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L-citrulline recycling in opossum internal anal sphincter relaxation by nonadrenergic, noncholinergic nerve stimulation
GASTROENTEROLOGY 1997;112:1250–1259
L-Citrulline Recycling in Opossum Internal Anal Sphincter
Relaxation by Nonadrenergic, Noncholinergic Nerve Stimulation SATISH RATTAN and SUSHANTA CHAKDER Division of Gastroenterology/Hepatology, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania
Background & Aims: L-citrulline formed stoichiometrically along with nitric oxide (1:1) from L-arginine may be enzymatically converted to L-arginine. The possibility of L-citrulline recycling in the maintenance of nitrergic neurotransmission in the opossum internal anal sphincter (IAS) smooth muscle strips was investigated. Methods: Responses to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) (either short-train or continuous stimulation) on the basal IAS tension were recorded before and after the NO synthase inhibitor N v-nitro-L-arginine (L-NNA), LNNA plus L-citrulline, or L-arginine. During continuous EFS, when the basal IAS tone after the initial relaxation had recovered to almost pre-EFS levels, the effects of L-citrulline or L-arginine were examined before and after L-glutamine, which is a putative blocker of L-citrulline uptake. Results: Inhibition of NANC nerve–mediated IAS relaxation by L-NNA was reversed by L-citrulline as well as L-arginine. L-Citrulline and L-arginine caused concentration-dependent relaxation of the IAS tone recovered during the prolonged EFS. L-Glutamine blocked the responses of L-citrulline but not of L-arginine. Furthermore, L-glutamine increased the speed of recovery of IAS tone during continuous EFS. Conclusions: L-citrulline recycling may be responsible for the maintenance of IAS relaxation during frequent short-train and prolonged NANC nerve stimulation.
T
he internal anal sphincter (IAS) relaxation in response to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) may involve both vasoactive intestinal polypeptide1,2 and nitric oxide.3 – 5 It has been shown that a major component of the IAS relaxation by EFS is mediated via the NO synthase (NOS) pathway.3 – 5 Furthermore, the IAS relaxation either by short-train or continuous EFS can be maintained for a long time. Interestingly, for prolonged continuous EFS, the IAS relaxation is characterized by a gradual fade and return of the tension toward the prestimulus level. The exact mechanism for the mainte/ 5e1b$$0027
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nance of the IAS relaxation and fade in the IAS smooth muscle relaxation during NANC nerve stimulation is not known. In a number of neuronal and nonneuronal cells, NO and L-citrulline are formed stoichiometrically (1:1) from the precursor L-arginine.6 – 8 NO serves as an inhibitory neurotransmitter, whereas L-citrulline has been regarded as a byproduct of the reaction. However, recent studies suggest that L-citrulline thus formed may be converted back to L-arginine.9 – 13 This occurs by the action of two enzymes: argininosuccinate synthetase (AS) and argininosuccinate lyase (AL).14,15 This scheme has considerable potential to support the NANC nerve–mediated relaxation of the gastrointestinal smooth muscle. Yet, there is limited information on the role of L-citrulline recycling in the gastrointestinal smooth muscle relaxation. To date, the role of L-citrulline recycling in the NANC nerve– mediated gut smooth muscle relaxation has only been reported in the canine colon.16 Furthermore, because of the lack of an appropriate and reproducible model for the fade of the relaxation, the role of L-citrulline recycling and arginine depletion in the NANC nerve–mediated relaxation of the gastrointestinal smooth muscle has not been investigated. Therefore, the purpose of the present investigation was twofold: to examine the role of L-citrulline recycling in the maintenance of nitrergic inhibitory neurotransmission and the mechanism of fade of the IAS smooth muscle relaxation during the continuous EFS.
Materials and Methods Preparation of IAS Smooth Muscle Strips Adult opossums (Didelphis virginiana) of either sex were killed by exsanguination after pentobarbital anesthesia (40 mg/ Abbreviations used in this paper: AL, argininosuccinate lyase; AS, argininosuccinate synthetase; D-NNA, Nv-nitro-D-arginine; EFS, electrical field stimulation; IAS, internal anal sphincter; L-NNA, Nv-nitroL-arginine; NANC, nonadrenergic, noncholinergic; NOS, nitric oxide synthase. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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kg intraperitoneally). After identifying and marking the landmarks of the IAS, the entire anal canal and a part of the rectum were dissected. The dissected tissue was then transferred to a Sylgard–coated Petri dish containing oxygenated Krebs’ solution of the following composition (in mmol/L): NaCl, 118.07; KCl, 4.69; CaCl2 , 2.52; MgSO4 , 1.16; NaH2PO4 , 1.01; NaHCO3 , 25; and glucose, 11.10. The anal canal was freed of all extraneous tissues including the small blood vessels and adhering skeletal muscles. The anal canal was then opened and pinned flat on the Petri dish. The mucosa along with the submucosal layers were carefully removed using sharp surgical dissection. From the specific region of the IAS, circular smooth muscle strips (Ç2 mm wide and 10 mm long) were prepared. The care and use of the laboratory animals were in accordance with the institutional guidelines.
Recording of Isometric Tension The smooth muscle strips were tied at both ends with silk sutures (3-0). One of the tied ends was attached to a tissue holder and transferred to a muscle bath (2 mL) containing Krebs’ solution bubbled continuously with a mixture of 95% oxygen and 5% carbon dioxide. The other end of the smooth muscle strip was attached to a force transducer (model FT03; Grass Instruments Co., Quincy, MA) for measurement of isometric tensions of the smooth muscle strips. The tensions were recorded on a Dynograph recorder (model R411; Beckman Instruments, Schiller Park, IL). An initial tension of 1 g was applied to the muscle strips and allowed to equilibrate for an hour with washings at every 20 minutes. During this period, the IAS smooth muscle strips developed spontaneous steady tension and relaxed in response to EFS. Only those strips that developed spontaneous tension and relaxed in response to EFS were included in the study. The optimal length (L0 ) and the baseline tension of the smooth muscle strips were determined as described previously.17
NANC Nerve Stimulation by EFS EFS was performed using a Grass stimulator (model S88; Grass Instruments Co.), and two platinum electrodes were placed parallel to each other at both sides of the smooth muscle strips. Two EFS protocols were used for the study. In the first protocol, the NANC nerves were stimulated with short-train EFS (4-second train, 0.5-millisecond pulse duration, 20–30 V) at varying frequencies (0.5–20.0 Hz) before and after treatment with different drugs. In the second protocol, long-train EFSs (30–60 minutes) of 5, 10, and 20 Hz (with the other parameters being same) were used. In these experiments, the smooth muscle strips showed transient relaxation. In the second protocol, continuous EFS (long train; 0.5millisecond pulse duration, 20–30 V) was used. On the application of the continuous EFS, the smooth muscle strips responded with an immediate relaxation. The immediate and
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full relaxation in the presence of ongoing EFS was followed by a gradual recovery of the basal tone toward the prestimulus level. A recovery ranging from 70% to 80% was observed in 15–30 minutes. At this time, the basal IAS tension became stable and the smooth muscle strips were treated with different drugs. All experiments were performed in the presence of atropine (1 1 1006 mol/L) and guanethidine (3 1 1006 mol/L).
Drugs L-Arginine hydrochloride, D-arginine, L-citrulline (L2-amino-5-ureidovaleric acid), L-argininosuccinate (argininosuccinic acid, and barium salt), L-glutamine (L-2-aminoglutaramic acid), D-glutamine, L-glutamic acid monosodium salt (L-glutamate), and N v-nitro-L-arginine (L-NNA) were from Sigma Chemical Co. (St. Louis, MO). Ethylenediaminetetraacetic acid (EDTA)-tetrasodium salt was from Fisher Scientific (Pittsburgh, PA). All chemicals were dissolved and diluted in Krebs’ solution, prepared fresh each day, kept on ice, and protected from light. The muscle baths were pretreated with 2.5% bovine serum albumin one night before the experiment.
