Na absorption in the colon plays an important role

Transcription

1 Japanese Journal of Physiology, 51, , 2001 The Effect of camp on Electrogenic Na Absorption in the Rat Distal Colon Yo TSUCHIYA and Yuichi SUZUKI Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan Abstract: Cyclic AMP is a ubiquitous second messenger produced in cells in response to extracellular stimulants. The aim of this study was to examine its role in the regulation of amiloridesensitive electrogenic Na absorption in the rat distal colon by measuring the short-circuit current (I sc ) and 22 Na fluxes in a chamber-mounted mucosal sheet. Forskolin stimulated the amilorideinhibitable I sc and amiloride-inhibitable 22 Na absorption. 8Br-cAMP also stimulated the amiloride-inhibitable I sc. Furthermore, isoproterenol, acting via -adrenergic activation, stimulated the amiloride-inhibitable I sc. The isoproterenol-induced increase in the amiloride-sensitive I sc was largely suppressed by H89, a protein kinase A inhibitor. In conclusion, camp can upregulate amiloride-sensitive electrogenic Na absorption in the rat distal colon. [Japanese Journal of Physiology, 51, , 2001] Key words: camp-dependent protein kinase, isoproterenol, -adrenergic agonist, amiloride, Na absorption. Na absorption in the colon plays an important role in salt and water homeostasis. Two transport mechanisms involved in Na absorption have been described, one being an electroneutral pathway mediated by the apical Na /H exchanger, which is functionally coupled to the Cl /HCO 3 exchanger, and the other an electrogenic pathway (generating the short-circuit current, I sc ) mediated by the apical amiloride-sensitive epithelial-type Na channel (ENaC) [1]. These two Na transport pathways are under the control of extracellular neurotransmitters, hormones, and paracrine substances. Cyclic AMP is a ubiquitous second messenger produced in cells in response to extracellular stimulants. camp production in the colonic mucosa decreases electroneutral Na absorption mediated by the Na /H exchanger [1 4]. However, previous studies concerning the effect of camp on electrogenic Na absorption in the colon have provided conflicting results. In the intestinal cell line, Caco-2, an I sc component inhibitable by an amiloride analogue, is stimulated by secretin, which acts through the camp-signaling pathway [5]. In contrast, camp has hardly any effect on 22 Na absorption in the rabbit colon [6]. In the rat colon, forskolin, an adenylate cyclase activator, has no effect on the amiloride-sensitive I sc under normal conditions, but it can enhance the amiloride-sensitive I sc after the tissue is exposed to sonicated suspensions of nucleated cells [7]. Finally, camp and isoproterenol cause, at least initially, a decrease in the amiloride-sensitive I sc in the guinea pig and human colon [8, 9]. The purpose of this study was therefore to determine the role of camp in the regulation of electrogenic Na absorption in the rat distal colon. To this end we have examined the effect of forskolin and the -adrenergic agonist, isoproterenol, on the amiloridesensitive short-circuit current (I sc ) and unidirectional 22 Na fluxes in the chamber-mounted rat distal colon from animals pretreated with a low Na diet to enhance electrogenic Na absorption. MATERIALS AND METHODS Animals. Male Sprague-Dawley rats were housed in a temperature-controlled room on a 12:12-h Received on December 29, 2000; accepted on April 16, 2001 Correspondence should be addressed to: Yuichi Suzuki, Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka, Japan. Tel: , Fax: , yuichi@smails.u-shizuoka-ken.ac.jp Japanese Journal of Physiology Vol. 51, No. 4,

2 Y. TSUCHIYA and Y. SUZUKI light-dark cycle. To induce electrogenic Na absorption, the animals were maintained for about a week on a sodium-poor diet (Na 0%, K 0.75%). They were treated in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan. Short-circuit current and 22 Na flux measurements. The short-circuit current (I sc ) was measured in vitro in Ussing chambers. The animals were killed by a blow to the head followed by exsanguination, then a segment of distal colon was isolated and opened longitudinally into a flat sheet, and the serosa, submucosa, and muscular layer were stripped away with fine forceps to obtain a mucosa preparation. The tissue was mounted vertically between acrylic resin chambers with an internal surface area of 0.5 cm 2. The bathing solution in each chamber (10 ml) was maintained at 37 C in a water-jacketed reservoir and consisted of 119 mm NaCl, 21 mm NaHCO 3, 2.4 mm K 2 HPO 4, 0.6 mm KH 2 PO 4, 1.2 mm MgCl 2, 1.2 mm CaCl 2, and 10 mm glucose, gassed with 95% O 2 and 5% CO 