beta -Adrenergic agonists stimulate Na+-K+-Clminus cotransport by inducing intracellular Ca2+ liberation in crypt cells

Jesús R. del Castillo, Julio C. Arévalo, Luis Burguillos, and María C. Súlbaran-Carrasco

Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas 1020-A, Venezuela


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epinephrine and beta -adrenergic agonists (beta 1 and beta 2 for isoproterenol, beta 1 for dobutamine, beta 2 for salbutamol) stimulated K+ (or 86Rb) influx mediated by the Na+-K+-2Cl- cotransporter and the Na+-K+ pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na+-K+ pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 µM epinephrine with an EC50 of 91.6 ± 9.98 nM. Epinephrine-induced K+ transport was blocked by propranolol with an IC50 of 134 ± 28.2 nM. alpha -Adrenergic drugs did not modify K+ transport mechanisms. Neither Ba2+ nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition, epinephrine did not affect K+ efflux. Dibutyryl cAMP did not alter K+ transport. Reduction of extracellular Ca2+ to 30 nM did not influence the response to epinephrine. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished epinephrine-induced K+ transport. Ionomycin increased Na+-K+-2Cl- cotransport activity. Moreover, epinephrine increased intracellular Ca2+ concentration in a process inhibited by propranolol. In conclusion, epinephrine stimulated the Na+-K+-2Cl- cotransporter in a process mediated by beta 1- and beta 2-receptors and modulated by intracellular Ca2+ liberation.

potassium transport; distal colon; guinea pig


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN THE MAMMALIAN COLON, the K+ secretory process implicates several transport mechanisms at the luminal and basolateral plasma membrane of the epithelial cell (1, 11). The Na+-K+ pump and the Na+-K+-2Cl- cotransporter actively transport K+ at the basolateral membrane. This active process permits K+ to accumulate into the colonocyte above its electrochemical equilibrium. K+ exits the cell across the apical membrane, following its electrochemical gradient, through Ba2+-sensitive and tetraethylammonium (TEA)-sensitive K+ channels. Epinephrine and the beta -adrenergic agonist isoproterenol stimulate K+ secretion (13, 19). The enhancement of K+ secretion by epinephrine could be due to an increase in K+ permeability of the conductive pathway at the apical membrane or to the stimulation of K+ uptake across the basolateral membrane or both.

Recently, we demonstrated that isolated colonocytes actively transport K+ through at least three separate mechanisms: a Na+-dependent, ouabain-sensitive mechanism compatible with the Na+-K+ pump; a Na+-dependent, bumetanide-sensitive mechanism consistent with the Na+-K+-2Cl- cotransporter; and a Na+-independent, ouabain-sensitive mechanism identified as the colonic K+-H+ pump, in addition to passive flux (4).

The aim of the present study was to evaluate the effects of several adrenergic agonists on K+ transport mechanisms present in isolated guinea pig colonic cells, using 86Rb as a tracer, and to identify the second messengers involved in the response to the adrenergic drugs.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. 86Rb and [3H]polyethylene glycol 4000 were purchased from Amersham (Little Chalfont, UK). Epinephrine, dobutamide, clonidine, salbutamol, isoproterenol, phenylephrine, propranolol, and benextramine were obtained from Research Biochemicals International (Natick, MA). Fura 2-AM was purchased from Calbiochem (La Jolla, CA). All other chemicals of analytical grade were obtained from Sigma Chemical (St. Louis, MO) or from Merck (Rahway, NJ).

Animals. Male guinea pigs (weight range of 300-350 g) were used. Animals were maintained on regular laboratory diet, were allowed free access to water, and were starved for 24 h before being killed.

Cell isolation methods. Colonocytes were isolated by the procedure described by del Castillo and Sepúlveda (5). Briefly, the colon was excised from the colonic flexure to 3 cm above the anal orifice and then rinsed, filled, and incubated for 10 min at 37°C with solution 1 (in mM: 7 K2SO4, 44 K2HPO4, 9 NaHCO3, 10 HEPES-Tris, and 180 glucose, pH 7.4 and 340 mosmol/l). The luminal content was discarded, and the intestinal segment was refilled and incubated for 3 min at 37°C with solution 2, which contained 0.5 mM dithiothreitol and 0.25 mM EDTA in addition to the components of solution 1. The colon was then gently palpated, and the luminal solution, containing isolated cells, was collected in DMEM (100 ml) at 4°C, filtered through a nylon mesh (60 µm pore diameter), and centrifuged at 100 g for 5 min. This step was repeated six times to obtain six different fractions. Cells isolated during the first two palpations (fractions 1-2) were considered surface cells, and those isolated from the fifth and sixth palpation (fractions 5-6) were considered crypt cells. This was confirmed in experiments that assessed the incorporation of intraperitoneally injected [3H]thymidine and bromodeoxyuridine, which give both spatial and quantitative information about cell proliferation (2, 5, 17). The isolated cells were resuspended in DMEM and stored at 4°C in plastic tubes without agitation. DMEM was modified to contain (in mM) 116 NaCl, 5 KCl, 1 MgSO4, 1 NaH2PO4, 7.5 K2SO4, and 10 HEPES-Tris, pH 7.4 and 320 mosmol/l. All solutions were oxygenated with 100% O2 for 15 min before use. After isolation, cells were washed three times by centrifugation with the respective influx or efflux medium and resuspended in the same solution. For analysis, cells were separated from the incubation medium by centrifugation through an oil layer as previously described (3, 4).