Drug Responses Responses to different agents were examined using either single-dose or cumulative concentration responses. When the concentration-response curve to an agent was determined, the smooth muscle strips were washed six times, and the basal tension was allowed to recover. The effect of agents on the NANC nerve–mediated IAS relaxation was determined using both short-train and continuous EFS. To examine the effect of NOS inhibitor L-NNA on NANC nerve–mediated IAS relaxation, control frequency–response curves were first obtained and then repeated in the presence of maximal effective concentration of L-NNA (3 1 1005 mol/ L) as previously established in this preparation.3 The tissues were pretreated with L-NNA for 10 minutes before testing the effects of EFS. To examine the role of L-citrulline recycling in the IAS relaxation, the experiments were repeated in the presence of different concentrations of L-citrulline alone and L-NNA plus L-citrulline. These effects were compared with those of L-arginine. In these experiments, different concentrations of L-citrulline or L-arginine were allowed in contact with the tissues for 10 minutes before testing the effects of EFS. In the continuous EFS experiments, the time course of decrease in the basal IAS tension in the presence of ongoing EFS was determined before and after L-NNA, different concentrations of L-citrulline, or L-arginine plus L-NNA. We also performed studies to examine the influence of Lglutamine (putative blocker of L-citrulline uptake)10,18 on the time course of the decrease in IAS tension in the presence of continuous EFS. To determine the specificity of actions of LNNA and L-glutamine, we examined the effects of D-NNA and L-glutamate. The tissues were pretreated with L-glutamine or L-glutamate for 30 minutes before testing the effects of
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EFS and L-citrulline and L-arginine. The effects of different concentrations of L-citrulline or L-arginine on the recovered IAS tone in the presence of ongoing EFS were also determined before and after L-glutamine. The effects of different concentrations of L-citrulline or L-arginine alone were also examined on the basal IAS tone and on NANC nerve–mediated IAS relaxation. In these experiments, different concentrations of Lcitrulline and L-arginine were added systematically either 5 minutes apart or at the time of stabilization of the effects of a given concentration. In some experiments, the effects of Largininosuccinate on the IAS given in a similar manner were also investigated.
Data Analysis The results are expressed as means { SE for different experiments. The decrease in the basal IAS tension was expressed as percent of maximal decrease (100%) of tension caused by 5 mmol/L EDTA. Statistical significance between different groups were determined using the t test (paired or unpaired); a P value of õ0.05 was considered statistically significant.
Results Effect of L-Citrulline and L-Arginine on NANC Nerve Stimulation by Short-Train EFS-Induced IAS Relaxation Short-train EFS caused a frequency–dependent decrease in the basal tension of the IAS. The IAS relaxation caused by the EFS was significantly suppressed by the NOS inhibitor L-NNA (3 1 1005 mol/L) (Figure 1A). The decrease in the IAS tension caused by the lower frequencies was obliterated by L-NNA, and there was a small residual decrease in the IAS tension by the higher frequencies of EFS in the presence of L-NNA. In control experiments, the decrease in IAS tension caused by 10 Hz was 76.8 { 4.4 and, after administration of L-NNA, 17.4% { 3.0% (P õ 0.05). D-NNA, however, failed to modify the IAS relaxation. The L-NNA–suppressed IAS relaxation was reversed by L-citrulline in a concentration-dependent manner so that the overall EFS–frequency response curves obtained during control experiments and after administration of 03 L-NNA plus L-citrulline (1 1 10 mol/L) were not significantly different from each other (P ú 0.05; n Å 5). Interestingly, the reversal of the L-NNA–suppressed IAS relaxation by L-citrulline was almost similar to that by L-arginine (Figure 1B). D-Arginine had no effect on the L-NNA–suppressed IAS relaxation. Effect of Continuous EFS on IAS Tone Similar to the short train, continuous EFS also caused a decrease in the IAS tension. In addition, the continuous EFS allowed the study of detailed time course / 5e1b$$0027
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Figure 1. Inhibition of neurally mediated (by different frequencies of EFS shown in log scale) decrease in IAS tension by the NOS inhibitor L-NNA and its reversal by different concentrations of (A ) L-citrulline and (B ) L-arginine. Preincubation of the IAS smooth muscle strips with L-NNA (3 1 1005 mol/L) caused significant attenuation (P õ 0.05) of neurally mediated decrease in the basal tension of IAS; this attenuation was reversed by L-citrulline and L-arginine in a concentrationdependent manner (n Å 5).
of the initial and the later components of the IAS relaxation. Figure 2 shows the time course of the decrease in IAS tension by 5, 10, and 20 Hz of continuous EFS. Note that, on application of EFS, there was an immediate WBS-Gastro
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decrease in the IAS tension followed by a gradual recovery toward the prestimulus basal levels during the continuous EFS. Interestingly, although the amplitudes of initial decrease in the IAS tension by these frequencies were not significantly different, the speed of recovery during the continuous EFS was dependent on the frequency of EFS. For example, the recovery of the basal tone during continuous EFS was significantly protracted when 5-Hz EFS was used compared with that of 10 and 20 Hz (P õ 0.05; n Å 5). During 5-, 10-, and 20- Hz EFS, the durations of 50% recovery of the basal IAS tone were 11.63 { 1.84, 5.37 { 1.26, and 4.0 { 1.05 minutes, respectively. In most of the experiments, especially with frequencies of õ20 Hz, the maximal recovery of the basal tone in the presence of continuous EFS was usually incomplete and was Ç70%–80% of the original tone. Furthermore, the speed of fade in the IAS relaxation was found to be frequency dependent only with the frequencies causing maximal decrease in the IAS tension. The decrease in the IAS tension with 1 Hz of EFS was only 48.1% { 1.1%, and maximal recovery of the response in the presence of continuous EFS occurred in 9.5 { 1.0 minutes. Influence of L-NNA on the IAS Relaxation Caused by Continuous EFS and Effect of L-Citrulline and L-Arginine The NOS inhibitor L-NNA caused significant suppression of the IAS relaxation caused by continuous EFS. This was reflected in the suppression of the initial IAS relaxation as well as by the shortening of the duration of the total time of IAS relaxation (Figure 3). The suppressed IAS relaxations (especially in the initial stages of continuous EFS) were significantly restored by the administration of L-citrulline (Figure 3A) and L-arginine (Figure 3B). Interestingly, in the latter phase of the continuous EFS, the reversal of the IAS relaxation by either L-citrulline or L-arginine was incomplete or absent. DArginine had no significant effect on L-NNA–suppressed IAS relaxations. Effect of L-Citrulline and L-Arginine on the Basal IAS Tone Recovered During Continuous EFS Figure 4A and B provide the time course of decrease in IAS tension from one representative experiment during 10 Hz of continuous EFS. Note that during continuous EFS, after the initial full relaxation of the IAS, there was complete recovery of the basal tone toward the prestimulus levels. The data show 47%, 65%, 74%, 82%, 88%, and 100% recovery of the basal IAS tone at 5, 6, 7, 8, 9, and 10 minutes, respectively, after the initial / 5e1b$$0027
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Figure 2. Time course (shown in linear scale) of the decrease in IAS tension and its recovery during continuous EFS at different frequencies (s, 5; ●, 10; and n, 20 Hz). Note a rapid and same degree of decrease in the basal tension of the IAS in the initial phase of the application of continuous EFS at different frequencies. In the later phase, this decrease in IAS tension began to recover in the presence of continuous EFS. However, in the later phase, the speed of recovery of the decreased basal tone was different based on the frequency of EFS used. The recovery of the decreased basal tone in the presence of 5 Hz of EFS was significantly slower (P õ 0.05) than with 10 and 20 Hz (n Å 8). At the end of 10 minutes, the basal tone in the presence of 20 Hz had almost returned to the prestimulus level, whereas in the presence of 5 Hz, the recovery was only approximately 50%.