2 (ph 7.4). The tissues were continuously short-circuited, with compensation for fluid resistance between the two potential-sensing bridges, by use of a voltage-clamping amplifier (CEZ9100, Nihon Kohden, Tokyo). The transepithelial potential was measured through 1 M KCl agar bridges connected to a pair of calomel halfcells, the transepithelial current being applied across the tissue through a pair of Ag AgCl electrodes kept in contact with the mucosal and serosal bathing solutions by use of a pair of 1 M NaCl agar bridges. The value of the I sc was expressed as positive when the current flowed from the mucosa to the serosa. The transmural tissue resistance (G t ) was calculated from the current change in response to voltage pulses according to Ohm s law. Unidirectional transmural 22 Na fluxes were measured in Ussing chambers under short-circuit conditions. The mucosal-to-serosal (J ms ) and serosal-to-mucosal (J sm ) fluxes were measured in adjacent tissues that had G t values differing by 30%. After labeling the bathing solution on either the serosal or mucosal side with 111 kbq of 22 Na, an interval of 30 min was allowed to achieve an isotopic steady state. Nine samples (0.5 ml each) were taken from the unlabeled side at 10 min intervals, the volume being made up with an equal volume of unlabeled solution. 22 Na in the samples was counted in a gamma-well spectrometer. Chemicals. Amiloride, 8-bromoadenosine 3 :5 cyclic monophosphate (8Br-cAMP), bumetanide, forskolin, indomethacin, tetrodotoxin, propranolol, and isoproterenol were purchased from Sigma (St. Louis, MO, USA). H89 and H85 were purchased from Seikagaku Cogyo (Tokyo). Amiloride, indomethacin, and isoproterenol were prepared as a concentrated stock solution dissolved in water, and 8Br-cAMP, bumetanide, forskolin, H89, and H85 were dissolved in dimethyl sulfoxide (DMSO), the final concentration of DMSO in the experimental solution being 0.1% (v/v). 22 Na was purchased from Du Pont NEN (Boston, MA, USA). Statistics. Values are presented as the mean SE. The number of animals is represented by n. Statistical comparisons between two means were made by using the paired or unpaired Student s t-test, a p value of 0.05 being taken as statistically significant. RESULTS I sc and G t responses to forskolin In the present study, the rats were placed on a low Na diet before the experiments to enhance electrogenic Na absorption. Furthermore, measurements were performed in the presence of indomethacin (10 M) added to the solutions bathing both the mucosal and serosal surfaces to suppress prostaglandin production. Under these conditions, the distal colon exhibited a basal I sc of A cm 2 and a G t of ms cm 2 (n 24). We first examined the effect of camp on the amiloride-sensitive component of the I sc. The addition of forskolin (10 M), an adenylate cyclase activator, to both the mucosal and serosal sides resulted in an increase in the I sc, which reached a maximum within 20 min and remained elevated thereafter (Fig. 1A, upper trace). The increase in the I sc was accompanied by an increase in the G t (Fig. 1A, lower trace). When the mucosal side of the tissue was pretreated with amiloride (100 M), the forskolin-induced I sc increase was markedly suppressed (Fig. 1B, upper trace), but the forskolin-induced G t increase was not markedly affected (Fig. 1B, lower trace). Table 1 summarizes the results for four experiments, showing that the forskolin-induced I sc increase was significantly greater in the absence of amiloride. These results suggest that forskolin can stimulate amiloride-sensitive, electrogenic Na absorption. 22 Na flux measurements To confirm that forskolin stimulates amiloride-sensitive, electrogenic Na absorption, we determined the effects of forskolin and 100 M amiloride on unidirectional 22 Na fluxes from the mucosa to the serosa (J ms ) and from the serosa to the mucosa (J sm ) (Table 436 Japanese Journal of Physiology Vol. 51, No. 4, 2001

3 Regulation of Na Absorption in the Colon 2). Luminal amiloride at a concentration of 100 M almost completely suppressed the amiloride-sensitive I sc (data not shown), and it has previously been reported to have little effect on Na absorption mediated by Na /H exchanger [10]. When 1,9-deoxyforskolin was used as an inactive forskolin analogue [11], it produced a slight but significant decrease in the J ms, possibly a result of a time-dependent decrease in Na absorption. In contrast, the addition of forskolin resulted in a slight increase in the J ms and net 22 Na absorption (J net ), as well as producing the I sc and G t increases described above. The changes in the Fig. 1. Effect of forskolin on the short-circuit current (I sc ) and transmucosal conductance (G t ) in the rat distal colon mounted in Ussing chambers. In this and the following experiments, indomethacin (10 M) was added to the mucosal and serosal sides at the start of incubation. (A) After the basal I sc and G t had stabilized, forskolin (10 M) was added to the mucosal and serosal sides (first arrow); then amiloride (100 M) was added to the mucosal side (second arrow). (B) Amiloride was added before forskolin. Representative results from two tissues with similar basal I sc s. J ms and J net induced by forskolin were significantly greater than those induced by 1,9-deoxy-forskolin ( 1,9-Deoxy-forskolin and Forskolin in Table 2). More important, the J net decrease induced by 100 M amiloride, mainly because of the decrease in the J ms, was significantly greater in the presence of forskolin than in the presence of 1,9-deoxy-forskolin. Moreover, the magnitude of the J net decrease induced by amiloride was similar to that of the I sc decrease induced by amiloride for 1,9-deoxy-forskolin or forskolin treatment ( Amiloride in Table 2). Thus amiloride-sensitive electrogenic Na absorption is stimulated by forskolin. Effect of isoproterenol on the amiloride-sensitive I sc We next tested the effect of a -adrenergic agonist, isoproterenol, which probably causes an increase in intracellular camp (Fig. 2 and Table 3). The addition of isoproterenol (1 M) to the serosal side caused a biphasic I sc change, consisting of an initial transient decrease, followed by an increase (Fig. 2A, upper trace), and this was accompanied by a monophasic G t increase (Fig. 2A, lower trace). When the mucosal side of the tissue was pretreated with amiloride (100 M), the addition of isoproterenol resulted only in the initial I sc decrease (Fig. 2B, upper trace), but the isoproterenol-induced G t increase was not markedly affected (Fig. 2B, lower trace). These results suggest that isoproterenol activates amiloride-sensitive, electrogenic Na absorption. The isoproterenol-induced I sc decrease and G t increase that were not inhibited by amiloride probably result from the activation of electrogenic K secretion [12]. Table 1. Effects of forskolin on the I sc and G t in the rat distal colon. I sc ( A/cm 2 ) G t (ms/cm 2 ) I sc ( A/cm 2 ) G t (ms/cm 2 ) A (n 4) Basal Forskolin * Amiloride B (n 4) Basal Amiloride Forskolin Values are the means SE. In this and the following experiments, indomethacin (10 M) was added to the mucosal and serosal sides at the start of incubation. The effects of forskolin and amiloride were determined as described in Fig. 1. (A) After the basal I sc and G t had stabilized, forskolin (10 M, mucosa and serosa) was added ( Forskolin); 60 min later amiloride (100 M, mucosa) was added ( Amiloride). (B) Amiloride was added before forskolin: After the basal I sc and G t had stabilized, amiloride was added ( Amiloride); 5 min later forskolin was added ( Forskolin). * p 0.05 comparing the changes in the I sc ( I sc ) and G t ( G t ) induced by amiloride or forskolin in groups A and B. The basal I sc and G t did not differ significantly between the two groups. Japanese Journal of Physiology Vol. 51, No. 4,

4 Y. TSUCHIYA and Y. SUZUKI Table 2. Effects of forskolin on unidirectional 22 Na fluxes in the rat distal colon. J ms J sm J net I sc G t ( Eq cm 2 h 1 ) ( Eq cm 2 h 1 ) ( Eq cm 2 h 1 ) ( Eq cm 2 h 1 ) (ms cm 2 ) 1,9-Dideoxy-forskolin Basal Diterpene * * Amiloride * * * * Diterpene Amiloride Forskolin Basal Diterpene * * Amiloride * * * * Diterpene Amiloride Values are the means SE. The mucosa-to-serosa (J ms ) and serosal-to-mucosal (J sm ) 22 Na fluxes were determined by using adjacent tissues and the net flux (J net J ms J sm ) calculated. After basal fluxes were determined for three 10 min periods (Basal), the diterpene, forskolin, or its inactive analogue, 1,9-dideoxy-forskolin, was added (10 M, mucosa and serosa) and the fluxes again determined for three 10 min periods ( Diterpene). Finally, amiloride (100 M, mucosa) was added and the fluxes determined for another three 10 min periods ( Amiloride). * p 0.05 compared with the value before treatment. p 0.05 comparing the magnitude of changes induced by diterpene ( Diterpene) or by amiloride ( Amiloride) in the 1,9-dideoxy-forskolin and forskolin groups. Effects of camp and isoproterenol in the presence of serosal bumetanide Intracellular camp can stimulate electrogenic K and Cl secretion in the colon [1, 3, 4, 12]. Because bumetanide, an Na /K /2Cl contransporter inhibitor, is known to inhibit electrogenic K secretion almost totally and electrogenic Cl secretion at least partially, the following experiments were performed in the presence of