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Constitutive nitric oxide release modulates neurally-evoked chloride secretion in guinea pig colon
Autonomic Neuroscience: Basic and Clinical 86 (2000) 47–57 www.elsevier.com / locate / autneu
Constitutive nitric oxide release modulates neurally-evoked chloride secretion in guinea pig colon a, b b a a Rhoda A. Reddix *, Xiaoping Liu , Mark J.S. Miller , Xiaomei Niu , Adrianne Powell a
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA b Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA Received 30 June 1999; received in revised form 22 May 2000; accepted 23 August 2000
Abstract The role of constitutive nitric oxide (NO) release in enteric neural pathways regulating ion transport was examined in guinea pig distal colon, in vitro and ex vivo. In in vitro studies, 43% of colonic preparations exhibited oscillations in baseline short-circuit current (Isc ), which were reduced by tetrodotoxin (TTX). The NO chelator, hemoglobin (Hb), and neuronal NO synthase inhibitor, 7-nitroindazole (7-NI), significantly increased the baseline Isc in these tissues, which was reduced by TTX. In tissues without oscillations in baseline Isc , Hb reduced the Isc , while 7-NI had little effect. In all tissues, electrical field stimulation (EFS; 15 V/ 10 Hz) caused a biphasic increase in the Isc which was enhanced by both Hb and 7-NI. In the ex vivo studies, basal release of nitric oxide was significantly lower in colonic segments isolated from guinea pigs administered N v -nitro-L-arginine methyl ester ( L-NAME) i.p. compared to control tissues. Moreover, carbachol, caused a 10-fold increase in NO release in control tissues, but had no effect in tissues isolated from the L-NAME group. L-NAME increased tissue conductance and EFS-induced changes in Isc , which were reversed by L-arginine. However, carbachol-induced ion secretion was unaltered in the L-NAME group compared to control animals. The results suggest that, in guinea pig colon, constitutive enteric NO release tonically suppresses submucous neural activity and it is involved in the maintenance of basal epithelial chloride secretion and mucosal permeability. Hence, constitutive NO promotes a delicate balance between pro-absorptive and pro-secretory processes in guinea pig colon. 2000 Elsevier Science B.V. All rights reserved. Keywords: Large intestine; Submucosal plexus; Nitrogen oxide species; Ion transport
1. Introduction The colon plays an important role in fine tuning the amount of fluid and electrolytes absorbed by the intestine (Debongnie and Phillips, 1978; Kaunitz et al., 1995). The human colon may absorb as much as 6 litres of fluid in an effort to prevent excess fluid loss (Debongnie and Phillips, 1978). The absorptive capacity of the colon is influenced by changes in gut motility, blood flow, and neurocrine, endocrine and paracrine factors controlling ion transport (Debongnie and Phillips, 1978). Recent evidence suggests that nitric oxide (NO) may contribute to the absorptive capacity of the colon. This is based on the fact that NO relaxes intestinal smooth muscle (Sanders and Ward, 1992) and increases transit time (Martinez-Cuesta et al., 1997; Mizuta et al., 1999), thereby allowing more contact time *Corresponding author. Tel.: 11-504-568-4740; fax: 11-504-5682361. E-mail address: [email protected] (R.A. Reddix).
for the absorption of fluid and electrolytes across intestinal epithelia. Additionally, we recently reported that inhibition of constitutive NO production enhances endothelin-1-induced absorption in guinea pig colon (Reddix et al., 1998). It is well established that NO is a noncholinergic nonadrenergic inhibitory neurotransmitter in myenteric neurons regulating intestinal motility and causes relaxation of vascular smooth muscle within the gastrointestinal tract (Sanders and Ward, 1992; Rattan et al., 1995). A growing body of evidence from immunohistochemical and functional studies in various species suggests that NO is also important in enteric pathways regulating fluid and electrolyte transport. The nature of its role in submucosal pathways is not clear. Several reports have shown that exogenous NO released from NO donors stimulates ion secretion in rat, guinea pig, dog and human intestine (MacNaughton, 1993; Tamai and Gaginella, 1993; Rhoads et al., 1995; Stack et al., 1996; Wilson et al., 1996). Under these conditions, NO evokes ion secretion by a prostaglandin-dependent mechanism (MacNaughton, 1993;
1566-0702 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1566-0702( 00 )00206-X
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Tamai and Gaginella, 1993; Wilson et al., 1996) and activation of enteric neurons in rat (Tamai and Gaginella, 1993) and human (Stack et al., 1996) colon. In other studies NO donors have been shown to promote ion absorption by both a direct effect on the mucosa as well as inhibition of secretagogue-induced secretion (SchirgiDegen and Beubler, 1995, 1996; Beubler and SchirgiDegen, 1997). Water and ion absorption was also increased in Thirty-Vella fistulas of canine ileum (Barry et al., 1995) in response to NO donors. Similarly, results concerning the role of constitutive NO release in enteric neural pathways regulating ion transport have been conflicting. In some studies, nitric oxide synthase inhibitors have been shown to attenuate secretagogueinduced ion secretion. Izzo et al. (1994) showed that the nitric oxide synthase inhibitor, N v -nitro-L-arginine methyl ester ( L-NAME) blunted carbachol-induced ion secretion in the small intestine of mice. Similarly, L-NAME (i.a.) attenuated 5-HT-induced ion secretion in the large intestine of anesthetized rats (Franks et al., 1994). In addition, Rhoads et al. (1995) showed an inhibition of asparagineinduced chloride secretion in pig jejunum by the NOS inhibitor, N G -monomethyl-L-arginine ( L-NMMA). On the other hand, inhibition of basal intestinal NO release increased ion secretion in other studies. The NOS inhibitors L-NAME, N v -nitro-L-arginine ( L-NA) and N v nitro-L-methyl arginine ( L-NMA) evoked an increase in baseline chloride secretion and the potential difference (PD) across segments of mouse ileum (Rao et al., 1994). L-Arginine reversed the effects of L-NMA and L-NNA, while D-arginine had no effect. Moreover, chlorisondamine and the neurotoxin, tetrodotoxin (TTX) nearly abolished the L-NMA-induced increase in Isc . A pro-absorptive role for NO was also demonstrated in ligated loops of rat jejunum (Schirgi-Degen and Beubler, 1995). In this study, pretreatment of jejunal loops with the NOS inhibitors L-NAME and L-NNA stimulated fluid secretion and enhanced Escherichia coli Sta-induced fluid secretion. Subsequently, the authors showed that the pro-absorptive effects of NO was partially mediated by maturation of K 1 channels (Schirgi-Degen and Beubler, 1996). The effects of L-NAME and L-NA were reversed by sodium nitroprusside and L-arginine. Takeuchi et al. (1993) showed that constitutively produced NO evokes ion absorption in the stomach and ileum. Inhibition of constitutive NO release caused an increase in bicarbonate secretion from rat gastric and duodenal mucosa (Takeuchi et al., 1993). Although contradictory, these results suggest that NO is involved in the control of ion transport across the small intestine of various species; however, the nature of its role remains to be determined. The small and large intestine are functionally distinct segments (Kaunitz et al., 1995). Differences in NOS distribution and the percentage of NOS-immunoreactive neurons between these two regions have been reported (Furness et al., 1994; Wang et al., 1995; Sang and Young, 1996). In guinea pig colon, NOS-
immunoreactivity has been identified in the myenteric plexus and submucous neurons projecting to circular muscles and muscularis mucosae and it is localized within interneurons (Furness et al., 1994). It was recently shown that, in guinea pig intestine, the density of NOS-positive neurons is greater in the large intestine than in the small intestine. Previous studies employing NOS inhibitors have focused primarily on the small intestine (Wang et al., 1995). Although there is a growing body of evidence demonstrating constitutive NOS-IR in enteric pathways regulating ion transport in the large intestine of various species, very little is known concerning the role of constitutive NO release in these pathways. Therefore, the focus of the present study was to examine the effect of constitutive NO release on ion transport in guinea pig colon, in vitro and ex vivo. We hypothesize that, in addition to its effects on smooth muscle, constitutive NO release may tonically inhibit submucosal secretomotor neurons in guinea pig colon. To test this hypothesis, experiments are designed to determine the effect of inhibiting constitutive NOS, in vitro and ex vivo, on baseline and electrical field stimulation (EFS)induced changes in the short-circuit current (Isc ) of guinea pig colon.
2. Methods
2.1. Tissue preparation Male albino guinea pigs (Harlan Sprague-Dawley, Indianapolis, IN, USA) weighing 350–500 g were housed in metal cages with food and water ad libitum. The animals were stunned and exanguinated. This method of euthanasia has been approved by the LSU Institutional Animal Care Committee and complies with federal regulations. Segments of distal colon were removed and opened along the mesenteric border. The intraluminal contents were removed and the segments were pinned with the mucosal side down onto a Sylgard-coated Petri dish. Tissues were perfused with a chilled Krebs–Ringer solution (in mM): 120 sodium chloride, 6 potassium chloride, 1.2 magnesium chloride, 6 H 2 O, 1.3 sodium phosphate, 14.4 sodium bicarbonate, 2.5 calcium chloride, 12.5 glucose. The longitudinal and circular muscle layers along with the myenteric plexus were removed by blunt dissection leaving the submucosa / mucosa intact.
2.2. Short-circuit current ( Isc ) measurements Colonic segments were divided into 2–3 cm sheets and mounted in Ussing flux chambers with an area of 0.785 cm 2 . Three to four adjacent sheets were obtained from the most distal portion of the colon from each animal. The baseline Isc values for all tissues were similar. Additionally, tissues were subject to submaximal stimulation (15
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V/ 10 Hz). The EFS-induced ion transport was of similar magnitude in tissues with and without on-going activity. Peak 1: (1) on-going, 321.1627.8 mA / cm 2 , n 5 23 and (2) on-going, 318.1622.7 mA / cm 2 , n 5 24; peak 2: (1) on-going, 307.2637.8 mA / cm 2 , n 5 24 and (2) on-going, 270.7624.2 mA / cm 2 , n 5 24. Tissues were bathed in a Krebs–Ringer solution maintained at 378C and aerated with 95% O 2 and 5% CO 2 . The chambers were designed with ports for Ringer-agar bridges and calomel half-cells for measurement of transmural potential difference (PD). Throughout the experiment, tissues were short circuited with a voltage clamp apparatus (Physiological Instruments, CA, USA) to abolish changes in PD. The short-circuit current (Isc ) was measured in mA and normalized to the tissue surface area (cm 2 ). Prior to the experimental session, all tissues were paired according to baseline Isc , Gt and PD. Any changes in the Isc during either electrical or chemical stimulation of tissues are due to alterations in active ion transport. Forty-three percent of all tissues exhibited on-going neural activity characterized by oscillations in baseline Isc . These oscillations are mediated by active cholinergic secretomotor neurons (Reddix et al., 1994). For tissues with (1) on-going neural activity, the baseline Isc was calculated as the mean value.