90% relaxation in the case of L-citrulline experiments (Figure 4A). After the basal IAS tone recovery had plateaued, L-citrulline caused a concentration–dependent decrease in the IAS tension. A similar decrease in IAS tension was observed with L-arginine (Figure 4B). L-Argininosuccinate, examined in various concentrations, was found to have no significant effect on the recovered basal IAS tone (data not shown). We also investigated the effect of L-arginine (3 1 1003 mol/L) pretreatment for 30 minutes on the decrease in IAS tension by continuous EFS. In these experiments, the duration of maximal IAS relaxation significantly increased from 2.3 { 0.8 to 5.1 { 0.6 minutes (P õ 0.05) and the IAS tone recovered to a level that was significantly lower than that in the control experiments, suggesting a lower speed and degree of fade in the IAS relaxation in the presence of L-arginine. However, the decrease in IAS tone in the presence of continuous EFS was not maintained. The exact reason for this remains to be determined. The effects of L-citrulline in such experiments were similar to those of L-arginine. Influence of L-Glutamine on the IAS Relaxation by L-Citrulline and L-Arginine Figure 5A and B show the quantitative data of the influence of L-glutamine on the IAS relaxation by LWBS-Gastro
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citrulline and L-arginine on the recovered IAS tone in the presence of continuous EFS. The decrease in the IAS tension caused by L-citrulline but not L-arginine was significantly inhibited by L-glutamine. L-Glutamate in contrast to L-glutamine had no significant effect on the L-citrulline–induced decrease in tension. The decrease in IAS tension after its plateau recovery in the presence of continuous EFS (10 Hz) by 3 1 1004, 1 1 1003, and 3 1 1003 mol/L L-citrulline in control experiments was 32.0% { 4.7%, 49.4% { 6.4%, and 57.5% { 7.6%, respectively. These values in the presence of L-glutamine were significantly reduced to 11.1% { 5.7%, 21.0% { 8.5%, and 40.9% { 8.6%, respectively (P õ 0.05). Conversely, a similar decrease in IAS tension by different concentrations of L-arginine on the recovered IAS tone was not significantly modified by Lglutamine (Figure 5B; P ú 0.05). In a separate series of experiments examining the arginine levels, L-glutamine was found to block the restoration of arginine levels depleted by continuous EFS and arginase pretreatment (data not shown), suggesting the specificity of actions of L-glutamine. Effect of L-Citrulline and L-Arginine Alone on the Basal IAS Tone and NANC Nerve– Mediated IAS Relaxation Although L-citrulline and L-arginine caused the reversal of L-NNA–suppressed IAS relaxation and decrease in the basal IAS tension after it had recovered during the continuous EFS, these agents themselves had no significant effect on either the basal IAS tension (Figure 6) or on the NANC nerve–mediated IAS relaxation (Figure 7; data are shown for L-citrulline only). Conversely, however, sodium nitroprusside caused a concentration–dependent decrease in the IAS tension (Figure 6), showing the viability of the preparations. Influence of L-Glutamine on the Speed of Recovery of the IAS Tone During Continuous EFS Figure 8A shows the time course of the decrease in IAS tension in response to continuous EFS (5 Hz) before and after L-glutamine. L-Glutamine caused a significant increase in the speed of recovery of the IAS tone in the presence of ongoing EFS. For example, 5 minutes after the onset of continuous EFS (5 Hz), the decrease in IAS tension in control experiments and after pretreatment of L-glutamine (3 1 1003 mol/L) was 78.3% { 7.3% and 51.1% { 7.7%, respectively (P õ 0.05). Corresponding times for 50% recovery of the IAS tone after the initial EFS–induced relaxation before and after L/ 5e1b$$0027
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Figure 3. Detailed time course (shown in log scale) of the decrease in IAS tension in the presence of continuous EFS (10 Hz) and its inhibition by L-NNA (3 1 1005 mol/L). In the initial phase of continuous EFS, maximal decrease in the IAS tension was observed in 30 seconds followed by a significant recovery in the later phase of continuous EFS. L-NNA caused a significant suppression (P õ 0.05) of decrease in the initial as well as in the later phase of the IAS relaxation during continuous EFS. Also note a delay in the maximal decrease in the IAS tension and rapid recovery in the decreased IAS tone during continuous EFS in the presence of L-NNA. The effects of L-NNA were reversed by different concentrations of (A ) L-citrulline and (B ) L-arginine. s, EFS (10 Hz) control; ●, EFS (L-NNA; 3 1 1005 mol/L); n, (L-NNA / L-cit or L-arg; 1 1 1004 mol/L); m, EFS (L-NNA / L-cit or L-arg; 1 1 1003 mol/L); h, EFS (L-NNA / L-cit or L-arg; 3 1 1003 mol/L).
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glutamine were 12.75 { 2.75 and 4.75 { 0.63 minutes, respectively (P õ 0.05). Similar data were obtained with 10 Hz of continuous EFS (Figure 8B).
Discussion The present study suggests that recycling of Lcitrulline to L-arginine can largely support the IAS relaxation caused by NANC nerve stimulation by either repe-
Figure 5. Quantitative data showing the effect of different concentrations of (A ) L-citrulline and (B ) L-arginine on the recovered IAS tone during continuous EFS (10 Hz) before and after L-glutamine pretreatment. After the recovery of the IAS tone during ongoing EFS, both Lcitrulline and L-arginine caused a concentration-dependent decrease of the tension. The pretreatment of L-glutamine (3 1 1003 mol/L), caused significant inhibition (P õ 0.05; n Å 6) of L-citrulline responses but not those of L-arginine. h, Control; , L-glutamine.
Figure 4. Time course (shown in linear scale) depicting the effects of different concentrations of (A ) L-citrulline and (B ) L-arginine on the recovered IAS tone during continuous EFS (10 Hz). EFS caused almost complete relaxation of the IAS tension followed by its recovery during ongoing stimulation. On stabilization of the basal tone, the smooth muscle strips were treated with cumulative concentrations (1 1 1004, 3 1 1004, 1 1 1003, and 3 1 1003 mol/L) of either L-arginine or Lcitrulline represented by arrows. Both L-arginine and L-citrulline caused a concentration-dependent decrease of the recovered tone.
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titious short-train or continuous EFS. The evidence was fourfold. One, L-citrulline caused concentration–dependent reversal of the IAS relaxation attenuated by the NOS inhibitor L-NNA. Furthermore, L-citrulline was nearly as potent as L-arginine in causing the reversal of IAS relaxation suppressed by the NOS inhibitor. This was observed both in the case of short-train EFS and prolonged continuous EFS. The reversal of the inhibition of NO–dependent inhibitory neurotransmission in the IAS by L-citrulline was similar to that reported by Shuttleworth et al. using the canine colon.16 Our previous studies have shown that a major part of NANC nerve–mediated IAS relaxation is via the NOS pathway and the NOS inhibitor L-NNA caused specific inhibition of IAS relaxation. Moreover, WBS-Gastro
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Figure 6. Effects of different concentrations of L-arginine, L-citrulline, and sodium nitroprusside on the basal tension of the IAS. These experiments were performed in the absence of any EFS. L-Arginine or L-citrulline added up to 3 1 1003 mol/L had no significant effect on the basal IAS tone, whereas SNP caused a concentration-dependent decrease in the basal tone of the IAS (n Å 6). SNP, sodium nitroprusside. h, L-Arginine; , L-citrulline; j, SNP.
the reversal of L-NNA-suppressed IAS relaxation was enantiomer specific and was specifically reversed by NOS substrate L-arginine. In the studies using continuous EFS, we found that the NOS inhibitor L-NNA (a nitro deriva-
Figure 8. Time course (in linear scale) showing the effect of L-glutamine on the speed of recovery of decrease in IAS tension in the presence of (A ) 5 Hz and (B ) 10 Hz of continuous EFS. Note a significant increase in the speed of recovery of the IAS tone, after L-glutamine pretreatment. SE were omitted for clarity. s, EFS control; ●, EFS L-glutamine (3 1 1004 mol/L); n, EFS L-glutamine (3 1 1003 mol/L).
Figure 7. Effect of different concentrations of L-citrulline on the neurally mediated (by different frequencies of EFS shown in log scale) decrease of the IAS tension caused by different frequencies of EFS. Overall, L-citrulline had no significant effect on the decrease in the IAS tension caused by different frequencies of EFS (P ú 0.05; n Å 4). s, Control; ●, L-citrulline (1 1 1004 mol/L); n, L-citrulline (3 1 1004 mol/L); m, L-citrulline (1 1 1003 mol/L).