The cell viability was evaluated by trypan blue exclusion, oxygen consumption, and intracellular ion concentration as previously described (3). Cells were used only when viability was >95%.

Transport experiments. For influx experiments, cells were preincubated in DMEM for 30 min at 25°C. Uptake was initiated by adding 300 µl of preincubated cells to 30 µl of incubation medium containing the isotopic tracer. One 250-µl sample was obtained at a time, diluted in 800 µl of incubation medium at 4°C, and immediately centrifuged at 13,000 g for 20 s. During time course experiments, cell and incubation volumes were adjusted to obtain the desired sample numbers, maintaining the relationship indicated above. The intracellular radioactivity was determined after correction for the radioactivity present in the trapped volume, measured using [3H]polyethylene glycol as an extracellular space marker. Control experiments showed that fluxes measured with 86Rb, as a tracer, were equivalents to those determined with 42K. 86Rb influx was linear for at least 2 min at 25°C under all evaluated conditions. Influx was measured after a 1-min incubation. As shown before (4), isolated colonocytes transport K+ by at least three different mechanisms: a Na+-dependent, ouabain-sensitive mechanism compatible with the Na+-K+ pump; a Na+-dependent, bumetanide-sensitive system consonant with the Na+-K+-2Cl- cotransporter; and a Na+-independent, ouabain-sensitive mechanism consistent with K+-H+ pump, in addition to a passive residual flux. To identify K+ transport mechanisms present in isolated colonic surface and crypt cells separately, 86Rb was measured in Na+-containing and Na+-free media (where Na+ was substituted by N-methyl-D-glucamine), and the effects of 50 µM bumetanide and 1 mM ouabain were evaluated.

For efflux experiments, isolated cells were preincubated in DMEM containing 0.5 µCi/ml of 86Rb for 60 min at 25°C. Then, cells were centrifuged at 100 g for 5 min, the supernatant was removed, and the cellular pellet was resuspended in a modified DMEM without 86Rb (and without K+), containing 1 mM ouabain and 50 µM bumetanide. The intracellular radioactivity was determined at different times, and the results were expressed as a percentage of the intracellular content at the initial time.

Intracellular Ca2+ measuring. Isolated crypt cells were loaded with 2 µM fura 2-AM during 30 min at room temperature. Loaded colonocytes were stuck over a glass coverslip with 0.1% polylysine mounted on a perfusion chamber and observed under a microscope at ×1,000. Intracellular Ca2+ concentrations were obtained with an SLS-100 IonOptix StepperSwitch dual-excitation light for fura 2, using an ICD-1000 intensified, charge-coupled device camera coupled to an IonOptix fluorescence image acquisition and analysis software. Images were acquired at 0.5 s at 340 and 380 nm. Intracellular Ca2+ was calculated by using a fluorescence ratio at 340:380 nm and an affinity constant (Kd) for the fura 2-to-Ca2+ ratio of 225 nM.

Protein determination. Cellular proteins were determined by a modified Coomassie blue method (10).

Statistics. Results are expressed as means ± SE. Differences between means were evaluated by ANOVA and considered significant at P < 0.05. Adjustment of experimental values to functions was made by nonlinear regression (Marquandt-Levenberg algorithm) using a commercial program (Origen 5.0, Microcal Software, Northampton, MA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

