production and PKA stimulation, whereas EP3 receptor couples to a Gi -type G protein that inhibits PKA [31,32]. Of the four EP receptors, EP1 receptors have the lowest affinity for PGE2 and are expected to be active only at high concentrations. Lower PGE2 concentrations involve receptors with higher affinities like EP2 and EP4. Thus, at low concentrations, PGE2 is expected to signal through cAMP/PKA. The effect of cAMP on the Na+ –K+ pump was highly studied but is still controversial. It varies from stimulation to activation and is species-dependent and tissue-specific. It can be direct through phosphorylation of the ␣ subunit on Serine residues 35 or indirect through a change in the activity of other Na+ transporters, leading to changes in intracellular Na+ concentrantion [33,34]. It can be speculated that the observed increase in the activity of the pump at low doses of PGE2, may be caused by an elevation in cAMP levels which are expected to increase the gradient needed for NaCl uptake through the Na+ /H+ exchanger and thus increase electrolyte and water absorption. However no increase in net water absorption was observed at low PGE2 concentrations, probably because absorption was masked by a greater increase in secretion or because the NHE was already inhibited. The most abundant NHE isoform on the apical side of the surface intestinal epithelial cells is NHE3. cAMP was previously shown to inhibit NHE3 [22], activate apical chloride channels [10], and up-regulate the NaKCl cotransproter [26]. As a consequence, although the stimulation of the pump caused an increase in sodium gradient, no increase in Na+ uptake resulted because the exchanger was inhibited by cAMP. On the other hand, cAMP is expected to stimulate indirectly the Na+ –K+ 2Cl− cotransporter through an activation of Cl− channels leading to a decrease in intracellular Cl− levels, an increase in the Cl− gradient across the membrane and a stimulation of the cotransporter [35]. The stimulation of the pump is also another factor contributing to the activation of the cotransporter because it increases the Na gradient. Activation of the pump could be also a consequence of the activation of the cotransporter. Activation of Cl− channels and the Na+ –K+ 2Cl− symporter leads to an increase in Cl− secretion and water retention in the lumen of the colon resulting in a decrease in net water absorption. It can be concluded that at low concentrations, PGE2 activates in surface cells, receptors that increase cAMP and cause a decrease in absorption and an increase in secretion. In crypt cells low doses of PGE2 decreased the activity of the pump and down-regulated the cotransproter. This may be ascribed to the presence of other types of PGE2 receptors that activate different signaling pathways. At high concentrations the net water and Cl− absorption was significantly increased. The activity of the pump was enhanced in surface cells but unchanged in crypt colonocytes. The protein expression of the cotransporter was also not affected. At high doses, PGE2 binds to receptors with high and low affinity and thus would signal through both PKA and PKC/Ca2+ . PKC can activate or inhibit the Na+ –K+ pump depending on the tissue and animal species [36] PKC is known also to inhibit NHE3 by translocating the exchanger from the membrane to sub-apical cytoplasmic compartments [37]. The outcome on the pump, of a simultaneous activation of both PKA and PKC, has not been reported. In this work, high doses of PGE2, which signal via both kinases, stimulated the pump in surface cells, but were without any effect on crypt colonocytes. This may be due to a different distribution of EP receptors and NHE transporter isoforms in the two types of cells. Similarly no change in the protein expression of the cotransporter was observed. To determine the type of receptors involved in the effect of PGE2 on the pump, isolated superficial and crypt colonocytes were incubated with different PGE2 agonists and antagonists. In superficial cells (Fig. 9), an increase in the pump activity was observed with the EP2 agonist butaprost, inferring that EP2 receptors are present in these cells, and their activation, which causes an increase in cAMP levels, leads to a stimulation of the Na+ –K+ ATPase. On the other hand, sulprostone, a PGE2 agonist at EP1 and EP3 receptors, reduced the activity of the pump and its effect disappeared in presence of SC-19220, a specific antagonist for EP1 receptors, suggesting that the effect of sulprostone is mediated via EP1 receptors. The increase in the pump activity observed with PGE2 was not affected by the simultaneous presence of AH-6809, a PGE2 antagonist with equal affinity for EP1, EP2 and EP3 receptors, inferring that the effect of the prostaglandin is not mediated via any of these receptors, but rather via EP4 receptors.