2.3. Electrical field stimulation ( EFS) Tissues were equilibrated for 40 min and then electrically stimulated for 90 s by a pair of aluminum foil electrodes placed adjacent to the serosal surface. The electrodes were connected to a Grass SD 88 stimulator (Grass Instruments, Quincy, MA, USA) which generated pulses at 0.5 ms, with a strength of 15 V (3A) and a frequency of 10 Hz. Changes in Isc were continuously monitored by a Kipp and Zonen 2 chart recorder. Measurements of Isc (mA / cm ) were calculated as the difference between the peak Isc and baseline Isc before stimulation (Isc peak 2 Isc baseline ). There was no significant difference in the EFS-induced secretory response between tissues with or without on-going neural activity. In a separate set of tissues, we examined the effect of chloride-free buffer consisting of (in mM): 120 sodium gluconate, 6 potassium gluconate, 1.2 magnesium gluconate, 2.5 calcium gluconate, 14.4 sodium bicarbonate, 12.5 glucose and 1.3 sodium phosphate on EFS-induced secretion. The chloride-free solution was replaced in both the mucosal and serosal compartments.
2.4. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on baseline short-circuit current ( Isc ) measurements Changes in Isc were monitored in colonic segments exposed to vehicle or the following agents: Hb (2.3 mM), an NO scavenger and the NOS inhibitors, N v -nitro-Larginine methyl ester ( L-NAME; 0.1 mM), and 7-NI (0.1 mM). These agents were added to both mucosal and
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serosal compartments. The concentration of each drug used in this study is based on previously published results (Bland-Ward and Moore, 1995; Rattan et al., 1995; Miller et al., 1996). In a separate set of experiments, the effect of Hb (2.3 mM) and 7-NI (0.1 mM) on Isc in tissues with on-going neural activity was examined. To determine whether the Hb- and 7-NI-induced secretory responses were partially mediated by activation of secretomotor neurons, we examined the effect of the neurotoxin, tetrodotoxin (TTX; 0.2 mM) on peak secretory effects of Hb and 7-NI.
2.5. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on the electrical field stimulation ( EFS)-induced secretion To further examine the role of submucous secretomotor neurons, we investigated the effect of NO inhibitors on EFS-induced ion secretion. All tissues were electrically stimulated 10 min prior to addition of vehicle, Hb (2.3 mM) or 7-NI (0.1 mM) to the bathing solution. Another EFS response was measured 10 min following the vehicle, Hb or 7-NI treatment. The effects of vehicle, Hb and 7-NI on the EFS response were examined during the same experimental period. The vehicle had no effect on baseline Isc and EFS-induced ion secretion. Drug-induced changes in the EFS response were calculated as a percent of the original EFS-induced secretory response. These changes were compared to EFS responses obtained in vehicletreated tissues.
2.6. Effect of N v -nitro-L-arginine methyl ester ( L-NAME) in vivo on short-circuit current ( Isc ) measurements ex vivo Albino male guinea pigs weighing 300–350 g were administered saline, L-NAME (50 mg / kg), L-NAME (50 mg / kg)1 L-ARG (100 mg / kg) or L-NAME (50 mg / kg)1 L-ARG (200 mg / kg), intraperitoneally. Muscle-stripped colonic segments were prepared as previously described. This protocol has been shown to significantly inhibit constitutive NOS in vivo (Dwyer et al., 1991).
2.7. Measurements of nitric oxide release To confirm NOS inhibition, NO release from whole thickness colonic segments isolated from the saline and L-NAME groups were measured using an NO sensor developed by Dr. Xiaoping Liu (Ribbons et al., 1997). Nitric oxide synthase has been previously identified in the mucosal epithelia, interneurons of the submucous plexus, muscularis mucosae, myenteric plexus and smooth muscle of guinea pig intestine (Furness et al., 1994). In addition, we have previously reported that hemoglobin, chelates
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nitric oxide, significantly reduced the baseline Isc in isolated muscle-stripped guinea pig colon (Reddix et al., 1998). Based on these results, nitric oxide measurements were obtained from the mucosal surface. Moreover, NO measurements were performed to confirm effective blockade in vivo of intestinal NO production. Briefly, 2–3 cm whole thickness colonic segments isolated from the saline and L-NAME groups were pinned mucosal side up onto a sylgard-coated Petri dishes. Tissues were bathed in warmed (378C) RPMI-1640 culture medium (NIF Technologies) containing vehicle or the acetylcholine agonist carbachol (10 mM). The Petri dish was transferred to the NO detection apparatus. The concentration of NO released from the tissues was detected by an electrochemical measurement system consisting of three electrodes, a platinum disc microelectrode coated with nafion membrane (working electrode), a reference electrode and an auxillary electrode, respectively. The electrodes were connected to a BAS 100B Electrochemical Analyzer with a PA-1 PreAmplifier and C2 cell stand. To measure NO, the tip of the working electrode was placed at the surface of the bathing solution to establish a baseline. Since the focus of the this study was to assess the effect of mucosal NO production on ion transport, the microelectrode tip was lowered to the mucosal surface. Changes in current (nA) passing through the working electrode was used as an index of the NO concentration. Changes in NO levels were calculated as the difference between current (nA) at the solution surface I0 and tissue level (Itissue ). This value was converted to mM from a calibration with nitric oxide gas solution. This process was repeated three to four times at different areas on the tissue. The values were averaged for each tissue.
2.8. Effect of NOS inhibition on the EFS response ex vivo Baseline and EFS-induced changes in Isc were recorded in tissues isolated from each group. An earlier report suggested that L-NAME treatment in vivo degranulates mast cells in the small bowel of rats (Kanwar et al., 1994). Mast cells are located within the wall of the gastrointestinal tract (Kanwar et al., 1994; Gaboury et al., 1996). Histamine is a prominent transmitter released from mast cells and has been shown to be a potent secretagogue (Cooke and Reddix, 1994; Frieling et al., 1994). Therefore, one set of control experiments was designed to determine whether histamine mediated baseline changes in Isc observed in colonic segments of L-NAME-treated guinea pigs. Colonic segments isolated from the saline or LNAME treatment groups were exposed to the histamine H 1 and H 2 receptor antagonists, 10 mM pyrilamine (PYR) and 10 mM cimetidine (CIM), 10 min following the initial EFS response. Both cimetidine and pyrilamine attenuated histamine (10 mM)-induced secretion (data not shown).
Another EFS response was monitored 10 min after drug treatment.
2.9. Effect of NOS inhibition on carbachol-induced secretion ex vivo Since there is a relatively high density of muscarinic receptors on epithelial cells throughout the gastrointestinal tract, stimulation of these receptors served as a positive control to assess tissue viability and responsiveness. At the end of all experiments, the muscarinic agonist carbachol (0.1 mM) was added to the serosal compartment and changes in Isc were recorded in colonic segments isolated from each treatment group.
2.10. Effect of NOS inhibition on tissue conductance ( Gt ), ex vivo Previous reports have shown that L-NAME treatment in vivo increases epithelial permeability (Kanwar et al., 1994; Alican and Kubes, 1996). To examine the possibility that changes in baseline Isc are due to alterations in mucosal permeability, tissue conductance was monitored in colonic segments isolated from all treatment groups. Tissue conductance gives an assessment of magnitude changes in the movement of ions across colonic segments. As tissue conductance increases, tissue resistance to ionic flux decreases, indicating an increase in mucosal permeability to these ions. The tissue conductance may increase without alterations in the Isc if due to electroneutral ionic fluxes.
3. Materials and chemicals The following chemicals were purchased from Sigma (St. Louis, MO, USA): hemoglobin, N v -nitro-L-arginine methyl ester ( L-NAME), L-arginine, carbachol, 7-nitroindazole, tetrodotoxin, cimetidine and pyrilamine. All drugs were dissolved in Krebs–Ringer solution except the following: cimetidine was dissolved in 1 M HCl (0.001%)1Krebs–Ringer; 7-nitroindazole was dissolved in dimethyl sulfoxide (DMSO, 0.001%)1Krebs–Ringer; RPMI-1640 culture medium (NIF Technologies).
4. Statistics All data are expressed as mean6S.E.M. An unpaired Student’s t-test was used to test the significance between group means. A probability value less than 0.05 was considered statistically significant.
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5. Results
5.1. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on baseline short-circuit current ( Isc ) measurements Forty-three percent of muscle-stripped colonic segments exhibited on-going neural activity. There was no significant difference in the baseline Isc in tissues with and without on-going neural activity: (2) on-going activity: 40.364.9 mA / cm 2 (9) and (1) on-going activity 35.4613.0 mA / cm 2 (9). However, tissue conductance was significantly greater in tissues with on-going neural activity: (2) on-going activity 12.065.4 mA / cm 2 (12) and (1) on-going activity 20.462 mA / cm 2 ; n 5 10, *P , 0.05. In colonic segments we observed a consistent, but variable increase in ion secretion in response to Hb and 7-NI. The NO scavenger Hb (2.3 mM) increased baseline Isc in tissues with on-going neural activity (Fig. 1A). The vehicle had no effect on baseline Isc . The peak Hb-induced secretory response was attenuated by 0.2 mM TTX (Fig. 1A). Conversely, in tissues without on-going activity, 2.3 mM Hb caused a significant reduction in baseline Isc (Fig. 1B). Overall, both hemoglobin and 7-NI consistently increased the baseline Isc in tissues with on-going neural activity. However, we observed variability in the magnitude of the secretory responses to these agents. The mean changes in baseline Isc evoked by 2.3 mM Hb and 7-NI (0.1 mM) in tissues with and without on-going neural activity were Hb, (1) on-going 55.6623.3 (8) and (2) on-going 210.364.4 mA / cm 2 (11); and 7-NI, (1) ongoing 28.066.4 mA / cm 2 (5) and (2) on-going 0.0 mA /
Fig. 2. Representative tracings illustrating baseline changes in the shortcircuit current (Isc ; mA / cm 2 ) in muscle-stripped segments of guinea pig colon. Tissues with (1) on-going neural activity (superimposed oscillations) exposed to: (A) nitric oxide scavenger, hemoglobin (Hb; 2.3 mM), followed by the neurotoxin tetrodotoxin (TTX; 0.2 mM). Hb increased the Isc 208 mA / cm 2 ; TTX reduced the Hb-induced increase in Isc by 152 mA / cm 2 . (B) Tissues without (2) on-going neural activity exposed to Hb (2.3 mM) alone.
cm 2 (5). The NO synthase inhibitor 7-NI (0.1 mM) increased baseline Isc in tissues with on-going neural activity (Fig. 2A). The vehicle had no effect on baseline Isc . The peak 7-NI-induced secretory response was reduced by 0.2 mM TTX (Fig. 2A). 7-NI, 24.862.1 mA / cm 2 ,
Fig. 1. Representative tracings illustrating baseline changes in the short-circuit current (Isc ; mA / cm 2 ) in muscle-stripped segments of guinea pig colon. Tissues with on-going neural activity (superimposed oscillations) were exposed to: (A) the nitric oxide synthase inhibitor 7-nitroindazole (7-NI; 0.1 mM), followed by the neurotoxin tetrodotoxin (TTX; 0.2 mM); 7-NI increased the Isc by 51 mA / cm 2 ; TTX reduced the 7-NI-induced increase in Isc by 12.5 mA / cm 2 . (B) Tissues without on-going neural activity exposed to 0.1 mM 7-NI; 7-NI had no effect on the Isc .