serosal bumetanide (0.1 mm) to measure changes in the amiloride-sensitive I sc more precisely by excluding a large portion of the I sc caused by electrogenic K and Cl secretion. Under these conditions, almost all the basal I sc was the amiloride-sensitive I sc, because the addition of mucosal amiloride (100 M) almost completely abolished the I sc (Tables 4 6). A stimulation of the amiloride-sensitive I sc by forskolin was confirmed in the presence of bumetanide (Fig. 3 and Table 4). Furthermore, 8BrcAMP was shown to stimulate the amiloride-sensitive I sc in the presence of bumetadine (Fig. 4 and Table 5). Both agents caused an increase in the I sc, which was markedly reduced, but not abolished, in the presence of amiloride. The amiloride-sensitive component of the I sc was significantly larger in the stimulant-treated tissue than in the control tissue. Forskolin and 8BrcAMP also significantly increased the amiloride-sensitive I sc / G t ratio, which would be interpreted as the increase in the apparent electromotive force for the amiloride-sensitive I sc generating system by camp. In the presence of bumetanide, the serosal addition of isoproterenol (1 M) resulted in an increase in the I sc without a transient decrease and an increase in the G t (Fig. 5, and Table 6). As with camp, the addition of isoproterenol resulted in an increased amiloridesensitive I sc / G t ratio (Table 6). In contrast to forskolin and 8Br-cAMP, isoproterenol had hardly any effect on the I sc and G t in the presence of amiloride, probably because electrogenic Cl secretion is hardly stimulated by isoproterenol, and the stimulation of electrogenic K secretion is almost completely inhibited by bumetanide [1, 4]. Thus under these conditions, the I sc almost essentially represents the amiloride-sensitive I sc not only under basal conditions, but also during isoproterenol stimulation. In the following experiments, we characterized the activation of electrogenic Na absorption by measuring the isoproterenol-induced increase in the I sc in the presence of serosal bumetanide. Characterization of isoproterenol-induced activation of electrogenic Na absorption The stimulation of electrogenic Na absorption by isoproterenol was mediated mainly by -adrenergic receptor activation, as 1 M propranol, an -adrenergic blocker, almost completely abolished the isoproterenol (1 M)-induced electrical responses (data not shown). The concentration response curve for the increase in I sc induced by isoproterenol gave an ED Japanese Journal of Physiology Vol. 51, No. 4, 2001

5 Regulation of Na Absorption in the Colon (concentration giving the half-maximal response) of approximately 300 nm, the maximal stimulation being about 60% (Fig. 6). The I sc increase induced by isoproterenol (1 M) was not significantly affected by treatment of the tissue with the nerve conduction blocker, tetrodotoxin (300 nm, serosa) (tetrodotoxin, 63 9% increase, n 4; control, 61 7% increase, n 20). Furthermore, the percentage increase in the I sc induced by 1 M isoproterenol (i.e., the percentage increase in the amiloride-sensitive I sc ) appeared to be essentially independent of the basal I sc level (Fig. 7). Fig. 2. Effect of isoproterenol on the I sc and G t. (A) Isoproterenol (1 M) was added to the serosal side; amiloride (100 M) was then added to the mucosal side. Addition of isoproterenol resulted in a decrease in I sc (b: the value obtained at the bottom), followed by an increase (t: the value obtained just before the addition of amiloride). (B) Amiloride was added before isoproterenol. Representative results from two tissues with similar basal I sc s. Role of protein-kinase A in the isoproterenolinduced electrical response We examined whether camp-dependent protein kinase (PKA) was involved in the isoproterenol-induced increase in the amiloride-sensitive I sc by using a PKA inhibitor, H89, and an inactive analogue, H85 [13]. As shown in Fig. 8, A and B, the isoproterenol-induced increase in the amiloride-sensitive I sc was blocked by H89. This effect of H89 was seen at a concentration of 30 M or greater, and H89 was significantly more potent than H85 (Fig. 8C). This dose-dependency relationship is essentially similar to that seen for the inhibitory effect of H89 on PKA activity in PC12D cells when applied to the incubation medium [13]. The isoproterenol-induced G t increase was also inhibited by H89, although the effect was apparent only at 50 M (Fig. 8D). Moreover, the electromotive force in the presence of isoproterenol was significantly suppressed by both 30 M and 50 M H89, but not by the same concentrations of H85 (Fig. 8, E and F). Thus the effect of isoproterenol on amiloride-sensitive