K+ transport mechanisms in colonic surface and crypt cells. We have shown that isolated colonocytes transport K+ by at least three different mechanisms: a Na+-dependent, ouabain-sensitive mechanism consistent with the Na+-K+ pump; a Na+-dependent, bumetanide-sensitive mechanism compatible with the Na+-K+-2Cl- cotransporter; and a Na+-independent, ouabain-sensitive mechanism identified as the K+-H+ pump, in addition to a passive residual flux (4). The cell preparation used in those previous studies included surface and crypt cells together. In the present report, we separated surface and crypt cells, following the procedure described by del Castillo and Sepúlveda (5), and examined the K+ transport mechanisms present in each cell type. As shown in Fig. 1, surface cells transport K+ by two different mechanisms: a Na+-dependent, ouabain-sensitive mechanism consistent with the Na+-K+ pump and a Na+-independent, ouabain-sensitive mechanism compatible with the K+-H+ pump, in addition to passive influx. In contrast, crypt cells transport K+ by the Na+-K+ pump and a Na+-dependent, bumetanide-sensitive mechanism identified as the Na+-K+-2Cl- cotransporter, in addition to passive influx. Thus, as reported before (5), we were unable to demonstrate K+ transport mediated by the Na+-K+-2Cl- cotransporter in surface cells. On the other hand, we could not identify K+-H+ pump activity in crypt cells.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   86Rb (K+) influx in isolated surface (A) and crypt (B) cells from guinea pig distal colon. Surface and crypt cells were isolated as indicated in MATERIALS AND METHODS. Insets: K+ transport mechanisms identified in isolated cells: Na+-dependent, ouabain-sensitive influx was assumed to be mediated by the Na+-K+ pump; Na+-independent, ouabain-sensitive K+ influx was considered to be mediated by the K+-H+ pump; Na+-dependent, bumetanide-sensitive K+ influx was presumed to be mediated by the Na+-K+-2Cl- cotransporter; and the ouabain-insensitive, bumetanide-insensitive uptake was defined as residual influx. Values are means ± SE of 3 different experiments. ** P < 0.01; *** P < 0.001; ns, not significant, compared with controls.

Effects of epinephrine on K+ transport mechanisms. In the colon, K+ secretion is supported by K+ uptake at the basolateral plasma membrane of the colonic epithelial cell mediated by the Na+-K+-2Cl- cotransporter and the Na+-K+ pump and K+ exit across the luminal plasma membrane through TEA-sensitive and Ba2+-sensitive K+ channels (1, 11). In guinea pig distal colon, serosal bumetanide or ouabain (13, 20) inhibits epinephrine-stimulated K+ secretion. In the present study, we attempted to identify the K+ transport mechanisms involved in the stimulatory response to epinephrine.

Figure 2 shows the effect of epinephrine (1 µM) on K+ transport mechanism identified in isolated crypt cells from the guinea pig distal colon. K+ influx was measured using 86Rb as a tracer. Epinephrine stimulated the Na+-dependent K+ transport mechanisms [the Na+-K+ pump (1.5-fold) and the Na+-K+-2Cl- cotransporter (3-fold)] and did not modify the residual influx. Epinephrine did not alter K+ transport mechanisms identified in surface epithelial cells even under adrenergic stimulation (data not shown).


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of epinephrine on K+ transport mechanisms in isolated crypt cells from guinea pig distal colon. Cells were preincubated 15 min at room temperature with 1 µM epinephrine, and then K+ transport mechanisms were determined as indicated in MATERIALS AND METHODS and Fig. 1. Values are means ± SE of 3 different experiments. ** P < 0.01; *** P < 0.001; ns, not significant, compared with controls.

To discriminate if the primary effect of the epinephrine was on the Na+-K+ pump or the Na+-K+-2Cl- cotransporter, we preincubated isolated crypt cells with epinephrine and bumetanide for 15 min and then the K+ influx was determined. Results are presented in Fig. 3. Preincubation of the isolated cells with 50 µM bumetanide abolished the stimulatory effect of epinephrine on the activity of the Na+-K+ pump, suggesting that the increase in the activity of the Na+-K+ pump is secondary to the activation of the Na+-K+-2Cl- cotransporter.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of epinephrine on K+ transport mechanisms in isolated crypt cells preincubated with 50 µM bumetanide. Colonocytes were preincubated with 50 µM bumetanide and 1 µM epinephrine for 15 min at room temperature, and then K+ influx was determined as indicated in MATERIALS AND METHODS. Values are means ± SE of 3 different experiments. ns, Not significant, compared with controls.

Figure 4 illustrates the concentration dependence for the epinephrine effect on the bumetanide-sensitive K+ influx in isolated crypt cells from guinea pig distal colon. The stimulatory effect on the Na+-K+-2Cl- cotransporter depended on the epinephrine concentration in the incubation medium, reaching the maximal effect at 1 µM with an EC50 of 91.6 ± 9.98 nM.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Concentration dependence for the epinephrine effect on bumetanide-sensitive K+ influx in isolated colonic crypt cells. Points show experimental data and represent means ± SE of 4 different experiments. Line is the best fit to the following equation representing a sigmoidal curve: f = (a - d)/[1 + (x/c)b + d], where a = asymptotic maximum, b = slope, c = inflexion, and d = asymptotic minimum. EC50 is the value at inflection point and represents epinephrine concentration that produced 50% of the maximal stimulation. chi 2 = 11.62; r2 = 0.9997.