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tive of L-arginine) caused a significant impairment in the IAS relaxation. The L-NNA inhibition of the IAS relaxation was reversed by pretreatment with either Lcitrulline (Figure 3A) or L-arginine (Figure 3B). The reversal with L-citrulline and L-arginine was complete in the initial stages of continuous EFS. However, in the latter stage of the EFS, the reversal was either incomplete or absent. The exact reason for this phenomenon is not presently understood. There are two possible speculations. First, in the absence of continuous EFS or other conditions leading to L-arginine depletion, the tissues may not attain a state of L-arginine deficiency. L-Arginine deficiency may be an important factor for the uptake of exogenous L-arginine because in the absence of such deficiency, L-citrulline and L-arginine have no significant WBS-Gastro
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effect on either the basal tone or the relaxation. Second, Larginine pretreatment causes temporary and preferential displacement of the NOS inhibitor from NOS only in the initial stages of continuous EFS followed by the replacement of the precursor from the binding sites of NOS by the NOS inhibitor. Two, our data show that during prolonged NANC nerve stimulation, the basal IAS tone after the initial relaxation begins to recover toward the prestimulus levels. After the recovery of the IAS tone in the presence of ongoing EFS, exogenous administration of L-citrulline caused a concentration–dependent decrease in the tone. This decrease of recovered IAS tone by L-citrulline in the presence of continuous EFS was comparable with that of L-arginine. The decrease in the IAS tone by these amino acids was enantiomer specific because, D-arginine had no effect in these experimental protocols. Interestingly, neither L-citrulline nor L-arginine had any effect on the basal IAS tone and on NANC nerve–mediated IAS relaxation in the absence of any ongoing EFS. Three, decrease in the recovered IAS tone by L-citrulline was blocked by L-glutamine, a putative inhibitor of L-citrulline uptake. The uptake of L-citrulline by the appropriate unknown intracellular compartment responsible is a prerequisite for its conversion to L-arginine. The data suggest that L-glutamine blocked the uptake of L-citrulline and its conversion to L-arginine that was necessary for L-citrulline to cause IAS relaxation in the L-arginine–deficient preparation. L-Arginine–deficient preparations were created by the long-term continuous EFS, which was confirmed in a separate series of experiments by a decrease in the tissue levels of arginine. Four, L-glutamine increased the speed of recovery of the basal IAS tone during the continuous EFS. These observations with L-glutamine suggest that, during the continuous EFS under normal circumstances, the IAS remained relaxed for longer durations because of the continuous recycling of L-citrulline to L-arginine. When that recycling was interrupted by L-glutamine, the duration of the IAS relaxation was considerably shortened, leading to the earlier recovery of the basal IAS tone. The effects of L-glutamine on L-citrulline–induced decrease in the recovered IAS tone and on NANC nerve–mediated IAS relaxation were similar to those in porcine cerebral arteries.18 The experiments suggest that recovery of the basal IAS tone during the continuous EFS occurred primarily because of the depletion of the NO precursor L-arginine. In this experimental protocol, the recovered IAS tone responds with relaxation to the exogenous administration of both L-citrulline and L-arginine. Furthermore, the de/ 5e1b$$0027
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crease in IAS tone by L-citrulline was blocked by Lglutamine, whereas the decrease in the IAS tone by Larginine remained unaffected by the putative blocker of citrulline uptake. The data suggest that L-citrulline had to be taken and converted to L-arginine to be effective in causing the IAS relaxation in the L-arginine–depleted state. The data for L-glutamine confirm the site and specificity of action of L-citrulline because the relaxant effects of L-arginine that bypasses the uptake step were not modified by L-glutamine. According to the L-citrulline–arginine cycle, NO and L-citrulline are formed stoichiometrically (1:1) from Larginine by NOS; L-citrulline considered as a byproduct is converted to argininosuccinic acid by AS, which in turn recycles into L-arginine by the interaction of AL. After the schema, in L-arginine–deficient preparation (created by the continuous EFS when the IAS tone after the initial relaxation returns toward the prestimulus level), all three amino acids should restore the IAS relaxation if the recovery during the continuous EFS is caused by depletion of L-arginine. However, only L-citrulline and L-arginine, but not L-argininosuccinic acid, were successful in doing so in the current experimental setup. The lack of significant decrease in the IAS tension by Largininosuccinic acid in these experiments may be explained on the basis of the requirement for a specific transporter by this amino acid for its uptake into the myenteric neuronal cytosol. This study also suggests the presence of L-citrulline recycling enzymatic machinery (NOS, AS, and AL) in the IAS. The presence of NOS in the IAS19,20 and other regions of the gastrointestinal tract has been reviewed previously.21 – 25 Although the presence of AS and AL has been shown in a number of tissues,26 there is relatively little information on the presence of AS and AL in the peripheral nervous system of the gut.16 The latter studies showed the presence of AS and AL immunoreactivities in the varicose nerve fibers within the circular and longitudinal muscle layers of the canine proximal colon. Furthermore, such immunoreactivities were found to be colocalized with NOS. In our studies, although the presence of AS and AL immunoreactivities were not examined, the reversal of L-NNA–suppressed IAS relaxation by Lcitrulline, the restoration of decrease in the IAS tension by L-citrulline, and the blockade of the effect by L-glutamine strongly suggest the existence of the enzymatic machinery in the IAS. Furthermore, our recent studies showing the presence of AS and AL at the transcriptional level27 provide additional support to the concept. It has been frequently observed that NANC nerve – mediated IAS relaxation in in vivo2,4,28 and in vitro3,17 WBS-Gastro
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studies in response to short-term rectoanal inhibitory reflex by rectal balloon distention, sacral nerve stimulation and local intramural stimulation of the myenteric neurons, and EFS can sustain for several hours without any fade. It is possible that interstimulus intervals in these studies were such that the L-arginine stored in the appropriate compartments of the neurons may have been sufficient to cope with the demand or were just enough to allow for the continuous replenishment of L-arginine for the synthesis and release of the inhibitory neurotransmitter. However, in the present studies using continuous EFS, it was obvious that the IAS relaxation could be maintained for some time. However, the IAS tone began to recover in the presence of continuous EFS, and depending on the frequency of EFS, it took 15 – 30 minutes for the maximal recovery of 70% – 80%. We conclude that L-citrulline recycling may be largely responsible for the maintenance of the IAS relaxation in response to NANC nerve stimulation. Furthermore, the present studies in the IAS provide a novel L-arginine – deficient model to investigate the role of L-citrulline recycling in the nitrergic inhibitory neurotransmission in the enteric nervous system. Previous findings that certain analogues of L-arginine (that compete with endogenous L-arginine) block the NOS pathway responsible for the NANC nerve – mediated relaxation of human IAS that can be reversed by L-arginine29 suggest the importance of L-arginine – NOS pathway in anorectal reflex4 and in the pathophysiology of IAS relaxation.
10.
11.
12.
13.
14.
15. 16.
17.
18.
19.