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n 5 4; 7-NI1TTX (0.2 mM), 21.367.4 mA / cm 2 , n 5 4. 7-NI had no effect on baseline Isc in tissues without on-going neural activity (Fig. 2B). Although 7-NI and Hb evoked changes in the baseline Isc of guinea pig colonic segments, addition of the nitric oxide synthase inhibitor N v -nitro-L-arginine methyl ester (1 mM L-NAME) to both mucosal and serosal compartments had no effect in vitro (data not shown).
5.2. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on electrical field stimulation ( EFS)-induced secretion To assess changes in EFS-induced secretion in control tissues over the same time period as drug treatment, an initial EFS response (EFS-1) was recorded in colonic segments followed by a second EFS response (EFS-2) measured 20 min later. Tissues were exposed to vehicle or test drug 10 min prior to EFS-2. The EFS (15 V/ 10 Hz) evoked a biphasic increase in baseline Isc (peak 1, PK1 and peak 2, PK2; Fig. 3). Peak 1 was complete within 30 s followed by a second peak (PK2) within 1 min of the PK1 response. It must be noted that peak 2 of the EFS response is relatively the same magnitude during the 30 min experimental period. It was previously shown that, in guinea pig colon, EFS-induced secretion is primarily due to an increase in electrogenic chloride secretion (MartinezCuesta et al., 1997). Similarly, our results showed that both peaks 1 and 2 were abolished in tissues exposed to chloride-free solution (Fig. 3). Moreover, there was no significant difference in both peaks of the EFS response in
Fig. 3. Electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in the short-circuit current (Isc ; mA / cm 2 ) in muscle-stripped segments of guinea pig colon bathed in normal Krebs–Ringer solution (h), or chloride-free Krebs–Ringer solution (replaced Cl 2 with gluconate) (9). Short-circuit current measurements in response to EFS were obtained at time zero, EFS-1. The tissues were allowed to recover for 20 min and then a second EFS-induced Isc response was recorded (EFS-2). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1). Peak 1 was completed within 30 s followed by a second peak (PK2) within 1 min of PK1. **Significant from control peak 2 (PK2); P , 0.05 was considered significant.
Fig. 4. Effect of 7-nitroindazole (7-NI; 0.1 mM) on electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in Isc in colonic segments. Results are reported as a percent of EFS-1 (EFS-2 / EFS-13 100%). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1). P , 0.05 was considered statistically significant.
tissues with (peak 1, 321.1627.8 mA / cm 2 , n 5 23; peak 2, 307.2637.8 mA / cm 2 , n 5 23) or without (peak 1, 318.1622.7 mA / cm 2 , n 5 24; peak 2, 270.7624.2 mA / cm 2 , n 5 24) (1) on-going neural activity. For subsequent studies, drug-induced changes in the EFS response were expressed as a percent of the initial EFS response (EFS-2 / EFS-13100). Both peaks 1 and 2 of the EFS response were significantly greater in tissues pretreated with the NOS inhibitor 7-NI (Fig. 4) or nitric oxide scavenger Hb (Fig. 5) compared to vehicle-treated tissues. Addition of L-NAME to the bathing solution of both mucosal and serosal compartments had no effect on EFSinduced changes in Isc (Fig. 5). A possible explanation lies in the fact that in vitro preparations of muscle-stripped colonic segments may not have the esterases required to activate L-NAME.
Fig. 5. Effect of the nitric oxide scavenger hemoglobin (Hb; 2.3 mM) on electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in Isc in colonic segments exposed to vehicle (Veh) or the nitric oxide synthase inhibitor N v -nitro-L-arginine methyl ester ( L-NAME; 1 mM). Results are reported as a percent of EFS-1 (EFS-2 / EFS-13100%). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1) or **significant from control peak 2 (PK2). P , 0.05 was considered statistically significant.
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5.3. Effect of N v -nitro-L-arginine methyl ester ( L-NAME) in vivo upon short-circuit current measurements ex vivo To determine whether results from in vitro studies were similar to those obtained ex vivo, electrical measurements were recorded in colonic segments isolated from guinea pigs administered saline, L-NAME (50 mg / kg), LNAME1 L-ARG (100 mg / kg) and L-NAME1 L-ARG (200 mg / kg), intraperitoneally, i.p. The animals were sacrificed 3 h post-injection. Baseline colonic NO release was lower in L-NAME-treated animals compared to the saline group
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Table 1 Effect of basal nitric oxide inhibition in vivo a Treatment
Baseline Isc (mA / cm 2 )
Conductance (Gt ; mS / cm 2 )
Saline (n 5 22) L-NAME (50 mg / kg; n 5 22) L-NAME1 L-ARG (100 mg / kg; n 5 14) L-NAME1 L-ARG (200 mg / kg; n517)
57.369.4
12.364.0
77.0614.9
20.563.3*
47.768.2
22.263.8*
39.9614.6
11.661.3
a
Baseline short-circuit current (Isc ) and tissue conductance (Gt ) values in muscle-stripped segments of guinea pig colon. Tissues were isolated from guinea pigs administered saline, the nitric oxide synthase inhibitor N v nitro-L-arginine methyl ester ( L-NAME; 50 mg / kg), a combination of L-NAME1 L-arginine ( L-ARG, NO precursor; 100 mg / kg) or L-NAME1 LARG (200 mg / kg). All drugs were administered intraperitoneally, i.p., and the animals were sacrificed 3 h post-injection; n 5 14–22. *P , 0.05, significant from the saline group.
(Fig. 6). The nonselective cholinergic receptor agonist carbachol (10 mM) caused a 10-fold increase in colonic NO release in the saline group; however, it had no effect in animals administered L-NAME. Table 1 summarizes the baseline Isc and Gt in all groups. There was no significant difference in baseline Isc in muscle-stripped colonic segments isolated from animals in each group. However, Gt was significantly higher in the L-NAME group and this effect was reversed in animals administered L-NAME1 L-ARG (200 mg / kg). In Fig. 7, peaks 1 and 2 of the EFS chloride secretory response were significantly elevated in the L-NAME group compared to the saline group. L-Arginine reversed the effects of L-NAME on both peaks of the EFS secretory response at 100 and 200 mg / kg.
Fig. 6. (A) Representative tracings of NO measurements (mM) in whole thickness colonic segments isolated from guinea pigs administered saline or N v -nitro-L-arginine methyl ester ( L-NAME, 50 mg / kg) intraperitoneally. All tissues were pinned mucosal side up onto sylgard-coated Petri dishes and bathed in 15 ml RPMI-1640 medium containing vehicle or 10 mM carbachol. Changes in NO current (nA) were detected by a platinum microelectrode (potential held at 0.55 V vs. a Ag /AgCl electrode) at the solution surface, I0 , and tissue level, Itiss . All measurements were repeated at three different sites for each tissue. The concentration of NO in the bathing solution was calculated as the difference between I0 and Itiss ; this value was converted to mM. (B) Mean NO levels (mM) released from whole thickness colonic segments isolated from saline or L-NAMEtreated animals. Tissues were bathed in RPMI-1640 medium containing vehicle or the muscarinic receptor agonist carbachol (10 mM). Values are expressed as the mean 6S.E.M., n 5 4. *Significance from the saline vehicle. P , 0.05 was considered statistically significant.
Fig. 7. Electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in the short-circuit current (Isc ; mA / cm 2 ) across colonic segments isolated from guinea pigs administered saline, N v -nitro-L-arginine methyl ester ( L-NAME; 50 mg / kg), L-NAME1 L-arginine ( L-ARG; 100 mg / kg) or L-NAME1 L-ARG (200 mg / kg), intraperitoneally, i.p. Results are reported as DIsc (mA / cm 2 ). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1) or **significant from control peak 2 (PK2). P , 0.05 was considered statistically significant.
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Fig. 8. Carbachol (CARB; 0.1 mM)-induced changes in the short-circuit current (Isc ; mA / cm 2 ) across colonic segments isolated from guinea pigs administered saline, N v -nitro-L-arginine methyl ester ( L-NAME; 50 mg / kg) or L-NAME (50 mg / kg)1 L-arginine ( L-ARG; 100 mg / kg), intraperitoneally. Results are reported as DIsc (mA / cm 2 ). Values are expressed as the mean6S.E.M., n 5 9–10.
It has been suggested that L-NAME in vivo promotes degranulation of mast cells with the subsequent release of histamine, a potent secretagogue. The histamine receptor antagonists cimetidine (H 2 ; 10 mM) and pyrilamine (H 1 ; 10 mM) blocked histamine-induced secretion but had no effect on the EFS response in colonic segments isolated from guinea pigs in the L-NAME group (data not shown). The nonselective cholinergic receptor agonist carbachol (0.1 mM) was added to the serosal compartment at the end of all experiments to assess the viability of each tissue preparation. Carbachol (0.1 mM) caused an increase in Isc of similar magnitude in colonic segments isolated from control and L-NAME groups. However, it was significantly reduced in animals co-administered L-NAME and L-ARG (Fig. 8).