Effect of different adrenergic drugs on the bumetanide-sensitive K+ influx. The effect of different adrenergic agonists and antagonists on the Na+-K+-2Cl- cotransporter was examined at concentrations of 10-6 M or 10-5 M. Figure 5A shows the effect of different adrenergic agonists on the cotransporter. Phenylephrine (alpha 1-agonist) and clonidine (alpha 2-agonist) did not affect the bumetanide-sensitive K+ influx. However, isoproterenol (beta 1- and beta 2-agonist), dobutamine (beta 1-agonist), and salbutamol (beta 2-agonist) stimulated, as epinephrine did, the activity of the Na+-K+-2Cl- cotransporter, suggesting that the effect of the epinephrine on this transport mechanism be mediated by beta 1- and beta 2-receptors. Figure 5B presents the effects of adrenergic antagonists on the bumetanide-sensitive K+ influx in isolated colonic cells. Cells were initially preincubated for 10 min with the antagonist and then another 15 min with epinephrine. K+ influx was evaluated as indicated in MATERIALS AND METHODS. Propranolol (10 µM), a beta -antagonist, inhibited the stimulatory effect of epinephrine on the bumetanide-sensitive K+ influx, whereas benextramine (10 µM), an alpha -antagonist, did not affect the cotransporter.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of different adrenergic drugs on bumetanide-sensitive K+ influx in isolated colonic crypt cells. Colonocytes were preincubated with the adrenergic drugs for 15 min at room temperature, and then K+ influx was determined as indicated in MATERIALS AND METHODS. Antagonists were added to preincubation medium 10 min before epinephrine. Adrenergic agonists (A) were used at 1 µM and adrenergic antagonists (B) at 10 µM final concentration. Values are means ± SE of 3 different experiments. *** P < 0.001; ns, not significant, compared with controls.

Figure 6 shows a dose-response curve to dobutamine and salbutamol on bumetanide-sensitive K+ (86Rb) influx. EC50 for salbutamol was six times lower than that obtained for dobutamine, suggesting that the beta -stimulatory effect is mainly mediated by beta 2-receptors.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of dobutamine and salbutamol on bumetanide-sensitive K+ influx. Crypt cells were preincubated with the agonists for 15 min at 25°C. Data are means ± SE of 3 different experiments. Points represent experimental values and lines the best fit to a sigmoidal curve, as described in Fig. 4.

Concentration dependence for the propranolol effect on epinephrine-stimulated, bumetanide-sensitive K+ influx is shown in Fig. 7A. Maximal effect was obtained at 10 µM propranolol with an IC50 of 134 ± 28.2 nM. In addition, bumetanide-sensitive K+ influx was evaluated at different epinephrine concentrations in the presence of increasing propranolol concentrations (Fig. 7B). Experimental data were fitted to a competitive inhibitory equation: JK = (JK max · S)/[Kd(1 + I/Ki) + S], where JK is K+ influx, JK max is maximal K+ influx, S is epinephrine concentration, Kd is the affinity constant for epinephrine, I is propranolol concentration, and Ki is the inhibitory constant for propranolol. Using this model, we obtained a JK max of 218 ± 6.1 nmol · mg-1 · min-1, a Kd for epinephrine of 91 ± 9 nM, and a Ki for propranolol of 72 ± 10 nM. These results confirm that the effect of the epinephrine on the Na+-K+-2Cl- cotransporter in colonic crypt cells is mediated by beta -receptors.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 7.   A: concentration dependence for the propranolol effect on epinephrine-induced, bumetanide-sensitive K+ influx in isolated colonic crypt cells. Colonocytes were preincubated for 10 min with propranolol and 15 additional min with epinephrine. K+ influx was measured as described in MATERIALS AND METHODS. Points represent experimental data and are means ± SE of 6 different experiments. The line is the best fit to a sigmoidal curve. IC50 is the value at inflection point and represents propranolol concentration that produced 50% of the maximal inhibition. chi 2 = 46.07; r2 = 0.9998. B: effect of propranolol on bumetanide-sensitive K+ influx at different epinephrine concentrations. Experimental data were fitted to the following equation describing a competitive inhibition: JK = (JK max · S)/[Kd(1 + I/Ki) + S], where JK is K+ influx, JK max is maximal K+ influx, S is epinephrine concentration, Kd is the affinity constant for epinephrine, I is propranolol concentration, and Ki is inhibitory constant for propranolol. Results are means ± SE of 3 different experiments. chi 2 = 52.67; r2 = 0.9889.