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
51
It can be concluded that in superficial cells EP2 and EP4 receptors are present, and their activation stimulates the pump through a phosphorylation by PKA, since both receptors signal via cAMP. EP1 receptors are also present but cause a reduction in the pump activity. In crypt colonocytes (Fig. 10), butaprost did not exert any effect on the pump activity suggesting that either EP2 receptors are not present, or if present, they do not mediate the effect of PGE2 on the pump. PGE2 and sulprostone exerted an inhibitory effect on the pump, which was of a similar order of magnitude. In presence of SC-19220, sulprostone which activates only EP3 receptors, caused a smaller inhibitory effect on the pump than the one observed with sulprostone alone. The results suggest that both EP1 and EP3 decrease the activity of the pump, and when activated together, they exert additive effects. On the other hand the inhibitory effect of PGE2 disappeared in presence of AH6809, i.e. when EP1, EP2 and EP3 are blocked. Since EP2 receptors were shown not to be involved in the effect of PGE2 on the pump, then the prostaglandin is acting on the pump via EP1 and EP3 receptors. It can be concluded that at high concentrations, the increase in water and chloride absorption is due to stimulation of the pump in surface cells leading to an increase in Na+ absorption. Water secretion is not affected and crypt cells appear not to be involved at high doses. Thus, the reduction in net water and Cl− transport demonstrated in this work at low PGE2 concentrations is due to an increase in secretion, while the net increase in water and Cl− absorption observed at high doses resulted from an increase in absorption. In both cases crypt cells seemed not to be involved in these regulatory processes. Acknowledgement This work was supported by a grant from the University Research Board. References [1] Hatae N, Sugimoto Y, Ichikawa A. Prostaglandin receptors: Advances in the study of EP3 receptor signalling. J Biochem (Tokyo) 2002;131:781–4. [2] Sernka TJ, Rood RP, Mah MY, Tseng CH. Antiabsorptive effects of 16,16 dimethyl prostaglandin E2 in isolated rat colon. Prostaglandins 1982;23:411–26. [3] Traynor TR, Brown DR, O’Grady SM. Effects of inflammatory mediators on electrolyte transport across the porcine distal colon epithelium. J Pharmacol Exp Ther 1993;264:61–6. [4] Greger R, Bleich M, Riedemann N, van Driessche W, Ecke D, Warth R. The role of K+ channels in colonic Cl− secretion. Comp Biochem Physiol A Physiol 1997;118:271–5. [5] Frieling T, Dobreva G, Weber E, et al. Different tachykinin receptors mediate chloride secretion in the distal colon through activation of submucosal neurons. Naunyn Schmiedebergs Arch Pharmacol 1999;359:71–9. [6] Frieling T, Rupprecht C, Dobreva G, Schemann M. Prostaglandin E2 (PGE2)-evoked chloride secretion in guinea-pig colon is mediated by nerve- dependent and nerve-independent mechanisms. Neurogastroenterol Motil 1994;6:95–102. [7] Peterson JW, Ochoa LG. Role of prostaglandins and cAMP in the secretory effects of cholera toxin. Science 1989;245:857–9. [8] Schmitz H, Fromm M, Bode H, Scholz P, Riecken EO, Schulzke JD. Tumor necrosis factor-a induces Cl− and K+ secretion in human distal colon driven byprostaglandin E2. Am J Physiol Gastrointest Liver Physiol 1996;271:G669–74. [9] Curran PF, Schwartz GF. Na, Cl and water transport by rat colon. J Gen Physiol 1960;43:555–71. [10] Kunzelmann K, Mall M. Electrolyte transport in the Mammalian colon: mechanisms and implications for disease. Physiol Rev 2002;82:245–89. [11] Singh SK, Binder HJ, Boron WF, Geibel JP. Fluid absorption in isolated perfused colonic crypts. J Clin Invest 1995;96:2373–9. [12] Binder HJ, Foster ES, Budinger ME, Hayslett JP. Mehcanism of electroneutral sodium chloride absorption in distal colon of the rat. Gastroenterology 1987;93:449–55. [13] Hargis L. Analytical chemistry, principles and techniques. Prentice Hall; 1988. p. 314. [14] Homaidan FR, Zhao L, Donovan V, Shinowara NL, Burakoff R. Separation of pure populations of epithelial cells from rabbit distal colon. Anal Biochem 1995;224:134–9. [15] Taussky HH, Shorr E. Microcolorimetric Method for Determination of Inorganic Phosphorous. J Biol Chem 1953;202:675–85. [16] Jorgensen PL. Sodium and potassium ion pump in kidney tubules. Physiol Rev 1980;60:864–917. [17] Dudeja PK, Rao DD, Syed I, et al. Intestinal distribution of human Na+ /H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA. Am J Physiol Gastrointest Liver Physiol 1996;271:G483–93. [18] Sardet C, Franchi A, Pouyssegur J. Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+ /H+ antiporter. Cell 1989;56:271–80. [19] Tse CM, Ma AI, Yang VW, et al. Molecular cloning and expression of a cDNA encoding the rabbit ileal villus cell basolateral membrane Na+ /H+ exchanger. EMBO J 1991;10:1957–67. [20] Tyagi S, Joshi V, Alrefai WA, Gill RA, Ramaswamy K, Dudeja PK. Evidence for a Na+ -H+ exchange across human colonic basolateral plasma membranes purified from organ donor colons. Dig Dis Sci 2000;45:2282–9.