References 1. Biancani P, Walsh JH, Behar J. Vasoactive intestinal polypeptide: a neurotransmitter for relaxation of the rabbit internal anal sphincter. Gastroenterology 1985;89:867–874. 2. Nurko S, Rattan S. Role of vasoactive intestinal polypeptide in the internal anal sphincter relaxation of the opossum. J Clin Invest 1988;81:1146–1153. 3. Rattan S, Chakder S. Role of nitric oxide as a mediator of internal anal sphincter relaxation. Am J Physiol 1992;262:G107–G112. 4. Rattan S, Sarkar A, Chakder S. Nitric oxide pathway in rectoanal inhibitory reflex of opossum internal anal sphincter. Gastroenterology 1992;103:43–50. 5. Tottrup A, Glavind EB, Svane D. Involvement of the L-arginine– nitric oxide pathway in internal anal sphincter relaxation. Gastroenterology 1992;102:409–415. 6. Bredt DS, Snyder SH. Nitric oxide mediates glutamate-linked enhancement of cGMP levels in the cerebellum. Proc Natl Acad Sci U S A 1989;86:9030–9033. 7. Knowles RG, Palacios M, Palmer RMJ, Moncada S. Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc Natl Acad Sci U S A 1989;86:5159–5162. 8. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology,
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pathophysiology, and pharmacology. Pharmacol Rev 1991;43: 109–142. Hattori Y, Campbell EB, Gross SS. Argininosuccinate synthetase mRNA and activity are induced by immunostimulants in vascular smooth muscle. Role in the regeneration of arginine for nitric oxide synthesis. J Biol Chem 1994;269:9405–9408. Sessa WC, Hecker M, Mitchell JA, Vane JR. The metabolism of Larginine and its significance for the biosynthesis of endotheliumderived relaxing factor: L-glutamine inhibits the generation of Larginine by cultured endothelial cells. Proc Natl Acad Sci USA 1990;87:8607–8611. Wu W, Meininger CJ. Regulation of L-arginine synthesis from Lcitrulline by L-glutamine in endothelial cells. Am J Physiol 1993; 265:H1965–H1971. Pow DV. Immunocytochemical evidence for a glial localisation of arginine, and a neuronal localisation of citrulline in the rat neurohypophysis: implications for nitrergic transmission. Neurosci Lett 1994;181:141–144. Flodstro¨m M, Niemann A, Bedoya FJ, Morris SM Jr, Eizirik DL. Expression of the citrulline-nitric oxide cycle in rodent and human pancreatic b-cells: induction of argininosuccinate synthetase by cytokines. Endocrinology 1995;136:3200–3206. Nagasaki A, Gotoh T, Takeya M, Yu YJ, Takiguchi M, Matsuzaki H, Takatsuki K, Mori M. Coinduction of nitric oxide synthase, argininosuccinate synthetase, and argininosuccinate lyase in lipopolysaccharide-treated rats—RNA blot, immunoblot, and immunohistochemical analyses. J Biol Chem 1996;271:2658– 2662. Ratner S. Enzymes of arginine and urea synthesis. Adv Enzymol 1973;39:1–90. Shuttleworth CWR, Burns AJ, Ward SM, O’Brien WE, Sanders KM. Recycling of L-citrulline to sustain nitric oxide–dependent enteric neurotransmission. Neuroscience 1995;68:1295– 1304. Moummi C, Rattan S. Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation. Am J Physiol 1988; 255:G571–G578. Lee TJF, Sarwinski S, Ishine T, Lai CC, Chen FY. Inhibition of cerebral neurogenic vasodilation by L-glutamine and nitric oxide synthase inhibitors and its reversal by L-citrulline. J Pharmacol Exp Ther 1996;276:353–358. Lynn RB, Sankey SL, Chakder S, Rattan S. Colocalization of NADPH-diaphorase staining and VIP immunoreactivity in neurons in opossum internal anal sphincter. Dig Dis Sci 1995;40:781– 791. Lynn RB, Sengupta A, Chakder S, Rattan S. NADPH-diaphorase staining correlates with nitric oxide synthase (NOS) like immunoreactivity in internal anal sphincter (IAS) neurons (abstr). Gastroenterology 1995;108:A642. Goyal RK, Hirano I. The enteric nervous system. N Engl J Med 1996;334:1106–1115. Stark ME, Szurszewski JH. Role of nitric oxide in gastrointestinal and hepatic function and disease. Gastroenterology 1992;103: 1928–1949. Llewellyn–Smith IJ, Song Z-M, Costa M, Bredt DS, Snyder SH. Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons. Brain Res 1992;577:337– 342. Sanders KM, Ward SM. Nitric oxide as a mediator of nonadrenergic noncholinergic neurotransmission. Am J Physiol 1992;262: G379–G392. Furness JB, Li ZS, Young HM, Fo¨rstermann U. Nitric oxide synthase in the enteric nervous system of the guinea-pig: a quantitative description. Cell Tissue Res 1994;277:139–149. Yu Y, Terada K, Nagasaki A, Takiguchi M, Mori M. Preparation of recombinant argininosuccinate synthetase and argininosucci-
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nate lyase: expression of the enzymes in rat tissues. J Biochem (Tokyo) 1995;117:952–957. 27. Rattan S, Mori M, Bandyopadhyay A, Chakder S. Evidence for Lcitrulline recycling being responsible for the maintenance of internal anal sphincter (IAS) relaxation by nonadrenergic noncholinergic (NANC) nerve stimulation (abstr). Gastroenterology 1996; 110:A742. 28. Culver PJ, Rattan S. Genesis of anal canal pressures in the opossum. Am J Physiol Gastrointest Liver Physiol 1986;251: G765–G771. 29. Burleigh DE. NG-Nitro-L-arginine reduces nonadrenergic, noncho-
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linergic relaxations of human gut. Gastroenterology 1992;102: 679–683.
Received July 22, 1996. Accepted December 10, 1996. Address requests for reprints to: Satish Rattan, D.V.M., 901 College, Thomas Jefferson University, 1025 Walnut Street, Philadelphia, Pennsylvania. Supported by National Institutes of Diabetes and Digestive and Kidney Diseases grant DK-35385 and an institutional grant from Thomas Jefferson University.
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L-Citrulline Recycling in Opossum Internal Anal Sphincter
Relaxation by Nonadrenergic, Noncholinergic Nerve Stimulation SATISH RATTAN and SUSHANTA CHAKDER Division of Gastroenterology/Hepatology, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pennsylvania
Background & Aims: L-citrulline formed stoichiometrically along with nitric oxide (1:1) from L-arginine may be enzymatically converted to L-arginine. The possibility of L-citrulline recycling in the maintenance of nitrergic neurotransmission in the opossum internal anal sphincter (IAS) smooth muscle strips was investigated. Methods: Responses to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) (either short-train or continuous stimulation) on the basal IAS tension were recorded before and after the NO synthase inhibitor N v-nitro-L-arginine (L-NNA), LNNA plus L-citrulline, or L-arginine. During continuous EFS, when the basal IAS tone after the initial relaxation had recovered to almost pre-EFS levels, the effects of L-citrulline or L-arginine were examined before and after L-glutamine, which is a putative blocker of L-citrulline uptake. Results: Inhibition of NANC nerve–mediated IAS relaxation by L-NNA was reversed by L-citrulline as well as L-arginine. L-Citrulline and L-arginine caused concentration-dependent relaxation of the IAS tone recovered during the prolonged EFS. L-Glutamine blocked the responses of L-citrulline but not of L-arginine. Furthermore, L-glutamine increased the speed of recovery of IAS tone during continuous EFS. Conclusions: L-citrulline recycling may be responsible for the maintenance of IAS relaxation during frequent short-train and prolonged NANC nerve stimulation.
T
he internal anal sphincter (IAS) relaxation in response to nonadrenergic, noncholinergic (NANC) nerve stimulation by electrical field stimulation (EFS) may involve both vasoactive intestinal polypeptide1,2 and nitric oxide.3 – 5 It has been shown that a major component of the IAS relaxation by EFS is mediated via the NO synthase (NOS) pathway.3 – 5 Furthermore, the IAS relaxation either by short-train or continuous EFS can be maintained for a long time. Interestingly, for prolonged continuous EFS, the IAS relaxation is characterized by a gradual fade and return of the tension toward the prestimulus level. The exact mechanism for the mainte/ 5e1b$$0027
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nance of the IAS relaxation and fade in the IAS smooth muscle relaxation during NANC nerve stimulation is not known. In a number of neuronal and nonneuronal cells, NO and L-citrulline are formed stoichiometrically (1:1) from the precursor L-arginine.6 – 8 NO serves as an inhibitory neurotransmitter, whereas L-citrulline has been regarded as a byproduct of the reaction. However, recent studies suggest that L-citrulline thus formed may be converted back to L-arginine.9 – 13 This occurs by the action of two enzymes: argininosuccinate synthetase (AS) and argininosuccinate lyase (AL).14,15 This scheme has considerable potential to support the NANC nerve–mediated relaxation of the gastrointestinal smooth muscle. Yet, there is limited information on the role of L-citrulline recycling in the gastrointestinal smooth muscle relaxation. To date, the role of L-citrulline recycling in the NANC nerve– mediated gut smooth muscle relaxation has only been reported in the canine colon.16 Furthermore, because of the lack of an appropriate and reproducible model for the fade of the relaxation, the role of L-citrulline recycling and arginine depletion in the NANC nerve–mediated relaxation of the gastrointestinal smooth muscle has not been investigated. Therefore, the purpose of the present investigation was twofold: to examine the role of L-citrulline recycling in the maintenance of nitrergic inhibitory neurotransmission and the mechanism of fade of the IAS smooth muscle relaxation during the continuous EFS.