The stimulatory effect of epinephrine on the Na+-K+-2Cl- cotransporter could be a direct effect on the cotransport mechanism or the result of an increase in K+ or Cl- exit of the cells that indirectly stimulates K+ uptake through the cotransporter. To evaluate this possibility, we examined the effect of 10 mM TEA, 5 mM Ba2+, and 0.1 mM DIDS, inhibitors of K+ channels and Cl- channels, on the bumetanide-sensitive K+ influx in colonic isolated cells. The results are presented in Fig. 8. Neither Ba2+ nor TEA nor DIDS affected the epinephrine-stimulated, bumetanide-sensitive K+ influx, suggesting that the stimulatory effect of epinephrine on the Na+-K+-2Cl- cotransporter is not an indirect effect determined by the activation of K+ or Cl- channels. Channel blockers did not modify the Na+-K+ pump activity and had only a small effect on residual influx (data not shown). In addition, to discard the possibility that epinephrine could be activating K+ channels different from those inhibited by TEA or Ba2+, we examined the effect of epinephrine on K+ efflux, using 86Rb as a tracer. Isolated cells, previously loaded with the isotope and preincubated in the presence of epinephrine, were incubated in the absence of 86Rb (and K+). Epinephrine did not modify K+ efflux in colonic isolated cells (Fig. 9). These results confirm that epinephrine acts directly on the Na+-K+-2Cl- cotransporter.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 8.   Effect of Ba2+ (5 mM), tetraethylammonium (TEA; 10 mM), and DIDS (0.1 mM) on bumetanide-sensitive K+ influx in isolated colonic crypt cells. Colonocytes were preincubated 15 min with Ba2+, TEA, or DIDS and then with or without 1 µM epinephrine. K+ flux mediated by the Na+-K+-2Cl- cotransporter was measured as indicated in MATERIALS AND METHODS and Fig. 1. Values are means ± SE of 3 different experiments. *** P < 0.001 compared with control.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 9.   Effect of epinephrine on 86Rb (or K+) efflux in isolated colonic crypt cells from guinea pig distal colon. Cells were loaded with 86Rb for 60 min. During the last 15 min, epinephrine (1 µM) was added to the preincubation medium. Control cells were preincubated and incubated without epinephrine. Colonocytes were washed by centrifugation and incubated in the absence of 86Rb (or K+) and in the presence or not of 1 µM epinephrine. Intracellular radioactivity was determined at different intervals, and results are expressed as a percentage of the intracellular content at the initial time. Values are means ± SE of 4 different experiments.

Second messenger implicated in the stimulatory response to epinephrine. To identify the second messenger implicated in the stimulatory effect of epinephrine on the Na+-K+-2Cl- cotransport in isolated colonic crypt cells, we evaluated the effect of dibutyryl cAMP (DBcAMP), Ca2+ chelants [EGTA and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM], and the Ca2+ ionophore ionomycin on the activity of the cotransporter. The results are shown in Fig. 10. The DBcAMP (0.5 mM) did not affect the bumetanide-sensitive K+ influx. Reducing extracellular Ca2+ to 30 nM, by the addition of EGTA to the incubation medium, did not modify the stimulatory effect of epinephrine. However, preincubation of isolated cells with 50 µM BAPTA-AM abolished the increase induced by epinephrine on the activity of the cotransporter, suggesting that the response to epinephrine is mediated by intracellular Ca2+ liberation. Increasing the intracellular Ca2+ can reproduce the stimulatory effect of epinephrine. Ionomycin, a Ca2+ ionophore, induced a significant increase in the activity of the Na+-K+-2Cl- cotransporter. BAPTA-AM also produced an inhibitory effect on basal bumetanide-sensitive influx (Fig. 10), suggesting that the basal activity of the Na+-K+-2Cl- cotransporter also depends on the intracellular Ca2+ concentration. In addition, we determined the effect of epinephrine on intracellular Ca2+ concentrations in isolated colonic crypt cells (Fig. 11). Epinephrine (10-6 M), added to the perfusion medium, significantly increased intracellular Ca2+ concentration, in a process inhibited by propranolol.


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 10.   Effect of dibutyryl cAMP (0.5 mM), EGTA (3 mM), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (50 µM), and ionomycin (5 µM) on epinephrine-induced, bumetanide-sensitive K+ influx in isolated crypt cells from guinea pig distal colon. Cells were preincubated for 15 min without addition (control), with 1 µM epinephrine, 0.5 mM dibutyryl cAMP, 3 mM EGTA, or 3 mM EGTA and and 1 µM epinephrine or for 30 min with 50 µM BAPTA-AM or 50 µM BAPTA-AM and 1 µM epinephrine. Ionomycin (5 µM) was added to the incubation medium. Values are means ± SE of 3 different experiments. * P < 0.05; *** P < 0.001; ns, not significant, compared with control.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 11.   Effect of epinephrine on intracelullar Ca2+ in isolated crypt cells. Register represents a typical experiment showing the effect of 1 µM epinephrine on intracellular Ca2+ concentration in a single isolated crypt cell, loaded with fura 2-AM as indicated in MATERIALS AND METHODS. Colonocytes were perfused with DMEM (control condition). Epinephrine was added to the perfusion medium at 150 s after initiating the register. At 250 s, perfusion medium was changed to DMEM without epinephrine. This experiment was repeated 30 times with different cells and preparations with similar results (intracellular Ca2+ was 123 ± 7.9 nM in control and 650 ± 75 nM with epinephrine, n = 30).