6. Discussion The purpose of the current study was to investigate the physiological role of constitutive nitric oxide in enteric pathways regulating ion transport in guinea pig colon. The effect of nitric oxide inhibitors on ion transport across colonic epithelia was examined in vitro and ex vivo. Results from the current study indicate that the role of constitutive NO release in the regulation of ion transport across colonic epithelia is multifaceted. A large component of the NO effect involves tonic inhibition of submucous neural activity. In addition, NO plays a role in the maintenance of epithelial transport and permeability. Evidence supporting an inhibitory effect of basal NO on submucous neurons comes from the fact that sequestration of NO by Hb or inhibition of NOS by 7-NI in vitro increased baseline Isc only in muscle-stripped colonic segments with on-going neural activity (Figs. 1 and 2). The increase in Isc by Hb was two-fold greater than that by
7-NI. This may be due to the fact that 7-NI inhibits neuronal NOS which has been identified primarily in interneurons of guinea pig colon (Furness et al., 1994). Hence, the small increase reflects removal of the inhibitory effect of constitutive NO on secretomotor neurons. On the other hand, the nerve terminals of secretomotor neurons are exposed to NO released from several sources in the mucosal and sub-epithelial regions, i.e. muscularis mucosae, sub-epithelial fibroblasts and epithelial cells. The NO released has an inhibitory effect on the release of secretagogues from secretomotor neurons. Hence, Hb scavenges the NO, removing this inhibitory influence which enhances the release of neurotransmitters at the neuro-epithelial junction resulting in an increase in secretion. The on-going neural activity observed in colonic segments is mediated primarily by active cholinergic secretomotor neurons (Reddix et al., 1994). The neurotoxin tetrodotoxin attenuated the peak secretory effects of 7-NI and Hb on baseline Isc in these tissues (Figs. 1A and 2A). A similar observation was reported in mouse small intestine. Rao et al. (1994) showed an increase in baseline chloride secretion following nitric oxide inhibition in mouse ileal segments that was TTX-sensitive. Based on these results, it may be concluded that constitutive NO tonically suppresses submucous neural activity. However, additional studies are required to delineate the mechanisms involved. To further examine the effect of constitutive NO release on neurally evoked secretion, colonic segments were electrically stimulated to depolarize the submucous neurons within our preparation. Electrical field stimulation induced a biphasic change in Isc mediated by an increase in electrogenic chloride secretion (Fig. 3). Both peaks 1 and 2 of the EFS response were greater in tissues pretreated with Hb or 7-NI (Figs. 4 and 5) compared to control. This result provides additional evidence supporting the notion that NO tonically inhibits submucosal neural activity. Furthermore, we previously reported that, in guinea pig colon, the neurokinin 3 agonist [Pro 7 ]neurokinin-B caused an increase in ion secretion that was TTX-sensitive (Reddix and Niu, 1997). The NK3-induced secretory response was enhanced at a low concentration in tissues pretreated with the NO scavenger Hb; however, it was unaltered at a high concentration. One possible explanation is that tonic NO release modulates neurally-evoked secretion by altering neurotransmitter release. Additional studies are required to confirm this observation. To confirm our results from in vitro studies employing NO inhibitors, we examined the effect of inhibiting NOS in vivo on neurally-evoked secretion in isolated colonic segments. For this series of experiments, guinea pigs were administered saline, L-NAME (50 mg / kg), L-NAME1 LARG (100 mg / kg) or L-NAME1 L-ARG (200 mg / kg), intraperitoneally. Basal and carbachol-stimulated NO release were measured in colonic segments isolated from the saline and L-NAME-treated animals to verify NOS inhibi-
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tion. In the saline group, carbachol caused a 10-fold increase in colonic release of NO which was abolished in the L-NAME group (Fig. 7). Moreover, the concentration of NO released in response to carbachol exceeded physiological levels reported in the literature (Archer et al., 1995). This may be due to the fact that prolonged exposure of carbachol stimulates an increase in intracellular calcium that maximally activates constitutive NOS enzymes to produce NO levels similar to inducible NOS. In addition, NO is derived from several cellular sources in our preparation which are pooled to yield higher levels. It is also interesting to note that although carbachol did not increase NO levels in the L-NAME-treated animals, it caused an increase in chloride secretion in this group that was of similar magnitude in the saline group (Fig. 8). In our preparation, carbachol evokes a TTX-insensitive secretory response. Hence, carbachol-induced ion secretion in colonic epithelia is not dependent on the release of NO. In the current study, results from ex vivo experiments also showed an enhanced neurally evoked chloride secretion in colonic segments isolated from guinea pigs administered L-NAME, i.p. (Fig. 7). This response was blunted with simultaneous administration of L-arginine (Table 1). Similar results were obtained by Izzo et al. (1994), who showed an inhibitory effect of constitutive NO and the NO donor sodium nitrite on neurally-mediated ion secretion in mouse ileum. In addition, Rao et al. (1994) showed an inhibitory influence of tonic NO release on secretomotor neurons within mouse ileum. In the present study, increased tissue conductance observed with LNAME treatment was reversed by a high concentration of L-arginine. Additional evidence supporting an inhibitory influence of NO on enteric neurons was reported by Tumara et al. (1992). Their results from electrophysiological studies showed that the NO donor sodium nitroprusside (SNP) had no effect on basal electrical behavior of myenteric neurons, however it attenuated noncholinergic slow excitatory postsynaptic potentials (EPSPs). Hence, NO may have a presynaptic inhibitory effect on neurotransmitter release (Rhoads et al., 1995; Yuan et al., 1995). However, the effect of NO on the electrical properties of submucosal neurons is not known. Shuttleworth et al. (1993) showed that NO stimulated an increase in intracellular cGMP levels within myenteric neurons which was associated with an inhibition of the neural activity. In another study, the NO donor sodium nitroprusside (SNP) also increased cGMP levels within myenteric neurons but to a much greater extent in submucosal neurons of guinea pig intestine (Young et al., 1993). Hence, it seems possible that NO may inhibit submucosal neural activity via a cGMP-dependent mechanism. In contrast to our results and those of others demonstrating an inhibitory effect of constitutively produced NO on submucosal neurons, it has been shown that NO
55
released from NO donors stimulates electrogenic chloride secretion in rat (Wilson et al., 1996) and human (Stack et al., 1996) colon that is TTX-sensitive. MacNaughton (1993) also demonstrated a stimulatory effect of exogenous NO on chloride secretion in guinea pig intestine. The authors examined the effect of SNP and isosorbide dinitrate (ISDN) on guinea pig duodenum, jejunum, ileum, proximal and distal colon. SNP and ISDN evoked a dosedependent increase in Isc that was mediated in part by PGE 2 and 5-HT. However, TTX had no effect on the SNP-induced secretory response in their preparations. This is in contrast to the rat and human colon, in which NO stimulated submucosal secretomotor nerves. Another possible explanation for the enhanced neural response in the presence of L-NAME, in vivo, is based on the results of Kanwar et al. (1994), who reported that L-NAME, i.a., increases histamine release from enteric mast cells. Mast cells are present in the gastrointestinal tract (Handlinger and Rothwell, 1984; Kanwar et al., 1994; Gaboury et al., 1996). It has been shown that mast cell mediators such as histamine or platelet activating factor (PAF) increase epithelial cell permeability. Since it is well established that histamine is a potent secretagogue (Cooke and Reddix, 1994; Frieling et al., 1994), we examined the effect of a combination of the histamine receptor antagonists pyrilamine (PYR; H 1 ) and cimetidine (CIMET; H 2 ) on EFS-induced changes in Isc in tissues isolated from the L-NAME group. CIMET and PYR had no effect on baseline Isc or the EFS response (data not shown), suggesting that the enhanced EFS response in L-NAMEtreated animals is not mediated by histamine. It has previously been suggested that constitutively produced NO is important for the maintenance of mucosal integrity (Kanwar et al., 1994; Alican and Kubes, 1996), although it is not clear whether NO modulates epithelial permeability via a direct effect on the epithelium or via the release of intermediate factors. Moreover, it has been demonstrated that L-NAME, i.a., increased 51 Cr-EDTA clearance (estimate of mucosal permeability) across feline small intestine (Kanwar et al., 1994). Therefore, we monitored changes in tissue conductance in all groups. The conductance was significantly greater in the L-NAME animals. L-Arginine (200 mg / kg) reversed the effects of L-NAME on tissue conductance. It is also of interest to note that 100 mg / kg L-arginine reversed the effects of L-NAME on the neural response, but had no effect on Gt . This suggests that changes in the neurally-induced chloride secretory response were not due to an increase in epithelial permeability. Additional evidence supporting a direct effect of NO on epithelial function is based on the observation that Hb reduced baseline Isc and 7-NI had no effect in tissues without on-going activity. Furthermore, we previously showed a reduction in baseline Isc in TTX-treated colonic segments exposed to Hb and N-nitro-L-arginine (Reddix et al., 1998). The reduction in baseline Isc evoked by Hb was
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attenuated but not abolished in the presence of the NaK2Cl pump inhibitor bumetanide and chloride channel blocker DPC (Reddix et al., 1998). A plausible explanation for this reduction in baseline Isc is that basal NO release stimulates the uptake of Cl into the epithelial cell which is important in the maintenance of baseline chloride secretion. Removal of NO reduces the amount of chloride entering the cell thereby decreasing Isc . NO did not reduce baseline Isc by stimulating potassium secretion in this study because mucosal addition of the potassium channel inhibitors barium and tetraethyl ammonium had no effect on the Hb response (Reddix et al., 1998). However, the chloride channel blocker DPC attenuated the Hb-induced reduction in Isc (Reddix et al., 1998). This suggests that the NO response is not due to a direct effect on chloride channels in the apical membrane. Results from the present study and those previously reported suggest that the direct effect of NO on the epithelia is to stimulate an uptake of Cl 2 ions from the basolateral side in an effort to maintain basal chloride transport. The results suggest that, within guinea pig colon, constitutive NO release assists in maintaining a delicate balance in fluid and electrolyte transport. Constitutively produced NO promotes ion absorption by tonically suppressing submucosal secretomotor neural activity. This may be important in limiting fluid loss in the face of excessive stimulation of submucous secretomotor neural activity. In addition, the results support the notion that NO is involved in the regulation of transepithelial chloride secretion and mucosal permeability. In conclusion, NO may promote both a pro-absorptive and pro-secretory action on intestinal transport. The overall effect of NO may represent a balance between these effects.
Acknowledgements This research was supported by a Louisiana Education Quality Support Fund RD-A-17 (Rhoda A. Reddix, Ph.D.).
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R. A. Reddix et al. / Autonomic Neuroscience: Basic and Clinical 86 (2000) 47 – 57 submucosal glutamate receptor and nitric oxide. J. Pharm. Exp. Ther. 274 (1), 404–412. Ribbons, K.A., Thompson, J.H., Liu, X., Pennline, K., Clark, D.A., Miller, M.J., 1997. Anti-inflammatory properties of interleukin-10 administration in hapten-induced colitis. Eur. J. Pharm. 323 (2 / 3), 245–254. Sanders, K., Ward, S., 1992. Nitric oxide as a mediator of nonadrenergic, noncholinergic neurotransmission. Am. J. Phsyiol. 262, G379–G392. Sang, Q., Young, H.M., 1996. Chemical coding of neurons in the myenteric plexus and external muscle of the small and large intestine of the mouse. Cell Tissue Res. 284 (1), 39–53. Schirgi-Degen, A., Beubler, E., 1995. Significance of nitric oxide in the stimulation of intestinal fluid absorption in the rat jejunum, in vivo. Br. J. Pharmacol. 114, 13–14. Schirgi-Degen, A., Beubler, E., 1996. Involvement of K 1 channel modulation in the proabsorptive effect of nitric oxide in the rat jejunum in vivo. Eur. J. Pharm. 316 (2 / 3), 257–262. Shuttleworth, C.W., Xue, C., Ward, S.M., de Vente, J., Sanders, K.M., 1993. Immunohistochemical localization of 3959-cyclic guanosine monophosphate in the canine inhibitory neurons. Neuroscience 56 (2), 513–522. Stack, W.A., Filipowicz, B., Hawkey, C.J., 1996. Nitric oxide donating compounds stimulate human colonic ion transport in vitro. Gut 39 (1), 93–99.