Na absorption is largely, if not completely, mediated by PKA. Neither H89 nor H85 significantly affected the basal I sc for at least 30 min, the change in basal I sc 30 min after treatment with vehicle (DMSO), H85, or H89 being , 15 4, or 17 3 A cm 2, respectively (n 4), indicating that the activation of electrogenic Na absorption by the PKA-dependent pathway does not occur under basal conditions. DISCUSSION The present study provides evidence that camp can upregulate electrogenic Na absorption in the rat dis- Table 3. Effects of isoproterenol on the I sc and G t. I sc ( A/cm 2 ) G t (ms/cm 2 ) I sc ( A/cm 2 ) G t (ms/cm 2 ) A (n 6) Basal Isoproterenol b Isoproterenol t Amiloride B (n 7) Basal Amiloride Isoproterenol Values are the means SE. The effects of isoproterenol and amiloride were determined as described in Fig. 2. (A) After the basal I sc and G t had stabilized, isoproterenol (1 M, serosa) was added; then 20 min later, amiloride (100 M, mucosa) was added ( Amiloride). As shown in Fig. 2, the addition of isoproterenol resulted in a decrease in the I sc (isoproterenol b ), followed by an increase (isoproterenol t ). The I sc value for isoproterenol t was significantly higher than for isoproterenol b. (B) Amiloride was added before isoproterenol: After the basal I sc and G t had stabilized, amiloride was added ( Amiloride); 5 min later isoproterenol was added ( Isoproterenol). The secondary I sc increase in the presence of isoproterenol was abolished. The basal I sc and G t did not differ significantly between the two groups. Japanese Journal of Physiology Vol. 51, No. 4,

6 Y. TSUCHIYA and Y. SUZUKI Fig. 3. Effect of forskolin on the I sc and G t in the presence of bumetanide. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment. (A) Forskolin (10 M) was added to the mucosa and serosal sides; then amiloride (100 M) was added to the mucosal side. (B) Amiloride was added before forskolin. Representative results from two tissues with similar basal I sc s. tal colon. This evidence is based on the following findings: (1) forskolin stimulated the amiloride-sensitive I sc and 22 Na absorption; (2) 8Br-cAMP stimulated the amiloride-sensitive I sc ; (3) isoproterenol, a adrenergic agonist, which induces camp production, stimulated the amiloride-sensitive I sc. These effects of camp may be mainly mediated by PKA activation, since the PKA inhibitor, H89, strongly suppressed the isoproterenol-induced increase in the amiloride-sensitive I sc. In the rat colon mounted in Ussing chambers, forskolin is reported to have no effect on the amiloride-sensitive I sc and 22 Na absorption under normal conditions, but to enhance the amiloride-sensitive I sc after the tissue was exposed to sonicated suspensions of nucleated cells [7]. The reason for the discrepancy between these results and our own is not clear. It is possible, however, that in the previous experiments the time-dependent decrease in the amiloride-sensitive I sc obscured the forskolin-induced increase (see table 1 of Cuthbert and Spayne [7]). The absence of a change in 22 Na absorption in the presence of forskolin or epinephrine, reported in previous studies [7, 14], was also seen in the present study (Table 2) and can be explained by the camp-induced increase in 22 Na absorption being largely canceled out by the simultaneous decrease in amiloride-insensitive 22 Na absorption (see Table 2). A previous study using the rabbit colon showed that camp hardly affects 22 Na absorption [6] whereas in the guinea pig and human colon, camp causes, at least in its initial phase, a decrease in the amiloride-sensitive I sc [8, 9] and 22 Na absorption [9]. Thus camp regulation of electrogenic Na absorption in the colon appears to differ between mammalian species. The stimulation of amiloride-sensitive electrogenic Na absorption mediated by the camp signaling pathway has been described in several other epithelial cells, such as the intestinal cell line Caco-2 [5] and the distal nephron epithelium [15]. The membrane transport machinery responsible for electrogenic Na absorption involves the amiloridesensitive, epithelial-type Na channel (ENaC) in the apical membrane and the Na,K -ATPase and the K channel in the basolateral membrane [16]. Thus changes in the activity of any one of these three pathways could contribute to the camp-induced regulation of electrogenic Na absorption. In the present study, we showed that at least the isoproterenol-in- Table 4. Effects of forskolin on the I sc and G t in the presence of bumetanide. I sc G t I sc G t I sc / G t ( Amil) ( A/cm 2 ) (ms/cm 2 ) ( A/cm 2 ) (ms/cm 2 ) (mv) A (n 4) Basal Forskolin * Amiloride * * B (n 4) Basal Amiloride Forskolin Values are the means SE. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment, and the effects of forskolin and amiloride determined as described in Fig. 3. (A) After the basal I sc and G t had stabilized, forskolin (10 M, mucosa and serosa) was added ( Forskolin); 60 min later amiloride (100 M, mucosa) was added ( Amiloride). (B) Amiloride was added before forskolin: After the basal I sc and G t had stabilized, amiloride was added ( Amiloride); 5 min later forskolin was added ( Forskolin). * p 0.05 comparing the change in the I sc ( I sc ) and G t ( G t ) induced by amiloride or forskolin and the amiloride-sensitive I sc /G t ratio ( I sc / G t [ Amil]) in group A with those in group B. The basal I sc and G t did not differ significantly between the two groups. 440 Japanese Journal of Physiology Vol. 51, No. 4, 2001

7 Regulation of Na Absorption in the Colon duced increase in the amiloride-sensitive I sc was accompanied by an increase in the amiloride-sensitive G t (Table 6), suggesting the increase in apical Na channel activity, since Na entry through the amiloride-sensitive Na channel is generally believed to be the rate-limiting step for electrogenic Na absorption in the colon [17]. The rat colon ENaC expressed in Xenopus oocytes does not respond to camp [18], and it is therefore possible that PKA-mediated phosphorylation of protein(s) besides ENaC is required for ENaC activation. In another report, how- Fig. 4. Effect of 8-bromoadenosine 3 :5 -cyclic monophosphate (8Br-cAMP) on the I sc and G t in the presence of bumetanide. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment. (A) 8Br-cAMP (1 mm) was added to the serosal side; then amiloride (100 M) was added to the mucosal side. (B) Amiloride was added before 8Br-cAMP. Representative results from two tissues with similar basal I sc s. ever, the amiloride-sensitive Na conductance of rat colonic crypt cells was suppressed by forskolin [19], the result conflicting with ours. One possible explanation for the discrepancy between this result and our own is that amiloride-sensitive Na conductances in the surface epithelial cells and crypt cells respond differently to camp, the former being activated and the latter inhibited. Since most epithelial Na channels in the colon are found in the surface epithelial cells [20, 21], the amiloride-sensitive I sc determined in the chamber-mounted colon may mainly reflect the properties of Na channels in surface epithelial cells. The present results also showed that the stimulation of electrogenic Na absorption by camp is accompanied by an increase in the amiloride-sensitive I sc / G t ratio, the electromotive force for the amiloride-sensitive I sc pathway (Tables 4 6). Moreover, the increase in the amiloride-sensitive I sc / G t ratio in the presence of isoproterenol was significantly suppressed by a PKA inhibitor. A simple explanation for the increase in apparent electromotive force is activation of the basolateral Na,K -ATPase, the reasons being that the driving force for electrogenic Na absorption is believed to depend primarily on ATP hydrolysis by Na,K -ATPase and that activation of ion channels alone may be insufficient to cause an increase in the apparent electromotive force [16]. Activation and inhibition of Na,K -ATPase by PKA have both been demonstrated in many cell types [22]. In the rat distal colon, however, forskolin is reported to have no effect on the ouabain-sensitive Na pump-current in nystatin-treated tissue [23]. The present findings suggest that camp and isopro- Table 5. Effects of 8-bromoadenosine 3 :5 -cyclic monophosphate (8Br-cAMP) on the I sc and G t in the presence of bumetanide. I sc G t I sc G t I sc / G t ( Amil) ( A/cm 2 ) (ms/cm 2 ) ( A/cm 2 ) (ms/cm 2 ) (mv) A (n 4) Basal Br-cAMP * Amiloride * * B (n 4) Basal Amiloride Br-cAMP Values are the means SE. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment, and the effects of 8Br-cAMP and amiloride were determined as described in Fig. 4. (A) After the basal I sc and G t had stabilized, 8Br-cAMP (1 mm) was added to the serosal side ( 8Br-cAMP); 20 min later amiloride (100 M) was added to the mucosal side ( Amiloride). (B) Amiloride was added before 8Br-cAMP: After the basal I sc and G t had stabilized, amiloride was added ( Amiloride); 5 min later 8Br-cAMP was added ( 8Br-cAMP). * p 0.05 comparing the changes in the I sc ( I sc ) and G t ( G t ) induced by amiloride or 8Br-cAMP and the amiloride-sensitive I sc /G t ratio ( I sc / G t [ Amil]) in group A with those in group B. The basal I sc and G t did not differ significantly between the two groups. Japanese Journal of Physiology Vol. 51, No. 4,