These results suggest that the epinephrine-induced increase in the activity of the Na+-K+-2Cl- cotransporter is mediated by intracellular Ca2+ liberation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epinephrine stimulates active K+ secretion in mammalian distal colon as a result of an increase in serosal-to-mucosal flux and a decrease in mucosal-to-serosal flux (13, 20). Epinephrine-induced K+ secretion is inhibited by serosal ouabain or bumetanide and by mucosal Ba2+, suggesting the role of basolateral Na+-K+ pump and/or Na+-K+-2Cl- cotransporter, in addition to luminal K+ channels, in the secretory process. The aim of the present study was to evaluate the effects of several adrenergic agonists on the different K+ transport mechanisms in isolated guinea pig colonic cells, using 86Rb as a tracer, and to identify the second massager implicated in the response to epinephrine.

In crypt cells, epinephrine stimulated K+ uptake by increasing the activity of the Na+-K+-2Cl- cotransporter and the Na+-K+ pump, without changes in the passive K+ flux (Fig. 2). Preincubation of isolated crypt cells with bumetanide abolished the stimulatory effect of epinephrine on the Na+-K+ pump (Fig. 3). This increase in the activity of the Na+-K+ pump resulted in the activation of the Na+-K+-2Cl-cotransporter. However, if the epinephrine-stimulated increase in the Na+-K+ pump was only due to an increased Na+ delivery by the cotransporter, then a larger epinephrine-stimulated increase in the pump would be expected (assuming a 3:2 stoichiometry for Na+ and K+). Our results suggest a possible change in the stoichiometry of the pump or an epinephrine inhibition of a Na+ influx via a bumetanide-insensitive pathway or activation of an ouabain- and bumetanide-insensitive, K+-independent Na+ extrusion, as reported in the small intestine and renal proximal tubule (18). Maximal effect of epinephrine on the cotransporter was obtained with 1 µM epinephrine with an EC50 of 91.6 nM (Fig. 4). In contrast, we were unable to demonstrate K+ transport mediated by the Na+-K+-2Cl- cotransporter in surface cells, even under adrenergic stimulation (data not shown).

The stimulatory effect of epinephrine on the Na+-K+-2Cl-cotransporter seems mediated by beta 1- and beta 2-receptors, since isoproterenol (a beta 1- and beta 2-agonist), dobutamine (a beta 1-agonist), and salbutamol (a beta 2-agonist) had a stimulatory effect on the cotransporter (Fig. 5A). The beta 2-effect seems to be predominant, as indicated by the fact that the EC50 for salbutamol is six times lower than that obtained for dobutamine (Fig. 6). alpha -Adrenergic agonists, like phenylephrine and clonidine, did not affect it. In addition, propranolol (a beta -antagonist), but not benextramine (an alpha -antagonist), inhibited the stimulatory effect of epinephrine on the cotransporter (Fig. 5B). These results suggest that the effect of epinephrine on the Na+-K+-2Cl- cotransporter is mediated by beta 1- and beta 2-receptors.

Stimulation of K+ secretion by the beta -adrenergic agonist has been demonstrated in the guinea pig distal colon (13, 20) and rabbit distal colon (16, 21). In rabbit colon, epinephrine reduced the short-circuit current (Isc) and the transepithelial potential and increased the total conductance (Gt). IC50 was 5 µM. These changes were attributed to an increase in K+ secretion without changes in Cl- transport. alpha -Adrenergic agonists did not alter Isc or Gt. Isoproterenol had a similar effect to epinephrine, and propranolol blocked the change in Isc and Gt produced by epinephrine. Thus the effect of epinephrine seems to be due to a direct stimulation of colonic epithelial cells mediated by beta -adrenergic receptors (16, 21).

The inability of Ba2+, TEA, or DIDS to inhibit epinephrine enhancement of the activity of the cotransporter, in isolated crypt cells, indicates that this stimulatory response is not an indirect effect mediated by the opening of TEA-sensitive or Ba2+-sensitive K+ channels nor DIDS-sensitive Cl- channels (Fig. 7). Moreover, epinephrine did not modify K+ (86Rb) efflux in isolated crypt cells (Fig. 8). These results support the idea that the stimulatory effect of epinephrine on the Na+-K+-2Cl- cotransporter is not an indirect effect induced by the opening of K+ or Cl- channels.

cAMP or Ca2+ mediates ion secretion in many epithelial cells as second messengers. Usually, cAMP has been recognized as the second messenger involved in the cellular response to activation of beta -adrenergic receptors (22). However, in some cell systems, different second messengers (9, 14) can mediate the response to beta -adrenergic agonists. To identify the second messenger related to the stimulatory effect of epinephrine on the Na+-K+-2Cl- cotransporter in isolated crypt cells, we examined the effect of cAMP and Ca2+ on the Na+-K+-2Cl- cotransporter. As shown before (5), DBcAMP (0.5 mM) was unable to stimulate the cotransporter. Reduction of the extracellular Ca2+ to 30 nM, by the addition of EGTA to the incubation medium, did not significantly affect the response to epinephrine. In contrast, using BAPTA-AM as an intracellular Ca2+ chelator, it was possible to abolish the stimulatory response to epinephrine in isolated crypt cells, indicating that an increase in intracellular Ca2+ is essential to maintain the response to epinephrine. Furthermore, ionomycin, a Ca2+ ionophore, also stimulated the activity of the cotransporter. In addition, epinephrine increases intracellular Ca2+ concentration in isolated cells (Fig. 11). These results show that Ca2+ serves as second messenger in the stimulatory response to epinephrine in crypt cells.