Materials and Methods Preparation of IAS Smooth Muscle Strips Adult opossums (Didelphis virginiana) of either sex were killed by exsanguination after pentobarbital anesthesia (40 mg/ Abbreviations used in this paper: AL, argininosuccinate lyase; AS, argininosuccinate synthetase; D-NNA, Nv-nitro-D-arginine; EFS, electrical field stimulation; IAS, internal anal sphincter; L-NNA, Nv-nitroL-arginine; NANC, nonadrenergic, noncholinergic; NOS, nitric oxide synthase. q 1997 by the American Gastroenterological Association 0016-5085/97/$3.00
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kg intraperitoneally). After identifying and marking the landmarks of the IAS, the entire anal canal and a part of the rectum were dissected. The dissected tissue was then transferred to a Sylgard–coated Petri dish containing oxygenated Krebs’ solution of the following composition (in mmol/L): NaCl, 118.07; KCl, 4.69; CaCl2 , 2.52; MgSO4 , 1.16; NaH2PO4 , 1.01; NaHCO3 , 25; and glucose, 11.10. The anal canal was freed of all extraneous tissues including the small blood vessels and adhering skeletal muscles. The anal canal was then opened and pinned flat on the Petri dish. The mucosa along with the submucosal layers were carefully removed using sharp surgical dissection. From the specific region of the IAS, circular smooth muscle strips (Ç2 mm wide and 10 mm long) were prepared. The care and use of the laboratory animals were in accordance with the institutional guidelines.
Recording of Isometric Tension The smooth muscle strips were tied at both ends with silk sutures (3-0). One of the tied ends was attached to a tissue holder and transferred to a muscle bath (2 mL) containing Krebs’ solution bubbled continuously with a mixture of 95% oxygen and 5% carbon dioxide. The other end of the smooth muscle strip was attached to a force transducer (model FT03; Grass Instruments Co., Quincy, MA) for measurement of isometric tensions of the smooth muscle strips. The tensions were recorded on a Dynograph recorder (model R411; Beckman Instruments, Schiller Park, IL). An initial tension of 1 g was applied to the muscle strips and allowed to equilibrate for an hour with washings at every 20 minutes. During this period, the IAS smooth muscle strips developed spontaneous steady tension and relaxed in response to EFS. Only those strips that developed spontaneous tension and relaxed in response to EFS were included in the study. The optimal length (L0 ) and the baseline tension of the smooth muscle strips were determined as described previously.17
NANC Nerve Stimulation by EFS EFS was performed using a Grass stimulator (model S88; Grass Instruments Co.), and two platinum electrodes were placed parallel to each other at both sides of the smooth muscle strips. Two EFS protocols were used for the study. In the first protocol, the NANC nerves were stimulated with short-train EFS (4-second train, 0.5-millisecond pulse duration, 20–30 V) at varying frequencies (0.5–20.0 Hz) before and after treatment with different drugs. In the second protocol, long-train EFSs (30–60 minutes) of 5, 10, and 20 Hz (with the other parameters being same) were used. In these experiments, the smooth muscle strips showed transient relaxation. In the second protocol, continuous EFS (long train; 0.5millisecond pulse duration, 20–30 V) was used. On the application of the continuous EFS, the smooth muscle strips responded with an immediate relaxation. The immediate and
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full relaxation in the presence of ongoing EFS was followed by a gradual recovery of the basal tone toward the prestimulus level. A recovery ranging from 70% to 80% was observed in 15–30 minutes. At this time, the basal IAS tension became stable and the smooth muscle strips were treated with different drugs. All experiments were performed in the presence of atropine (1 1 1006 mol/L) and guanethidine (3 1 1006 mol/L).
Drugs L-Arginine hydrochloride, D-arginine, L-citrulline (L2-amino-5-ureidovaleric acid), L-argininosuccinate (argininosuccinic acid, and barium salt), L-glutamine (L-2-aminoglutaramic acid), D-glutamine, L-glutamic acid monosodium salt (L-glutamate), and N v-nitro-L-arginine (L-NNA) were from Sigma Chemical Co. (St. Louis, MO). Ethylenediaminetetraacetic acid (EDTA)-tetrasodium salt was from Fisher Scientific (Pittsburgh, PA). All chemicals were dissolved and diluted in Krebs’ solution, prepared fresh each day, kept on ice, and protected from light. The muscle baths were pretreated with 2.5% bovine serum albumin one night before the experiment.
Drug Responses Responses to different agents were examined using either single-dose or cumulative concentration responses. When the concentration-response curve to an agent was determined, the smooth muscle strips were washed six times, and the basal tension was allowed to recover. The effect of agents on the NANC nerve–mediated IAS relaxation was determined using both short-train and continuous EFS. To examine the effect of NOS inhibitor L-NNA on NANC nerve–mediated IAS relaxation, control frequency–response curves were first obtained and then repeated in the presence of maximal effective concentration of L-NNA (3 1 1005 mol/ L) as previously established in this preparation.3 The tissues were pretreated with L-NNA for 10 minutes before testing the effects of EFS. To examine the role of L-citrulline recycling in the IAS relaxation, the experiments were repeated in the presence of different concentrations of L-citrulline alone and L-NNA plus L-citrulline. These effects were compared with those of L-arginine. In these experiments, different concentrations of L-citrulline or L-arginine were allowed in contact with the tissues for 10 minutes before testing the effects of EFS. In the continuous EFS experiments, the time course of decrease in the basal IAS tension in the presence of ongoing EFS was determined before and after L-NNA, different concentrations of L-citrulline, or L-arginine plus L-NNA. We also performed studies to examine the influence of Lglutamine (putative blocker of L-citrulline uptake)10,18 on the time course of the decrease in IAS tension in the presence of continuous EFS. To determine the specificity of actions of LNNA and L-glutamine, we examined the effects of D-NNA and L-glutamate. The tissues were pretreated with L-glutamine or L-glutamate for 30 minutes before testing the effects of
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EFS and L-citrulline and L-arginine. The effects of different concentrations of L-citrulline or L-arginine on the recovered IAS tone in the presence of ongoing EFS were also determined before and after L-glutamine. The effects of different concentrations of L-citrulline or L-arginine alone were also examined on the basal IAS tone and on NANC nerve–mediated IAS relaxation. In these experiments, different concentrations of Lcitrulline and L-arginine were added systematically either 5 minutes apart or at the time of stabilization of the effects of a given concentration. In some experiments, the effects of Largininosuccinate on the IAS given in a similar manner were also investigated.
Data Analysis The results are expressed as means { SE for different experiments. The decrease in the basal IAS tension was expressed as percent of maximal decrease (100%) of tension caused by 5 mmol/L EDTA. Statistical significance between different groups were determined using the t test (paired or unpaired); a P value of õ0.05 was considered statistically significant.