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Takeuchi, K., Ohuchi, T., Miyake, H., Okabe, S., 1993. Stimulation by nitric oxide synthase inhibitors of gastric and duodenal HCO 2 3 secretion in rats. J. Pharmacol. Exp. Ther. 266 (3), 1512–1519. Tamai, H., Gaginella, T.S., 1993. Direct evidence for nitric oxide stimulation of electrolyte secretion in the rat colon. Free Radic. Res. Commun. 19, 229–239. Tumara, K., Schemann, M., Wood, J.D., 1992. Actions of nitric oxide generated sodium nitroprusside in myenteric plexus of guinea pig small intestine. Am. J. Physiol. 265, G887–G893. Wang, X.Y., Wong, W.C., Ling, E.A., 1995. Localization of NADPHdiaphorase activity in the submucous plexus of the guinea pig intestine: light and electron microscopic studies. J. Neurocytol. 24 (4), 271–281. Wilson, K.T., Vaandrager, A.B., deVente, J., Musch, M.W., De Jaye, H.R., Chang, E.B., 1996. Production and localization of cGMP and PGE 2 in nitroprusside stimulated rat colonic ion transport. Am. J. Physiol. 270 (3 Pt 1), C832–C840. Young, H.M., McConologue, K., Furness, J.B., De Vente, J., 1993. Nitric oxide targets in the guinea pig intestine identified by induction of cyclic GMP immunoreactivity. Neuroscience 55, 583–596. Yuan, S.Y., Bornstein, J.C., Furness, J.B., 1995. Pharmacological evidence that NO may be a retrograde messenger in the enteric nervous system. Br. J. Pharmacol. 114 (2), 428–432.
Constitutive nitric oxide release modulates neurally-evoked chloride secretion in guinea pig colon a, b b a a Rhoda A. Reddix *, Xiaoping Liu , Mark J.S. Miller , Xiaomei Niu , Adrianne Powell a
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA b Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA Received 30 June 1999; received in revised form 22 May 2000; accepted 23 August 2000
Abstract The role of constitutive nitric oxide (NO) release in enteric neural pathways regulating ion transport was examined in guinea pig distal colon, in vitro and ex vivo. In in vitro studies, 43% of colonic preparations exhibited oscillations in baseline short-circuit current (Isc ), which were reduced by tetrodotoxin (TTX). The NO chelator, hemoglobin (Hb), and neuronal NO synthase inhibitor, 7-nitroindazole (7-NI), significantly increased the baseline Isc in these tissues, which was reduced by TTX. In tissues without oscillations in baseline Isc , Hb reduced the Isc , while 7-NI had little effect. In all tissues, electrical field stimulation (EFS; 15 V/ 10 Hz) caused a biphasic increase in the Isc which was enhanced by both Hb and 7-NI. In the ex vivo studies, basal release of nitric oxide was significantly lower in colonic segments isolated from guinea pigs administered N v -nitro-L-arginine methyl ester ( L-NAME) i.p. compared to control tissues. Moreover, carbachol, caused a 10-fold increase in NO release in control tissues, but had no effect in tissues isolated from the L-NAME group. L-NAME increased tissue conductance and EFS-induced changes in Isc , which were reversed by L-arginine. However, carbachol-induced ion secretion was unaltered in the L-NAME group compared to control animals. The results suggest that, in guinea pig colon, constitutive enteric NO release tonically suppresses submucous neural activity and it is involved in the maintenance of basal epithelial chloride secretion and mucosal permeability. Hence, constitutive NO promotes a delicate balance between pro-absorptive and pro-secretory processes in guinea pig colon. 2000 Elsevier Science B.V. All rights reserved. Keywords: Large intestine; Submucosal plexus; Nitrogen oxide species; Ion transport
1. Introduction The colon plays an important role in fine tuning the amount of fluid and electrolytes absorbed by the intestine (Debongnie and Phillips, 1978; Kaunitz et al., 1995). The human colon may absorb as much as 6 litres of fluid in an effort to prevent excess fluid loss (Debongnie and Phillips, 1978). The absorptive capacity of the colon is influenced by changes in gut motility, blood flow, and neurocrine, endocrine and paracrine factors controlling ion transport (Debongnie and Phillips, 1978). Recent evidence suggests that nitric oxide (NO) may contribute to the absorptive capacity of the colon. This is based on the fact that NO relaxes intestinal smooth muscle (Sanders and Ward, 1992) and increases transit time (Martinez-Cuesta et al., 1997; Mizuta et al., 1999), thereby allowing more contact time *Corresponding author. Tel.: 11-504-568-4740; fax: 11-504-5682361. E-mail address: [email protected] (R.A. Reddix).
for the absorption of fluid and electrolytes across intestinal epithelia. Additionally, we recently reported that inhibition of constitutive NO production enhances endothelin-1-induced absorption in guinea pig colon (Reddix et al., 1998). It is well established that NO is a noncholinergic nonadrenergic inhibitory neurotransmitter in myenteric neurons regulating intestinal motility and causes relaxation of vascular smooth muscle within the gastrointestinal tract (Sanders and Ward, 1992; Rattan et al., 1995). A growing body of evidence from immunohistochemical and functional studies in various species suggests that NO is also important in enteric pathways regulating fluid and electrolyte transport. The nature of its role in submucosal pathways is not clear. Several reports have shown that exogenous NO released from NO donors stimulates ion secretion in rat, guinea pig, dog and human intestine (MacNaughton, 1993; Tamai and Gaginella, 1993; Rhoads et al., 1995; Stack et al., 1996; Wilson et al., 1996). Under these conditions, NO evokes ion secretion by a prostaglandin-dependent mechanism (MacNaughton, 1993;
1566-0702 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S1566-0702( 00 )00206-X
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Tamai and Gaginella, 1993; Wilson et al., 1996) and activation of enteric neurons in rat (Tamai and Gaginella, 1993) and human (Stack et al., 1996) colon. In other studies NO donors have been shown to promote ion absorption by both a direct effect on the mucosa as well as inhibition of secretagogue-induced secretion (SchirgiDegen and Beubler, 1995, 1996; Beubler and SchirgiDegen, 1997). Water and ion absorption was also increased in Thirty-Vella fistulas of canine ileum (Barry et al., 1995) in response to NO donors. Similarly, results concerning the role of constitutive NO release in enteric neural pathways regulating ion transport have been conflicting. In some studies, nitric oxide synthase inhibitors have been shown to attenuate secretagogueinduced ion secretion. Izzo et al. (1994) showed that the nitric oxide synthase inhibitor, N v -nitro-L-arginine methyl ester ( L-NAME) blunted carbachol-induced ion secretion in the small intestine of mice. Similarly, L-NAME (i.a.) attenuated 5-HT-induced ion secretion in the large intestine of anesthetized rats (Franks et al., 1994). In addition, Rhoads et al. (1995) showed an inhibition of asparagineinduced chloride secretion in pig jejunum by the NOS inhibitor, N G -monomethyl-L-arginine ( L-NMMA). On the other hand, inhibition of basal intestinal NO release increased ion secretion in other studies. The NOS inhibitors L-NAME, N v -nitro-L-arginine ( L-NA) and N v nitro-L-methyl arginine ( L-NMA) evoked an increase in baseline chloride secretion and the potential difference (PD) across segments of mouse ileum (Rao et al., 1994). L-Arginine reversed the effects of L-NMA and L-NNA, while D-arginine had no effect. Moreover, chlorisondamine and the neurotoxin, tetrodotoxin (TTX) nearly abolished the L-NMA-induced increase in Isc . A pro-absorptive role for NO was also demonstrated in ligated loops of rat jejunum (Schirgi-Degen and Beubler, 1995). In this study, pretreatment of jejunal loops with the NOS inhibitors L-NAME and L-NNA stimulated fluid secretion and enhanced Escherichia coli Sta-induced fluid secretion. Subsequently, the authors showed that the pro-absorptive effects of NO was partially mediated by maturation of K 1 channels (Schirgi-Degen and Beubler, 1996). The effects of L-NAME and L-NA were reversed by sodium nitroprusside and L-arginine. Takeuchi et al. (1993) showed that constitutively produced NO evokes ion absorption in the stomach and ileum. Inhibition of constitutive NO release caused an increase in bicarbonate secretion from rat gastric and duodenal mucosa (Takeuchi et al., 1993). Although contradictory, these results suggest that NO is involved in the control of ion transport across the small intestine of various species; however, the nature of its role remains to be determined. The small and large intestine are functionally distinct segments (Kaunitz et al., 1995). Differences in NOS distribution and the percentage of NOS-immunoreactive neurons between these two regions have been reported (Furness et al., 1994; Wang et al., 1995; Sang and Young, 1996). In guinea pig colon, NOS-
immunoreactivity has been identified in the myenteric plexus and submucous neurons projecting to circular muscles and muscularis mucosae and it is localized within interneurons (Furness et al., 1994). It was recently shown that, in guinea pig intestine, the density of NOS-positive neurons is greater in the large intestine than in the small intestine. Previous studies employing NOS inhibitors have focused primarily on the small intestine (Wang et al., 1995). Although there is a growing body of evidence demonstrating constitutive NOS-IR in enteric pathways regulating ion transport in the large intestine of various species, very little is known concerning the role of constitutive NO release in these pathways. Therefore, the focus of the present study was to examine the effect of constitutive NO release on ion transport in guinea pig colon, in vitro and ex vivo. We hypothesize that, in addition to its effects on smooth muscle, constitutive NO release may tonically inhibit submucosal secretomotor neurons in guinea pig colon. To test this hypothesis, experiments are designed to determine the effect of inhibiting constitutive NOS, in vitro and ex vivo, on baseline and electrical field stimulation (EFS)induced changes in the short-circuit current (Isc ) of guinea pig colon.
2. Methods
2.1. Tissue preparation Male albino guinea pigs (Harlan Sprague-Dawley, Indianapolis, IN, USA) weighing 350–500 g were housed in metal cages with food and water ad libitum. The animals were stunned and exanguinated. This method of euthanasia has been approved by the LSU Institutional Animal Care Committee and complies with federal regulations. Segments of distal colon were removed and opened along the mesenteric border. The intraluminal contents were removed and the segments were pinned with the mucosal side down onto a Sylgard-coated Petri dish. Tissues were perfused with a chilled Krebs–Ringer solution (in mM): 120 sodium chloride, 6 potassium chloride, 1.2 magnesium chloride, 6 H 2 O, 1.3 sodium phosphate, 14.4 sodium bicarbonate, 2.5 calcium chloride, 12.5 glucose. The longitudinal and circular muscle layers along with the myenteric plexus were removed by blunt dissection leaving the submucosa / mucosa intact.