The mechanism by which the adrenergic agonist modifies K+ secretion in mammalian colon has not been well defined. Both cAMP and Ca2+ have been shown to increase K+ secretion in colonic epithelium (1, 6, 11). However, isoproterenol does not increase intracellular cAMP levels in human colon (7) and Cl- secretion is not increased by epinephrine (20). These results suggest that the beta -adrenergic stimulation of K+ secretion, as well as the increase in the activity of the Na+-K+-2Cl- cotransporter in crypt cells, is not mediated by cAMP. On the other hand, removal of Ca2+ of the serosal solution partially inhibits the decrease in Isc produced by epinephrine (21) and the addition of A-23187 (19, 20) or ionomycin (8), Ca2+ ionophores, induces an increase in K+ secretion. These findings suggest that Ca2+ could mediate the enhancement of K+ secretion by beta -agonists.

It should be noted that cAMP did not stimulate the Na+-K+-2Cl- cotransport (Fig. 10). This observation raises the question about why the activation of cAMP-dependent Cl- channels did not stimulate the Na+-K+-2Cl- cotransporter. A possibility could be that a stimulation of Na+-K+-2Cl- cotransport is not observed because cAMP may also activate a basolateral K+ conductance, leading to underestimation of 86Rb influx due to a basolateral recycling. If this is the case, cAMP must stimulate 86Rb efflux. However, neither 0.5 mM DBcAMP, 0.5 mM bromo-cAMP, nor 100 µM forskolin produced any effect on 86Rb efflux (data not shown). In addition, Deiner et al. (6), using whole cell patch-clamp recordings, showed that forskolin, vasoactive intestinal polypeptide, or membrane-permeable cAMP analogs decreased K+ conductance in rat distal colon. Furthermore, Maguire et al. (15) observed an inhibition of basolateral K+ conductance in nystatin-permeabilized human colon by forskolin. These reports and our experiments suggest that cAMP did not significantly modify basolateral K+ conductance in colonic cells. A second possibility is that, under our experimental conditions, Cl- flux through Cl- channels must be low. Intracellular Cl- concentration in isolated crypt cells was 93 ± 10.5 mM, and the addition of 20 mM K+ to the medium induces cellular depolarization. These conditions may generate a relatively small electrochemical gradient for Cl- flux through cAMP-dependent Cl- channels, even if they are open.

Interestingly, epinephrine stimulated the cotransporter in crypt but not in surface cells (Fig. 2). This observation agrees with that reported by Halm and Rick (12), which indicates that the secretory process related with K+ secretory response to epinephrine, in guinea pig distal colon, was restricted to the lower two-thirds of the crypt of Lieberkühn.

In summary, we have shown that the K+ transport mechanism implicated in the response to epinephrine, in isolated crypt cells from guinea pig distal colon, is the Na+-K+-2Cl- cotransporter, in a process mediated by beta 1- and beta 2-receptors and modulated by intracellular Ca2+ liberation. Our results suggest that the stimulatory effect of epinephrine on K+ secretion seems primarily mediated by the activation of K+ entry across the basolateral plasma membrane through the Na+-K+-2Cl- cotransporter.


    ACKNOWLEDGEMENTS

This study formed part of J. C. Arévalo's Magister Scientiarum Thesis at Centro de Estudios Avanzados of the Instituto Venezolano de Investigaciones Científicas, 1997.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. R. del Castillo, Centro de Biofísica y Bioquímica, IVIC, Apartado 21827, Caracas 1020-A, Venezuela (E-mail: jdelcas{at}cbb.ivic.ve).

Received 9 February 1998; accepted in final form 25 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Binder, H. J., and G. I. Sandle. Electrolyte transport in the mammalian colon. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 2133-2171.

2.   Chwalinski, S., C. S. Potten, and G. Evans. Double labeling with bromodeoxyuridine and 3H-thymidine of proliferative cells in small intestinal epithelium in steady state and after irradiation. Cell Tissue Kinet. 21: 317-329, 1988[Medline].

3.   Del Castillo, J. R., B. Ricabarra, and M. C. Súlbaran-Carrasco. Intermediary metabolism and its relationship with ion transport in isolated guinea pig colonic epithelial cells. Am. J. Physiol. 260 (Cell Physiol. 29): C626-C634, 1991[Abstract/Free Full Text].