8 Y. TSUCHIYA and Y. SUZUKI Table 6. Effects of isoproterenol on the I sc and G t in the presence of bumetanide. I sc G t I sc G t I sc / G t ( Amil) ( A/cm 2 ) (ms/cm 2 ) ( A/cm 2 ) (ms/cm 2 ) (mv) A (n 7) Basal Isoproterenol * * Amiloride * * 107 7* B (n 5) Basal Amiloride Isoproterenol Values are the means SE. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment, and the effects of isoproterenol and amiloride were determined as described in Fig. 5. (A) After the basal I sc and G t had stabilized, isoproterenol (1 M) was added to the serosal side ( Isoproterenol); 20 min later amiloride (100 M) was added to the mucosal side ( Amiloride). (B) Amiloride was added before isoproterenol: After the basal I sc and G t had stabilized, amiloride was added ( Amiloride); 5 min later isoproterenol was added ( Isoproterenol): * p 0.05 comparing the changes in the I sc ( I sc ) and G t ( G t ) induced by amiloride or isoproterenol and the amiloride-sensitive I sc /G t ratio ( I sc / G t [ Amil]) in group A with those in group B. The basal I sc and G t did not differ significantly between the two groups. Fig. 5. Effect of isoproterenol on the I sc and G t in the presence of bumetanide. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment. (A) Isoproterenol (1 M) was added to the serosal side: amiloride (100 M) was then added to the mucosal side. (B) Amiloride was added before isoproterenol. Representative results from two tissues with similar basal I sc s. terenol can stimulate electrogenic Na absorption regardless of whether electrogenic Cl and K secretion is inhibited by bumetanide. Thus an upregulation of amiloride-sensitive electrogenic Na absorption by camp appears to occur largely independently of electrogenic Cl and K secretion. It remains to be determined, however, whether some interaction exists between the electrogenic K and Cl transport pathways and electrogenic Na absorption. Adrenergic regulation plays an important role in electrolyte transport in the colon, but is clearly complicated and requires further study [14]. The present and previous studies [1 4, 14] suggest that the activation of -adrenergic receptors in the rat distal colon mucosa stimulates electrogenic Na absorption and Fig. 6. Dose-dependency of the isoproterenol effect on the I sc in the presence of bumetanide. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment. Isoproterenol was added cumulatively to the serosal side and the percentage increase in the I sc was determined. In the presence of bumetanide, both the basal I sc and the isoproterenol-induced I sc are almost totally the amiloride-sensitive I sc. n 3. Fig. 7. Relationship between the basal I sc and the percentage increase in the I sc induced by isoproterenol. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment. The slope of linear regression ( ) was not significantly different from zero (p 0.12). 442 Japanese Journal of Physiology Vol. 51, No. 4, 2001

9 Regulation of Na Absorption in the Colon Fig. 8. Effects of camp-dependent protein kinase (PKA) inhibitors on the isoproterenol-induced increase in the I sc and G t. Bumetanide (0.1 mm) was added to the serosal side about 30 min before the test drug and throughout the experiment. The PKA inhibitor H89, the less active analogue H85 (both 30 M), or the vehicle (DMSO) was added to the mucosal and serosal sides 30 min before the addition of isoproterenol (1 M, serosa). A and B: Representative results from two adjacent tissues treated with DMSO (A) or H89 (B). C and D: Concentration-dependency of the K secretion and possibly the suppression of nonelectrogenic Na absorption. However, the role of adrenergic receptors in the regulation of other transport processes in the colon, such as Cl absorption, mediated by the Cl /HCO 3 exchanger [1], and K absorption, mediated by the H,K -ATPase [24], is inhibitory effects of H85 (hatched bar) and H89 (filled bar) on the isoproterenol-induced increase in the I sc (C) and G t (D). The increase in the I sc and G t induced by isoproterenol (1 M) in the presence of inhibitors is expressed as a percentage of the increase under control conditions. n 3 for 5 M, n 5 for 30 M, and n 5 for 50 M inhibitors. * p 0.05 compared with control. E and F: Effects of H89 and H85 on the amiloride-sensitive I sc / G t ratio at 30 M (E) or 50 M (F) inhibitors. * p 0.05 compared with control. largely unknown. Furthermore, -adrenergic receptors appear to be present at multiple sites in the colonic mucosa. Functional evidence for the presence of adrenergic receptors on submucosal neurons has been reported [14], but the present isoproterenol-induced activation of electrogenic Na absorption was not Japanese Journal of Physiology Vol. 51, No. 4,