2.2. Short-circuit current ( Isc ) measurements Colonic segments were divided into 2–3 cm sheets and mounted in Ussing flux chambers with an area of 0.785 cm 2 . Three to four adjacent sheets were obtained from the most distal portion of the colon from each animal. The baseline Isc values for all tissues were similar. Additionally, tissues were subject to submaximal stimulation (15
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V/ 10 Hz). The EFS-induced ion transport was of similar magnitude in tissues with and without on-going activity. Peak 1: (1) on-going, 321.1627.8 mA / cm 2 , n 5 23 and (2) on-going, 318.1622.7 mA / cm 2 , n 5 24; peak 2: (1) on-going, 307.2637.8 mA / cm 2 , n 5 24 and (2) on-going, 270.7624.2 mA / cm 2 , n 5 24. Tissues were bathed in a Krebs–Ringer solution maintained at 378C and aerated with 95% O 2 and 5% CO 2 . The chambers were designed with ports for Ringer-agar bridges and calomel half-cells for measurement of transmural potential difference (PD). Throughout the experiment, tissues were short circuited with a voltage clamp apparatus (Physiological Instruments, CA, USA) to abolish changes in PD. The short-circuit current (Isc ) was measured in mA and normalized to the tissue surface area (cm 2 ). Prior to the experimental session, all tissues were paired according to baseline Isc , Gt and PD. Any changes in the Isc during either electrical or chemical stimulation of tissues are due to alterations in active ion transport. Forty-three percent of all tissues exhibited on-going neural activity characterized by oscillations in baseline Isc . These oscillations are mediated by active cholinergic secretomotor neurons (Reddix et al., 1994). For tissues with (1) on-going neural activity, the baseline Isc was calculated as the mean value.
2.3. Electrical field stimulation ( EFS) Tissues were equilibrated for 40 min and then electrically stimulated for 90 s by a pair of aluminum foil electrodes placed adjacent to the serosal surface. The electrodes were connected to a Grass SD 88 stimulator (Grass Instruments, Quincy, MA, USA) which generated pulses at 0.5 ms, with a strength of 15 V (3A) and a frequency of 10 Hz. Changes in Isc were continuously monitored by a Kipp and Zonen 2 chart recorder. Measurements of Isc (mA / cm ) were calculated as the difference between the peak Isc and baseline Isc before stimulation (Isc peak 2 Isc baseline ). There was no significant difference in the EFS-induced secretory response between tissues with or without on-going neural activity. In a separate set of tissues, we examined the effect of chloride-free buffer consisting of (in mM): 120 sodium gluconate, 6 potassium gluconate, 1.2 magnesium gluconate, 2.5 calcium gluconate, 14.4 sodium bicarbonate, 12.5 glucose and 1.3 sodium phosphate on EFS-induced secretion. The chloride-free solution was replaced in both the mucosal and serosal compartments.
2.4. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on baseline short-circuit current ( Isc ) measurements Changes in Isc were monitored in colonic segments exposed to vehicle or the following agents: Hb (2.3 mM), an NO scavenger and the NOS inhibitors, N v -nitro-Larginine methyl ester ( L-NAME; 0.1 mM), and 7-NI (0.1 mM). These agents were added to both mucosal and
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serosal compartments. The concentration of each drug used in this study is based on previously published results (Bland-Ward and Moore, 1995; Rattan et al., 1995; Miller et al., 1996). In a separate set of experiments, the effect of Hb (2.3 mM) and 7-NI (0.1 mM) on Isc in tissues with on-going neural activity was examined. To determine whether the Hb- and 7-NI-induced secretory responses were partially mediated by activation of secretomotor neurons, we examined the effect of the neurotoxin, tetrodotoxin (TTX; 0.2 mM) on peak secretory effects of Hb and 7-NI.
2.5. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on the electrical field stimulation ( EFS)-induced secretion To further examine the role of submucous secretomotor neurons, we investigated the effect of NO inhibitors on EFS-induced ion secretion. All tissues were electrically stimulated 10 min prior to addition of vehicle, Hb (2.3 mM) or 7-NI (0.1 mM) to the bathing solution. Another EFS response was measured 10 min following the vehicle, Hb or 7-NI treatment. The effects of vehicle, Hb and 7-NI on the EFS response were examined during the same experimental period. The vehicle had no effect on baseline Isc and EFS-induced ion secretion. Drug-induced changes in the EFS response were calculated as a percent of the original EFS-induced secretory response. These changes were compared to EFS responses obtained in vehicletreated tissues.
2.6. Effect of N v -nitro-L-arginine methyl ester ( L-NAME) in vivo on short-circuit current ( Isc ) measurements ex vivo Albino male guinea pigs weighing 300–350 g were administered saline, L-NAME (50 mg / kg), L-NAME (50 mg / kg)1 L-ARG (100 mg / kg) or L-NAME (50 mg / kg)1 L-ARG (200 mg / kg), intraperitoneally. Muscle-stripped colonic segments were prepared as previously described. This protocol has been shown to significantly inhibit constitutive NOS in vivo (Dwyer et al., 1991).
2.7. Measurements of nitric oxide release To confirm NOS inhibition, NO release from whole thickness colonic segments isolated from the saline and L-NAME groups were measured using an NO sensor developed by Dr. Xiaoping Liu (Ribbons et al., 1997). Nitric oxide synthase has been previously identified in the mucosal epithelia, interneurons of the submucous plexus, muscularis mucosae, myenteric plexus and smooth muscle of guinea pig intestine (Furness et al., 1994). In addition, we have previously reported that hemoglobin, chelates
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nitric oxide, significantly reduced the baseline Isc in isolated muscle-stripped guinea pig colon (Reddix et al., 1998). Based on these results, nitric oxide measurements were obtained from the mucosal surface. Moreover, NO measurements were performed to confirm effective blockade in vivo of intestinal NO production. Briefly, 2–3 cm whole thickness colonic segments isolated from the saline and L-NAME groups were pinned mucosal side up onto a sylgard-coated Petri dishes. Tissues were bathed in warmed (378C) RPMI-1640 culture medium (NIF Technologies) containing vehicle or the acetylcholine agonist carbachol (10 mM). The Petri dish was transferred to the NO detection apparatus. The concentration of NO released from the tissues was detected by an electrochemical measurement system consisting of three electrodes, a platinum disc microelectrode coated with nafion membrane (working electrode), a reference electrode and an auxillary electrode, respectively. The electrodes were connected to a BAS 100B Electrochemical Analyzer with a PA-1 PreAmplifier and C2 cell stand. To measure NO, the tip of the working electrode was placed at the surface of the bathing solution to establish a baseline. Since the focus of the this study was to assess the effect of mucosal NO production on ion transport, the microelectrode tip was lowered to the mucosal surface. Changes in current (nA) passing through the working electrode was used as an index of the NO concentration. Changes in NO levels were calculated as the difference between current (nA) at the solution surface I0 and tissue level (Itissue ). This value was converted to mM from a calibration with nitric oxide gas solution. This process was repeated three to four times at different areas on the tissue. The values were averaged for each tissue.
2.8. Effect of NOS inhibition on the EFS response ex vivo Baseline and EFS-induced changes in Isc were recorded in tissues isolated from each group. An earlier report suggested that L-NAME treatment in vivo degranulates mast cells in the small bowel of rats (Kanwar et al., 1994). Mast cells are located within the wall of the gastrointestinal tract (Kanwar et al., 1994; Gaboury et al., 1996). Histamine is a prominent transmitter released from mast cells and has been shown to be a potent secretagogue (Cooke and Reddix, 1994; Frieling et al., 1994). Therefore, one set of control experiments was designed to determine whether histamine mediated baseline changes in Isc observed in colonic segments of L-NAME-treated guinea pigs. Colonic segments isolated from the saline or LNAME treatment groups were exposed to the histamine H 1 and H 2 receptor antagonists, 10 mM pyrilamine (PYR) and 10 mM cimetidine (CIM), 10 min following the initial EFS response. Both cimetidine and pyrilamine attenuated histamine (10 mM)-induced secretion (data not shown).
Another EFS response was monitored 10 min after drug treatment.
2.9. Effect of NOS inhibition on carbachol-induced secretion ex vivo Since there is a relatively high density of muscarinic receptors on epithelial cells throughout the gastrointestinal tract, stimulation of these receptors served as a positive control to assess tissue viability and responsiveness. At the end of all experiments, the muscarinic agonist carbachol (0.1 mM) was added to the serosal compartment and changes in Isc were recorded in colonic segments isolated from each treatment group.
2.10. Effect of NOS inhibition on tissue conductance ( Gt ), ex vivo Previous reports have shown that L-NAME treatment in vivo increases epithelial permeability (Kanwar et al., 1994; Alican and Kubes, 1996). To examine the possibility that changes in baseline Isc are due to alterations in mucosal permeability, tissue conductance was monitored in colonic segments isolated from all treatment groups. Tissue conductance gives an assessment of magnitude changes in the movement of ions across colonic segments. As tissue conductance increases, tissue resistance to ionic flux decreases, indicating an increase in mucosal permeability to these ions. The tissue conductance may increase without alterations in the Isc if due to electroneutral ionic fluxes.
3. Materials and chemicals The following chemicals were purchased from Sigma (St. Louis, MO, USA): hemoglobin, N v -nitro-L-arginine methyl ester ( L-NAME), L-arginine, carbachol, 7-nitroindazole, tetrodotoxin, cimetidine and pyrilamine. All drugs were dissolved in Krebs–Ringer solution except the following: cimetidine was dissolved in 1 M HCl (0.001%)1Krebs–Ringer; 7-nitroindazole was dissolved in dimethyl sulfoxide (DMSO, 0.001%)1Krebs–Ringer; RPMI-1640 culture medium (NIF Technologies).
4. Statistics All data are expressed as mean6S.E.M. An unpaired Student’s t-test was used to test the significance between group means. A probability value less than 0.05 was considered statistically significant.