4.   Del Castillo, J. R., M. C. Súlbaran-Carrasco, and L. Burguillos. K+ transport in isolated guinea pig colonocytes: evidence for a Na+-independent ouabain-sensitive K+ pump. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G1083-G1089, 1994[Abstract/Free Full Text].

5.   Del Castillo, J. R., and F. V. Sepúlveda. Activation of an Na+/K+/2Cl- cotransporter system by phosphorylation in crypt cells isolated from guinea pig distal colon. Gastroenterology 109: 387-396, 1995[Medline].

6.   Diener, M., F. Hug, D. Strabel, and E. Scharrer. Cyclic AMP-dependent regulation of K+ transport in the rat distal colon. Br. J. Pharmacol. 118: 1477-1487, 1996[Abstract].

7.   Dupont, C., M. Laburthe, J. P. Broyart, D. Bataille, and G. Rosselin. Cyclic AMP production in isolated colonic epithelial crypts: a highly sensitive model for the evaluation of vasoactive intestinal peptide action in human intestine. Eur. J. Clin. Invest. 10: 67-76, 1980[Medline].

8.   Duvall, M. D., and S. M. O'Grady. cGMP and Ca2+ regulation of ion transport across isolated porcine distal colon epithelium. Am. J. Physiol. 267 (Regulatory Integrative Comp. Physiol. 36): R1026-R1033, 1994[Abstract/Free Full Text].

9.   Erdos, J. J., G. Vuquelin, Y. C. Stella, W. C. Broaddus, P. L. Jacobs, and M. E. Maguire. Magnesium transport: an independenly regulated beta -adrenergic response not mediated by cyclic AMP. Adv. Nucleotide Res. 14: 69-81, 1981.

10.   Gaad, K. G. Protein estimation in spinal fluid using Coomassie blue reagent. Med. Lab. Sci. 38: 61-63, 1981[Medline].

11.   Halm, D. R., and R. A. Frizzell. Ion transport across the large intestine. In: Handbook of Physiology. Gastrointestinal System. Intestinal Absorption and Secretion. Bethesda, MD: Am. Physiol. Soc., 1991, sect. 6, , vol. IV, chapt. 8, p. 257-274.

12.   Halm, D. R., and R. Rick. Secretion of K+ and Cl- across colonic epithelium: cellular localization using electron microprobe analysis. Am. J. Physiol. 262 (Cell Physiol. 31): C1392-C1402, 1992[Abstract/Free Full Text].

13.   Ishida, H., and Y. Suzuki. Potassium secretion in guinea pig distal colon. Jpn. J. Physiol. 37: 33-48, 1987[Medline].

14.   James, S. R., C. Vaziri, T. R. Walker, G. Milligan, and C. P. Downes. The turkey erythrocyte beta -adrenergic receptors coupled to both adenylate cyclase and phospholipase C via distint G-protein alpha  subunits. Biochem. J. 304: 359-364, 1994[Medline].

15.   Maguire, D. D., G. C. O'Sulivan, and B. J. Harvey. Membrane and genomic mechanisms for aldosterone effect in human colon. Surg. Forum 46: 203-205, 1995.

16.   Plass, H., A. Gridl, and K. Turnheim. Absorption and secretion of potassium by rabbit descending colon. Pflügers Arch. 406: 509-519, 1986[Medline].

17.   Potten, C. S., M. Kellett, S. A. Roberts, D. A. Rew, and G. D. Wilson. The measurement of in vivo proliferation in human colorectal mucosa using bromodeoxyuridine. Gut 33: 71-78, 1992[Abstract].

18.   Proverbio, F., R. Marín, and T. Proverbio. The ouabain-insensitive sodium pump. Comp. Biochem. Physiol. A Physiol. 99: 279-283, 1991.

19.   Rechkemmer, G., R. A. Frizzell, and D. R. Halm. Active potassium transport across guinea-pig distal colon: action of secretagogues. J. Physiol. (Lond.) 493: 485-502, 1996[Abstract].

20.   Smith, P. L., and R. D. McCabe. A23187-induced changes in colonic K and Cl transport are mediated by separate mechanisms. Am. J. Physiol. 247 (Gastrointest. Liver Physiol. 10): G695-G702, 1984[Abstract/Free Full Text].

21.   Smith, P. L., and R. D. McCabe. Potassium secretion by rabbit descending colon: effects of adrenergic stimuli. Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G432-G439, 1986[Medline].

22.   Summers, R. J., and L. R. McMartin. Adrenoreceptors and their second messenger systems. J. Neurochem. 60: 10-23, 1993[Medline].


Am J Physiol Gastroint Liver Physiol 277(3):G563-G571
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by del Castillo, J. R.
Articles by Súlbaran-Carrasco, M. C.
Articles citing this Article
PubMed
PubMed Citation
Articles by del Castillo, J. R.
Articles by Súlbaran-Carrasco, M. C.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online