Results Effect of L-Citrulline and L-Arginine on NANC Nerve Stimulation by Short-Train EFS-Induced IAS Relaxation Short-train EFS caused a frequency–dependent decrease in the basal tension of the IAS. The IAS relaxation caused by the EFS was significantly suppressed by the NOS inhibitor L-NNA (3 1 1005 mol/L) (Figure 1A). The decrease in the IAS tension caused by the lower frequencies was obliterated by L-NNA, and there was a small residual decrease in the IAS tension by the higher frequencies of EFS in the presence of L-NNA. In control experiments, the decrease in IAS tension caused by 10 Hz was 76.8 { 4.4 and, after administration of L-NNA, 17.4% { 3.0% (P õ 0.05). D-NNA, however, failed to modify the IAS relaxation. The L-NNA–suppressed IAS relaxation was reversed by L-citrulline in a concentration-dependent manner so that the overall EFS–frequency response curves obtained during control experiments and after administration of 03 L-NNA plus L-citrulline (1 1 10 mol/L) were not significantly different from each other (P ú 0.05; n Å 5). Interestingly, the reversal of the L-NNA–suppressed IAS relaxation by L-citrulline was almost similar to that by L-arginine (Figure 1B). D-Arginine had no effect on the L-NNA–suppressed IAS relaxation. Effect of Continuous EFS on IAS Tone Similar to the short train, continuous EFS also caused a decrease in the IAS tension. In addition, the continuous EFS allowed the study of detailed time course / 5e1b$$0027
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Figure 1. Inhibition of neurally mediated (by different frequencies of EFS shown in log scale) decrease in IAS tension by the NOS inhibitor L-NNA and its reversal by different concentrations of (A ) L-citrulline and (B ) L-arginine. Preincubation of the IAS smooth muscle strips with L-NNA (3 1 1005 mol/L) caused significant attenuation (P õ 0.05) of neurally mediated decrease in the basal tension of IAS; this attenuation was reversed by L-citrulline and L-arginine in a concentrationdependent manner (n Å 5).
of the initial and the later components of the IAS relaxation. Figure 2 shows the time course of the decrease in IAS tension by 5, 10, and 20 Hz of continuous EFS. Note that, on application of EFS, there was an immediate WBS-Gastro
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decrease in the IAS tension followed by a gradual recovery toward the prestimulus basal levels during the continuous EFS. Interestingly, although the amplitudes of initial decrease in the IAS tension by these frequencies were not significantly different, the speed of recovery during the continuous EFS was dependent on the frequency of EFS. For example, the recovery of the basal tone during continuous EFS was significantly protracted when 5-Hz EFS was used compared with that of 10 and 20 Hz (P õ 0.05; n Å 5). During 5-, 10-, and 20- Hz EFS, the durations of 50% recovery of the basal IAS tone were 11.63 { 1.84, 5.37 { 1.26, and 4.0 { 1.05 minutes, respectively. In most of the experiments, especially with frequencies of õ20 Hz, the maximal recovery of the basal tone in the presence of continuous EFS was usually incomplete and was Ç70%–80% of the original tone. Furthermore, the speed of fade in the IAS relaxation was found to be frequency dependent only with the frequencies causing maximal decrease in the IAS tension. The decrease in the IAS tension with 1 Hz of EFS was only 48.1% { 1.1%, and maximal recovery of the response in the presence of continuous EFS occurred in 9.5 { 1.0 minutes. Influence of L-NNA on the IAS Relaxation Caused by Continuous EFS and Effect of L-Citrulline and L-Arginine The NOS inhibitor L-NNA caused significant suppression of the IAS relaxation caused by continuous EFS. This was reflected in the suppression of the initial IAS relaxation as well as by the shortening of the duration of the total time of IAS relaxation (Figure 3). The suppressed IAS relaxations (especially in the initial stages of continuous EFS) were significantly restored by the administration of L-citrulline (Figure 3A) and L-arginine (Figure 3B). Interestingly, in the latter phase of the continuous EFS, the reversal of the IAS relaxation by either L-citrulline or L-arginine was incomplete or absent. DArginine had no significant effect on L-NNA–suppressed IAS relaxations. Effect of L-Citrulline and L-Arginine on the Basal IAS Tone Recovered During Continuous EFS Figure 4A and B provide the time course of decrease in IAS tension from one representative experiment during 10 Hz of continuous EFS. Note that during continuous EFS, after the initial full relaxation of the IAS, there was complete recovery of the basal tone toward the prestimulus levels. The data show 47%, 65%, 74%, 82%, 88%, and 100% recovery of the basal IAS tone at 5, 6, 7, 8, 9, and 10 minutes, respectively, after the initial / 5e1b$$0027
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Figure 2. Time course (shown in linear scale) of the decrease in IAS tension and its recovery during continuous EFS at different frequencies (s, 5; ●, 10; and n, 20 Hz). Note a rapid and same degree of decrease in the basal tension of the IAS in the initial phase of the application of continuous EFS at different frequencies. In the later phase, this decrease in IAS tension began to recover in the presence of continuous EFS. However, in the later phase, the speed of recovery of the decreased basal tone was different based on the frequency of EFS used. The recovery of the decreased basal tone in the presence of 5 Hz of EFS was significantly slower (P õ 0.05) than with 10 and 20 Hz (n Å 8). At the end of 10 minutes, the basal tone in the presence of 20 Hz had almost returned to the prestimulus level, whereas in the presence of 5 Hz, the recovery was only approximately 50%.
90% relaxation in the case of L-citrulline experiments (Figure 4A). After the basal IAS tone recovery had plateaued, L-citrulline caused a concentration–dependent decrease in the IAS tension. A similar decrease in IAS tension was observed with L-arginine (Figure 4B). L-Argininosuccinate, examined in various concentrations, was found to have no significant effect on the recovered basal IAS tone (data not shown). We also investigated the effect of L-arginine (3 1 1003 mol/L) pretreatment for 30 minutes on the decrease in IAS tension by continuous EFS. In these experiments, the duration of maximal IAS relaxation significantly increased from 2.3 { 0.8 to 5.1 { 0.6 minutes (P õ 0.05) and the IAS tone recovered to a level that was significantly lower than that in the control experiments, suggesting a lower speed and degree of fade in the IAS relaxation in the presence of L-arginine. However, the decrease in IAS tone in the presence of continuous EFS was not maintained. The exact reason for this remains to be determined. The effects of L-citrulline in such experiments were similar to those of L-arginine. Influence of L-Glutamine on the IAS Relaxation by L-Citrulline and L-Arginine Figure 5A and B show the quantitative data of the influence of L-glutamine on the IAS relaxation by LWBS-Gastro
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citrulline and L-arginine on the recovered IAS tone in the presence of continuous EFS. The decrease in the IAS tension caused by L-citrulline but not L-arginine was significantly inhibited by L-glutamine. L-Glutamate in contrast to L-glutamine had no significant effect on the L-citrulline–induced decrease in tension. The decrease in IAS tension after its plateau recovery in the presence of continuous EFS (10 Hz) by 3 1 1004, 1 1 1003, and 3 1 1003 mol/L L-citrulline in control experiments was 32.0% { 4.7%, 49.4% { 6.4%, and 57.5% { 7.6%, respectively. These values in the presence of L-glutamine were significantly reduced to 11.1% { 5.7%, 21.0% { 8.5%, and 40.9% { 8.6%, respectively (P õ 0.05). Conversely, a similar decrease in IAS tension by different concentrations of L-arginine on the recovered IAS tone was not significantly modified by Lglutamine (Figure 5B; P ú 0.05). In a separate series of experiments examining the arginine levels, L-glutamine was found to block the restoration of arginine levels depleted by continuous EFS and arginase pretreatment (data not shown), suggesting the specificity of actions of L-glutamine. Effect of L-Citrulline and L-Arginine Alone on the Basal IAS Tone and NANC Nerve– Mediated IAS Relaxation Although L-citrulline and L-arginine caused the reversal of L-NNA–suppressed IAS relaxation and decrease in the basal IAS tension after it had recovered during the continuous EFS, these agents themselves had no significant effect on either the basal IAS tension (Figure 6) or on the NANC nerve–mediated IAS relaxation (Figure 7; data are shown for L-citrulline only). Conversely, however, sodium nitroprusside caused a concentration–dependent decrease in the IAS tension (Figure 6), showing the viability of the preparations. Influence of L-Glutamine on the Speed of Recovery of the IAS Tone During Continuous EFS Figure 8A shows the time course of the decrease in IAS tension in response to continuous EFS (5 Hz) before and after L-glutamine. L-Glutamine caused a significant increase in the speed of recovery of the IAS tone in the presence of ongoing EFS. For example, 5 minutes after the onset of continuous EFS (5 Hz), the decrease in IAS tension in control experiments and after pretreatment of L-glutamine (3 1 1003 mol/L) was 78.3% { 7.3% and 51.1% { 7.7%, respectively (P õ 0.05). Corresponding times for 50% recovery of the IAS tone after the initial EFS–induced relaxation before and after L/ 5e1b$$0027
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Figure 3. Detailed time course (shown in log scale) of the decrease in IAS tension in the presence of continuous EFS (10 Hz) and its inhibition by L-NNA (3 1 1005 mol/L). In the initial phase of continuous EFS, maximal decrease in the IAS tension was observed in 30 seconds followed by a significant recovery in the later phase of continuous EFS. L-NNA caused a significant suppression (P õ 0.05) of decrease in the initial as well as in the later phase of the IAS relaxation during continuous EFS. Also note a delay in the maximal decrease in the IAS tension and rapid recovery in the decreased IAS tone during continuous EFS in the presence of L-NNA. The effects of L-NNA were reversed by different concentrations of (A ) L-citrulline and (B ) L-arginine. s, EFS (10 Hz) control; ●, EFS (L-NNA; 3 1 1005 mol/L); n, (L-NNA / L-cit or L-arg; 1 1 1004 mol/L); m, EFS (L-NNA / L-cit or L-arg; 1 1 1003 mol/L); h, EFS (L-NNA / L-cit or L-arg; 3 1 1003 mol/L).