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5. Results
5.1. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on baseline short-circuit current ( Isc ) measurements Forty-three percent of muscle-stripped colonic segments exhibited on-going neural activity. There was no significant difference in the baseline Isc in tissues with and without on-going neural activity: (2) on-going activity: 40.364.9 mA / cm 2 (9) and (1) on-going activity 35.4613.0 mA / cm 2 (9). However, tissue conductance was significantly greater in tissues with on-going neural activity: (2) on-going activity 12.065.4 mA / cm 2 (12) and (1) on-going activity 20.462 mA / cm 2 ; n 5 10, *P , 0.05. In colonic segments we observed a consistent, but variable increase in ion secretion in response to Hb and 7-NI. The NO scavenger Hb (2.3 mM) increased baseline Isc in tissues with on-going neural activity (Fig. 1A). The vehicle had no effect on baseline Isc . The peak Hb-induced secretory response was attenuated by 0.2 mM TTX (Fig. 1A). Conversely, in tissues without on-going activity, 2.3 mM Hb caused a significant reduction in baseline Isc (Fig. 1B). Overall, both hemoglobin and 7-NI consistently increased the baseline Isc in tissues with on-going neural activity. However, we observed variability in the magnitude of the secretory responses to these agents. The mean changes in baseline Isc evoked by 2.3 mM Hb and 7-NI (0.1 mM) in tissues with and without on-going neural activity were Hb, (1) on-going 55.6623.3 (8) and (2) on-going 210.364.4 mA / cm 2 (11); and 7-NI, (1) ongoing 28.066.4 mA / cm 2 (5) and (2) on-going 0.0 mA /
Fig. 2. Representative tracings illustrating baseline changes in the shortcircuit current (Isc ; mA / cm 2 ) in muscle-stripped segments of guinea pig colon. Tissues with (1) on-going neural activity (superimposed oscillations) exposed to: (A) nitric oxide scavenger, hemoglobin (Hb; 2.3 mM), followed by the neurotoxin tetrodotoxin (TTX; 0.2 mM). Hb increased the Isc 208 mA / cm 2 ; TTX reduced the Hb-induced increase in Isc by 152 mA / cm 2 . (B) Tissues without (2) on-going neural activity exposed to Hb (2.3 mM) alone.
cm 2 (5). The NO synthase inhibitor 7-NI (0.1 mM) increased baseline Isc in tissues with on-going neural activity (Fig. 2A). The vehicle had no effect on baseline Isc . The peak 7-NI-induced secretory response was reduced by 0.2 mM TTX (Fig. 2A). 7-NI, 24.862.1 mA / cm 2 ,
Fig. 1. Representative tracings illustrating baseline changes in the short-circuit current (Isc ; mA / cm 2 ) in muscle-stripped segments of guinea pig colon. Tissues with on-going neural activity (superimposed oscillations) were exposed to: (A) the nitric oxide synthase inhibitor 7-nitroindazole (7-NI; 0.1 mM), followed by the neurotoxin tetrodotoxin (TTX; 0.2 mM); 7-NI increased the Isc by 51 mA / cm 2 ; TTX reduced the 7-NI-induced increase in Isc by 12.5 mA / cm 2 . (B) Tissues without on-going neural activity exposed to 0.1 mM 7-NI; 7-NI had no effect on the Isc .
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n 5 4; 7-NI1TTX (0.2 mM), 21.367.4 mA / cm 2 , n 5 4. 7-NI had no effect on baseline Isc in tissues without on-going neural activity (Fig. 2B). Although 7-NI and Hb evoked changes in the baseline Isc of guinea pig colonic segments, addition of the nitric oxide synthase inhibitor N v -nitro-L-arginine methyl ester (1 mM L-NAME) to both mucosal and serosal compartments had no effect in vitro (data not shown).
5.2. Effect of 7 -nitroindazole (7 -NI) and hemoglobin ( Hb) on electrical field stimulation ( EFS)-induced secretion To assess changes in EFS-induced secretion in control tissues over the same time period as drug treatment, an initial EFS response (EFS-1) was recorded in colonic segments followed by a second EFS response (EFS-2) measured 20 min later. Tissues were exposed to vehicle or test drug 10 min prior to EFS-2. The EFS (15 V/ 10 Hz) evoked a biphasic increase in baseline Isc (peak 1, PK1 and peak 2, PK2; Fig. 3). Peak 1 was complete within 30 s followed by a second peak (PK2) within 1 min of the PK1 response. It must be noted that peak 2 of the EFS response is relatively the same magnitude during the 30 min experimental period. It was previously shown that, in guinea pig colon, EFS-induced secretion is primarily due to an increase in electrogenic chloride secretion (MartinezCuesta et al., 1997). Similarly, our results showed that both peaks 1 and 2 were abolished in tissues exposed to chloride-free solution (Fig. 3). Moreover, there was no significant difference in both peaks of the EFS response in
Fig. 3. Electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in the short-circuit current (Isc ; mA / cm 2 ) in muscle-stripped segments of guinea pig colon bathed in normal Krebs–Ringer solution (h), or chloride-free Krebs–Ringer solution (replaced Cl 2 with gluconate) (9). Short-circuit current measurements in response to EFS were obtained at time zero, EFS-1. The tissues were allowed to recover for 20 min and then a second EFS-induced Isc response was recorded (EFS-2). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1). Peak 1 was completed within 30 s followed by a second peak (PK2) within 1 min of PK1. **Significant from control peak 2 (PK2); P , 0.05 was considered significant.
Fig. 4. Effect of 7-nitroindazole (7-NI; 0.1 mM) on electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in Isc in colonic segments. Results are reported as a percent of EFS-1 (EFS-2 / EFS-13 100%). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1). P , 0.05 was considered statistically significant.
tissues with (peak 1, 321.1627.8 mA / cm 2 , n 5 23; peak 2, 307.2637.8 mA / cm 2 , n 5 23) or without (peak 1, 318.1622.7 mA / cm 2 , n 5 24; peak 2, 270.7624.2 mA / cm 2 , n 5 24) (1) on-going neural activity. For subsequent studies, drug-induced changes in the EFS response were expressed as a percent of the initial EFS response (EFS-2 / EFS-13100). Both peaks 1 and 2 of the EFS response were significantly greater in tissues pretreated with the NOS inhibitor 7-NI (Fig. 4) or nitric oxide scavenger Hb (Fig. 5) compared to vehicle-treated tissues. Addition of L-NAME to the bathing solution of both mucosal and serosal compartments had no effect on EFSinduced changes in Isc (Fig. 5). A possible explanation lies in the fact that in vitro preparations of muscle-stripped colonic segments may not have the esterases required to activate L-NAME.
Fig. 5. Effect of the nitric oxide scavenger hemoglobin (Hb; 2.3 mM) on electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in Isc in colonic segments exposed to vehicle (Veh) or the nitric oxide synthase inhibitor N v -nitro-L-arginine methyl ester ( L-NAME; 1 mM). Results are reported as a percent of EFS-1 (EFS-2 / EFS-13100%). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1) or **significant from control peak 2 (PK2). P , 0.05 was considered statistically significant.
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5.3. Effect of N v -nitro-L-arginine methyl ester ( L-NAME) in vivo upon short-circuit current measurements ex vivo To determine whether results from in vitro studies were similar to those obtained ex vivo, electrical measurements were recorded in colonic segments isolated from guinea pigs administered saline, L-NAME (50 mg / kg), LNAME1 L-ARG (100 mg / kg) and L-NAME1 L-ARG (200 mg / kg), intraperitoneally, i.p. The animals were sacrificed 3 h post-injection. Baseline colonic NO release was lower in L-NAME-treated animals compared to the saline group
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Table 1 Effect of basal nitric oxide inhibition in vivo a Treatment
Baseline Isc (mA / cm 2 )
Conductance (Gt ; mS / cm 2 )
Saline (n 5 22) L-NAME (50 mg / kg; n 5 22) L-NAME1 L-ARG (100 mg / kg; n 5 14) L-NAME1 L-ARG (200 mg / kg; n517)
57.369.4
12.364.0
77.0614.9
20.563.3*
47.768.2
22.263.8*
39.9614.6
11.661.3
a
Baseline short-circuit current (Isc ) and tissue conductance (Gt ) values in muscle-stripped segments of guinea pig colon. Tissues were isolated from guinea pigs administered saline, the nitric oxide synthase inhibitor N v nitro-L-arginine methyl ester ( L-NAME; 50 mg / kg), a combination of L-NAME1 L-arginine ( L-ARG, NO precursor; 100 mg / kg) or L-NAME1 LARG (200 mg / kg). All drugs were administered intraperitoneally, i.p., and the animals were sacrificed 3 h post-injection; n 5 14–22. *P , 0.05, significant from the saline group.
(Fig. 6). The nonselective cholinergic receptor agonist carbachol (10 mM) caused a 10-fold increase in colonic NO release in the saline group; however, it had no effect in animals administered L-NAME. Table 1 summarizes the baseline Isc and Gt in all groups. There was no significant difference in baseline Isc in muscle-stripped colonic segments isolated from animals in each group. However, Gt was significantly higher in the L-NAME group and this effect was reversed in animals administered L-NAME1 L-ARG (200 mg / kg). In Fig. 7, peaks 1 and 2 of the EFS chloride secretory response were significantly elevated in the L-NAME group compared to the saline group. L-Arginine reversed the effects of L-NAME on both peaks of the EFS secretory response at 100 and 200 mg / kg.
Fig. 6. (A) Representative tracings of NO measurements (mM) in whole thickness colonic segments isolated from guinea pigs administered saline or N v -nitro-L-arginine methyl ester ( L-NAME, 50 mg / kg) intraperitoneally. All tissues were pinned mucosal side up onto sylgard-coated Petri dishes and bathed in 15 ml RPMI-1640 medium containing vehicle or 10 mM carbachol. Changes in NO current (nA) were detected by a platinum microelectrode (potential held at 0.55 V vs. a Ag /AgCl electrode) at the solution surface, I0 , and tissue level, Itiss . All measurements were repeated at three different sites for each tissue. The concentration of NO in the bathing solution was calculated as the difference between I0 and Itiss ; this value was converted to mM. (B) Mean NO levels (mM) released from whole thickness colonic segments isolated from saline or L-NAMEtreated animals. Tissues were bathed in RPMI-1640 medium containing vehicle or the muscarinic receptor agonist carbachol (10 mM). Values are expressed as the mean 6S.E.M., n 5 4. *Significance from the saline vehicle. P , 0.05 was considered statistically significant.
Fig. 7. Electrical field stimulation (EFS; 15 V/ 10 Hz)-induced changes in the short-circuit current (Isc ; mA / cm 2 ) across colonic segments isolated from guinea pigs administered saline, N v -nitro-L-arginine methyl ester ( L-NAME; 50 mg / kg), L-NAME1 L-arginine ( L-ARG; 100 mg / kg) or L-NAME1 L-ARG (200 mg / kg), intraperitoneally, i.p. Results are reported as DIsc (mA / cm 2 ). Values are expressed as the mean 6S.E.M., n 5 8–10. *Significant from control peak 1 (PK1) or **significant from control peak 2 (PK2). P , 0.05 was considered statistically significant.
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Fig. 8. Carbachol (CARB; 0.1 mM)-induced changes in the short-circuit current (Isc ; mA / cm 2 ) across colonic segments isolated from guinea pigs administered saline, N v -nitro-L-arginine methyl ester ( L-NAME; 50 mg / kg) or L-NAME (50 mg / kg)1 L-arginine ( L-ARG; 100 mg / kg), intraperitoneally. Results are reported as DIsc (mA / cm 2 ). Values are expressed as the mean6S.E.M., n 5 9–10.
It has been suggested that L-NAME in vivo promotes degranulation of mast cells with the subsequent release of histamine, a potent secretagogue. The histamine receptor antagonists cimetidine (H 2 ; 10 mM) and pyrilamine (H 1 ; 10 mM) blocked histamine-induced secretion but had no effect on the EFS response in colonic segments isolated from guinea pigs in the L-NAME group (data not shown). The nonselective cholinergic receptor agonist carbachol (0.1 mM) was added to the serosal compartment at the end of all experiments to assess the viability of each tissue preparation. Carbachol (0.1 mM) caused an increase in Isc of similar magnitude in colonic segments isolated from control and L-NAME groups. However, it was significantly reduced in animals co-administered L-NAME and L-ARG (Fig. 8).