10 Y. TSUCHIYA and Y. SUZUKI suppressed by either tetrodotoxin or indomethacin, suggesting the presence of -adrenergic receptors on epithelial cells. Furthermore, adrenergic stimulation may well be mediated not only by -adrenergic receptors, but also by -adrenergic receptors, the role of which remains largely unknown in the colon. Further studies are therefore required to elucidate the physiological role of colonic mucosal adrenergic receptors in the regulation of colonic electrolyte transport. Finally, camp is known to be produced by a variety of neurohumoral agents besides adrenergic agents, and the physiological significance of the possible stimulation of electrogenic Na absorption in the colon by these agents remains to be elucidated. We thank Dr. Thomas Barkas for helping us to edit the English text. REFERENCES 1. Binder HJ and Sandle GI: Electrolyte transport in the mammalian colon. In: Physiology of the Gastrointestinal Tract, 3rd ed, pp , ed. Johnson LR, Raven Press, New York, Foster ES, Zimmerman TW, Hayslett JP, and Binder HJ: Corticosteroid alteration of active electrolyte transport in rat distal colon. Am J Physiol 245: G668 G675, Bridges RJ, Rummel W, and Simon B: Forskolin induced chloride secretion across the isolated mucosa of rat colon descendens. Naunyn Schmiedebergs Arch Pharmacol 323: , Diener MH, Strabel F, and Scharrer DE: Cyclic AMP dependent regulation of K transport in the rat distal colon. Br J Pharmacol 118: , Fukuda M, Ohara A, Bamba T, and Saeki Y: Activation of transepithelial ion transport by secretin in human intestinal Caco-2 cells. Jpn J Physiol 50: , Frizzell RA, Koch MJ, and Schultz SG: Ion transport by rabbit colon. I. Active and passive components. J Membr Biol 27: , Cuthbert AW and Spayne JA: Conversion of sodium channels to a form sensitive to cyclic AMP by component(s) from red cells. Br J Pharmacol 79: , Mall M, Bleich M, Kuehr J, Brandis M, Greger R, and Kunzelmann K: CFTR-mediated inhibition of epithelial Na conductance in human colon is defective in cystic fibrosis. Am J Physiol 277: G709 G716, Yamamoto T, Ikuma M, and Suzuki Y: Regulation of electrogenic Na absorption by camp in guinea pig distal colon. Jpn J Physiol 46 (Suppl): S31, Binder HJ, Foster ES, Budinger ME, and Hayslett JP: Mechanism of electroneutral sodium chloride absorption in distal colon of the rat. Gastroenterology 93: , Seamon KB, Daly JW, Metzger H, de Souza NJ, and Reden J: Structure activity relationships for activation of adenylate cyclase by the diterpene forskolin and its derivatives. J Med Chem 26: , Grotjohann I, Gitter AH, Kockerling A, Bertog M, Schulzke JD, and Fromm M: Localization of camp- and aldosterone-induced K secretion in rat distal colon by conductance scanning. J Physiol (Lond) 507: , Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, and Hidaka H: Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H- 89), of PC12D pheochromocytoma cells. J Biol Chem, 265: , Horger S, Schultheiss G, and Diener M: Segment-specific effects of epinephrine on ion transport in the colon of the rat. Am J Physiol 275: G1367 G1376, Marunaka Y: Hormonal and osmotic regulation of NaCl transport in renal distal nephron epithelium. Jpn J Physiol 47: , Shultz SG and Hudson RL: Biology of sodium-absorbing epithelial cells: dawning of a new era. In: Handbook of Physiology, Sec 6, The Gastrointestinal System, Vol 4, Intestinal Absorption and Secretion, ed. Shultz SG, American Physiological Society, Bethesda, MD, pp 45 81, Thompson SM, Suzuki Y, and Schultz SG: The electrophysiology of rabbit descending colon. I. Instantaneous transepithelial current voltage relations and the current voltage relations of the Na-entry mechanism. J Membr Biol 66: 41 54, Awayda MS, Ismailov II, Berdiev BK, Fuller CM, and Benos DJ: Protein kinase regulation of a cloned epithelial Na channel. J Gen Physiol 108: 49 65, Ecke D, Bleich M, and Greger R: The amiloride inhibitable Na conductance of rat colonic crypt cells is suppressed by forskolin. Pflügers Arch 431: , Kockerling A, Sorgenfrei D, and Fromm M: Electrogenic Na absorption of rat distal colon is confined to surface epithelium: a voltage-scanning study. Am J Physiol 264: C1285 C1293, Duc C, Farman N, Canessa CM, Bonvalet JP, and Rossier BC: Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol: , Therien AG and Blostein R: Mechanisms of sodium pump regulation. Am J Physiol Cell 279: C541 C566, Schultheiss G and Diener M: Regulation of apical and basolateral K conductances in rat colon. Br J Pharmacol 122: 87 94, Jaisser F and Beggah AT: The nongastric H -K -AT- Pases: molecular and functional properties. Am J Physiol 276: F812 F824, Japanese Journal of Physiology Vol. 51, No. 4, 2001