52
S.I. Kreydiyyeh et al. / Prostaglandins & other Lipid Mediators 79 (2006) 43–52
[21] Tse M, Levine S, Yun C, et al. Structure/function studies of the epithelial isoforms of the mammalian Na+ /H+ exchanger gene family. J Membr Biol 1993;135:93–108. [22] Kandasamy RA, Yu FH, Harris R, Boucher A, Hanrahan JW, Orlowski J. Plasma membrane Na+ /H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. JBC 1995;270:29209–16. [23] Janecki AJ, Montrose MH, Zimniak P, et al. Subcellular redistribution is involved in acute regulation of the brush border Na+ /H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 1998;273:8790–8. [24] Bachmann O, Riederer B, Rossmann H, et al. The Na+ /H+ exchanger isoform 2 is the predominant NHE isoform in murine coIonic crypts and its lack causes NHE3 upregulation. Am J Physiol Gastrointest Liver Physiol 2004;287:G125–33. [25] Andres H, Rock R, Bridges RJ, Rummel W, Schneider J. Sub-mucosal plexus and electrolyte transport across rat colonic mucosa. J Physiol (London) 1985;364:301–12. [26] D’Andrea L, Lytle C, Matthews JB, Hofman P, Forbush B, Madar JL. Na-K-2Cl cotransporter (NKCC) of intestinal epithelial cells. Surface expression in response to cAMP. J Biol Chem 1996;271:28969–76. [27] Layne J, Yip S, Crook RB. Down-regulation of Na-K-Cl cotransport by protein kinase C is mediated by protein phosphatase 1 in pigmented ciliary epithelial cells. Exp Eye Res 2001;72:371–9. [28] Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors: structures, properties, and functions. Physiol Rev 1999;79:1193–226. [29] Hata AN, Breyer RM. Pharmacology and signaling of prostaglandin receptors: Multiple roles in inflammation and immune modulation. Pharmacol Therapeut 2004;103:147–66. [30] Hebert RL, Jacobson HR, Breyer MD. PGE2 inhibits AVP-induced water flow in cortical collecting ducts by protein kinase C activation. Am J Physiol 1990;259:F318–25. [31] Bos CL, Richel DJ, Ritsema T, Peppelenbosch MP, Versteeg HH. Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 2004;36:1187–205. [32] Abramovitz M, Adam M, Boie Y, et al. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta 2000;1483:285–93. [33] Fisone G, Cheng SX, Nairn AC, et al. Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+ , K(+)-ATPase and effects of site-directed mutagenesis. J Biol Chem 1994;269:9368–73. [34] Stewart WC, Pekala PH, Lieberman EM. Acute and chronic regulation of Na+ /K+ ATPase transport activity in the RN22 Schwann cell line in response to stimulation of cyclic AMP production. Glia 1998;23:349–60. [35] Russel JM. Sodium-potassium-chloride cotransport. Physiol Rev 2000;80:211–76. [36] Therien AG, Rhoda B. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 2000;279:C541–66. [37] Janecki AJ, Montrose MH, Zimniak P, et al. Subcellular redistribution is involved in acute regulation of the brush border Na+ /H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 1998;173:8790–8.