6. Discussion The purpose of the current study was to investigate the physiological role of constitutive nitric oxide in enteric pathways regulating ion transport in guinea pig colon. The effect of nitric oxide inhibitors on ion transport across colonic epithelia was examined in vitro and ex vivo. Results from the current study indicate that the role of constitutive NO release in the regulation of ion transport across colonic epithelia is multifaceted. A large component of the NO effect involves tonic inhibition of submucous neural activity. In addition, NO plays a role in the maintenance of epithelial transport and permeability. Evidence supporting an inhibitory effect of basal NO on submucous neurons comes from the fact that sequestration of NO by Hb or inhibition of NOS by 7-NI in vitro increased baseline Isc only in muscle-stripped colonic segments with on-going neural activity (Figs. 1 and 2). The increase in Isc by Hb was two-fold greater than that by
7-NI. This may be due to the fact that 7-NI inhibits neuronal NOS which has been identified primarily in interneurons of guinea pig colon (Furness et al., 1994). Hence, the small increase reflects removal of the inhibitory effect of constitutive NO on secretomotor neurons. On the other hand, the nerve terminals of secretomotor neurons are exposed to NO released from several sources in the mucosal and sub-epithelial regions, i.e. muscularis mucosae, sub-epithelial fibroblasts and epithelial cells. The NO released has an inhibitory effect on the release of secretagogues from secretomotor neurons. Hence, Hb scavenges the NO, removing this inhibitory influence which enhances the release of neurotransmitters at the neuro-epithelial junction resulting in an increase in secretion. The on-going neural activity observed in colonic segments is mediated primarily by active cholinergic secretomotor neurons (Reddix et al., 1994). The neurotoxin tetrodotoxin attenuated the peak secretory effects of 7-NI and Hb on baseline Isc in these tissues (Figs. 1A and 2A). A similar observation was reported in mouse small intestine. Rao et al. (1994) showed an increase in baseline chloride secretion following nitric oxide inhibition in mouse ileal segments that was TTX-sensitive. Based on these results, it may be concluded that constitutive NO tonically suppresses submucous neural activity. However, additional studies are required to delineate the mechanisms involved. To further examine the effect of constitutive NO release on neurally evoked secretion, colonic segments were electrically stimulated to depolarize the submucous neurons within our preparation. Electrical field stimulation induced a biphasic change in Isc mediated by an increase in electrogenic chloride secretion (Fig. 3). Both peaks 1 and 2 of the EFS response were greater in tissues pretreated with Hb or 7-NI (Figs. 4 and 5) compared to control. This result provides additional evidence supporting the notion that NO tonically inhibits submucosal neural activity. Furthermore, we previously reported that, in guinea pig colon, the neurokinin 3 agonist [Pro 7 ]neurokinin-B caused an increase in ion secretion that was TTX-sensitive (Reddix and Niu, 1997). The NK3-induced secretory response was enhanced at a low concentration in tissues pretreated with the NO scavenger Hb; however, it was unaltered at a high concentration. One possible explanation is that tonic NO release modulates neurally-evoked secretion by altering neurotransmitter release. Additional studies are required to confirm this observation. To confirm our results from in vitro studies employing NO inhibitors, we examined the effect of inhibiting NOS in vivo on neurally-evoked secretion in isolated colonic segments. For this series of experiments, guinea pigs were administered saline, L-NAME (50 mg / kg), L-NAME1 LARG (100 mg / kg) or L-NAME1 L-ARG (200 mg / kg), intraperitoneally. Basal and carbachol-stimulated NO release were measured in colonic segments isolated from the saline and L-NAME-treated animals to verify NOS inhibi-
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tion. In the saline group, carbachol caused a 10-fold increase in colonic release of NO which was abolished in the L-NAME group (Fig. 7). Moreover, the concentration of NO released in response to carbachol exceeded physiological levels reported in the literature (Archer et al., 1995). This may be due to the fact that prolonged exposure of carbachol stimulates an increase in intracellular calcium that maximally activates constitutive NOS enzymes to produce NO levels similar to inducible NOS. In addition, NO is derived from several cellular sources in our preparation which are pooled to yield higher levels. It is also interesting to note that although carbachol did not increase NO levels in the L-NAME-treated animals, it caused an increase in chloride secretion in this group that was of similar magnitude in the saline group (Fig. 8). In our preparation, carbachol evokes a TTX-insensitive secretory response. Hence, carbachol-induced ion secretion in colonic epithelia is not dependent on the release of NO. In the current study, results from ex vivo experiments also showed an enhanced neurally evoked chloride secretion in colonic segments isolated from guinea pigs administered L-NAME, i.p. (Fig. 7). This response was blunted with simultaneous administration of L-arginine (Table 1). Similar results were obtained by Izzo et al. (1994), who showed an inhibitory effect of constitutive NO and the NO donor sodium nitrite on neurally-mediated ion secretion in mouse ileum. In addition, Rao et al. (1994) showed an inhibitory influence of tonic NO release on secretomotor neurons within mouse ileum. In the present study, increased tissue conductance observed with LNAME treatment was reversed by a high concentration of L-arginine. Additional evidence supporting an inhibitory influence of NO on enteric neurons was reported by Tumara et al. (1992). Their results from electrophysiological studies showed that the NO donor sodium nitroprusside (SNP) had no effect on basal electrical behavior of myenteric neurons, however it attenuated noncholinergic slow excitatory postsynaptic potentials (EPSPs). Hence, NO may have a presynaptic inhibitory effect on neurotransmitter release (Rhoads et al., 1995; Yuan et al., 1995). However, the effect of NO on the electrical properties of submucosal neurons is not known. Shuttleworth et al. (1993) showed that NO stimulated an increase in intracellular cGMP levels within myenteric neurons which was associated with an inhibition of the neural activity. In another study, the NO donor sodium nitroprusside (SNP) also increased cGMP levels within myenteric neurons but to a much greater extent in submucosal neurons of guinea pig intestine (Young et al., 1993). Hence, it seems possible that NO may inhibit submucosal neural activity via a cGMP-dependent mechanism. In contrast to our results and those of others demonstrating an inhibitory effect of constitutively produced NO on submucosal neurons, it has been shown that NO
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released from NO donors stimulates electrogenic chloride secretion in rat (Wilson et al., 1996) and human (Stack et al., 1996) colon that is TTX-sensitive. MacNaughton (1993) also demonstrated a stimulatory effect of exogenous NO on chloride secretion in guinea pig intestine. The authors examined the effect of SNP and isosorbide dinitrate (ISDN) on guinea pig duodenum, jejunum, ileum, proximal and distal colon. SNP and ISDN evoked a dosedependent increase in Isc that was mediated in part by PGE 2 and 5-HT. However, TTX had no effect on the SNP-induced secretory response in their preparations. This is in contrast to the rat and human colon, in which NO stimulated submucosal secretomotor nerves. Another possible explanation for the enhanced neural response in the presence of L-NAME, in vivo, is based on the results of Kanwar et al. (1994), who reported that L-NAME, i.a., increases histamine release from enteric mast cells. Mast cells are present in the gastrointestinal tract (Handlinger and Rothwell, 1984; Kanwar et al., 1994; Gaboury et al., 1996). It has been shown that mast cell mediators such as histamine or platelet activating factor (PAF) increase epithelial cell permeability. Since it is well established that histamine is a potent secretagogue (Cooke and Reddix, 1994; Frieling et al., 1994), we examined the effect of a combination of the histamine receptor antagonists pyrilamine (PYR; H 1 ) and cimetidine (CIMET; H 2 ) on EFS-induced changes in Isc in tissues isolated from the L-NAME group. CIMET and PYR had no effect on baseline Isc or the EFS response (data not shown), suggesting that the enhanced EFS response in L-NAMEtreated animals is not mediated by histamine. It has previously been suggested that constitutively produced NO is important for the maintenance of mucosal integrity (Kanwar et al., 1994; Alican and Kubes, 1996), although it is not clear whether NO modulates epithelial permeability via a direct effect on the epithelium or via the release of intermediate factors. Moreover, it has been demonstrated that L-NAME, i.a., increased 51 Cr-EDTA clearance (estimate of mucosal permeability) across feline small intestine (Kanwar et al., 1994). Therefore, we monitored changes in tissue conductance in all groups. The conductance was significantly greater in the L-NAME animals. L-Arginine (200 mg / kg) reversed the effects of L-NAME on tissue conductance. It is also of interest to note that 100 mg / kg L-arginine reversed the effects of L-NAME on the neural response, but had no effect on Gt . This suggests that changes in the neurally-induced chloride secretory response were not due to an increase in epithelial permeability. Additional evidence supporting a direct effect of NO on epithelial function is based on the observation that Hb reduced baseline Isc and 7-NI had no effect in tissues without on-going activity. Furthermore, we previously showed a reduction in baseline Isc in TTX-treated colonic segments exposed to Hb and N-nitro-L-arginine (Reddix et al., 1998). The reduction in baseline Isc evoked by Hb was
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attenuated but not abolished in the presence of the NaK2Cl pump inhibitor bumetanide and chloride channel blocker DPC (Reddix et al., 1998). A plausible explanation for this reduction in baseline Isc is that basal NO release stimulates the uptake of Cl into the epithelial cell which is important in the maintenance of baseline chloride secretion. Removal of NO reduces the amount of chloride entering the cell thereby decreasing Isc . NO did not reduce baseline Isc by stimulating potassium secretion in this study because mucosal addition of the potassium channel inhibitors barium and tetraethyl ammonium had no effect on the Hb response (Reddix et al., 1998). However, the chloride channel blocker DPC attenuated the Hb-induced reduction in Isc (Reddix et al., 1998). This suggests that the NO response is not due to a direct effect on chloride channels in the apical membrane. Results from the present study and those previously reported suggest that the direct effect of NO on the epithelia is to stimulate an uptake of Cl 2 ions from the basolateral side in an effort to maintain basal chloride transport. The results suggest that, within guinea pig colon, constitutive NO release assists in maintaining a delicate balance in fluid and electrolyte transport. Constitutively produced NO promotes ion absorption by tonically suppressing submucosal secretomotor neural activity. This may be important in limiting fluid loss in the face of excessive stimulation of submucous secretomotor neural activity. In addition, the results support the notion that NO is involved in the regulation of transepithelial chloride secretion and mucosal permeability. In conclusion, NO may promote both a pro-absorptive and pro-secretory action on intestinal transport. The overall effect of NO may represent a balance between these effects.
Acknowledgements This research was supported by a Louisiana Education Quality Support Fund RD-A-17 (Rhoda A. Reddix, Ph.D.).
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