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glutamine were 12.75 { 2.75 and 4.75 { 0.63 minutes, respectively (P õ 0.05). Similar data were obtained with 10 Hz of continuous EFS (Figure 8B).
Discussion The present study suggests that recycling of Lcitrulline to L-arginine can largely support the IAS relaxation caused by NANC nerve stimulation by either repe-
Figure 5. Quantitative data showing the effect of different concentrations of (A ) L-citrulline and (B ) L-arginine on the recovered IAS tone during continuous EFS (10 Hz) before and after L-glutamine pretreatment. After the recovery of the IAS tone during ongoing EFS, both Lcitrulline and L-arginine caused a concentration-dependent decrease of the tension. The pretreatment of L-glutamine (3 1 1003 mol/L), caused significant inhibition (P õ 0.05; n Å 6) of L-citrulline responses but not those of L-arginine. h, Control; , L-glutamine.
Figure 4. Time course (shown in linear scale) depicting the effects of different concentrations of (A ) L-citrulline and (B ) L-arginine on the recovered IAS tone during continuous EFS (10 Hz). EFS caused almost complete relaxation of the IAS tension followed by its recovery during ongoing stimulation. On stabilization of the basal tone, the smooth muscle strips were treated with cumulative concentrations (1 1 1004, 3 1 1004, 1 1 1003, and 3 1 1003 mol/L) of either L-arginine or Lcitrulline represented by arrows. Both L-arginine and L-citrulline caused a concentration-dependent decrease of the recovered tone.
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titious short-train or continuous EFS. The evidence was fourfold. One, L-citrulline caused concentration–dependent reversal of the IAS relaxation attenuated by the NOS inhibitor L-NNA. Furthermore, L-citrulline was nearly as potent as L-arginine in causing the reversal of IAS relaxation suppressed by the NOS inhibitor. This was observed both in the case of short-train EFS and prolonged continuous EFS. The reversal of the inhibition of NO–dependent inhibitory neurotransmission in the IAS by L-citrulline was similar to that reported by Shuttleworth et al. using the canine colon.16 Our previous studies have shown that a major part of NANC nerve–mediated IAS relaxation is via the NOS pathway and the NOS inhibitor L-NNA caused specific inhibition of IAS relaxation. Moreover, WBS-Gastro
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Figure 6. Effects of different concentrations of L-arginine, L-citrulline, and sodium nitroprusside on the basal tension of the IAS. These experiments were performed in the absence of any EFS. L-Arginine or L-citrulline added up to 3 1 1003 mol/L had no significant effect on the basal IAS tone, whereas SNP caused a concentration-dependent decrease in the basal tone of the IAS (n Å 6). SNP, sodium nitroprusside. h, L-Arginine; , L-citrulline; j, SNP.
the reversal of L-NNA-suppressed IAS relaxation was enantiomer specific and was specifically reversed by NOS substrate L-arginine. In the studies using continuous EFS, we found that the NOS inhibitor L-NNA (a nitro deriva-
Figure 8. Time course (in linear scale) showing the effect of L-glutamine on the speed of recovery of decrease in IAS tension in the presence of (A ) 5 Hz and (B ) 10 Hz of continuous EFS. Note a significant increase in the speed of recovery of the IAS tone, after L-glutamine pretreatment. SE were omitted for clarity. s, EFS control; ●, EFS L-glutamine (3 1 1004 mol/L); n, EFS L-glutamine (3 1 1003 mol/L).
Figure 7. Effect of different concentrations of L-citrulline on the neurally mediated (by different frequencies of EFS shown in log scale) decrease of the IAS tension caused by different frequencies of EFS. Overall, L-citrulline had no significant effect on the decrease in the IAS tension caused by different frequencies of EFS (P ú 0.05; n Å 4). s, Control; ●, L-citrulline (1 1 1004 mol/L); n, L-citrulline (3 1 1004 mol/L); m, L-citrulline (1 1 1003 mol/L).
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tive of L-arginine) caused a significant impairment in the IAS relaxation. The L-NNA inhibition of the IAS relaxation was reversed by pretreatment with either Lcitrulline (Figure 3A) or L-arginine (Figure 3B). The reversal with L-citrulline and L-arginine was complete in the initial stages of continuous EFS. However, in the latter stage of the EFS, the reversal was either incomplete or absent. The exact reason for this phenomenon is not presently understood. There are two possible speculations. First, in the absence of continuous EFS or other conditions leading to L-arginine depletion, the tissues may not attain a state of L-arginine deficiency. L-Arginine deficiency may be an important factor for the uptake of exogenous L-arginine because in the absence of such deficiency, L-citrulline and L-arginine have no significant WBS-Gastro
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effect on either the basal tone or the relaxation. Second, Larginine pretreatment causes temporary and preferential displacement of the NOS inhibitor from NOS only in the initial stages of continuous EFS followed by the replacement of the precursor from the binding sites of NOS by the NOS inhibitor. Two, our data show that during prolonged NANC nerve stimulation, the basal IAS tone after the initial relaxation begins to recover toward the prestimulus levels. After the recovery of the IAS tone in the presence of ongoing EFS, exogenous administration of L-citrulline caused a concentration–dependent decrease in the tone. This decrease of recovered IAS tone by L-citrulline in the presence of continuous EFS was comparable with that of L-arginine. The decrease in the IAS tone by these amino acids was enantiomer specific because, D-arginine had no effect in these experimental protocols. Interestingly, neither L-citrulline nor L-arginine had any effect on the basal IAS tone and on NANC nerve–mediated IAS relaxation in the absence of any ongoing EFS. Three, decrease in the recovered IAS tone by L-citrulline was blocked by L-glutamine, a putative inhibitor of L-citrulline uptake. The uptake of L-citrulline by the appropriate unknown intracellular compartment responsible is a prerequisite for its conversion to L-arginine. The data suggest that L-glutamine blocked the uptake of L-citrulline and its conversion to L-arginine that was necessary for L-citrulline to cause IAS relaxation in the L-arginine–deficient preparation. L-Arginine–deficient preparations were created by the long-term continuous EFS, which was confirmed in a separate series of experiments by a decrease in the tissue levels of arginine. Four, L-glutamine increased the speed of recovery of the basal IAS tone during the continuous EFS. These observations with L-glutamine suggest that, during the continuous EFS under normal circumstances, the IAS remained relaxed for longer durations because of the continuous recycling of L-citrulline to L-arginine. When that recycling was interrupted by L-glutamine, the duration of the IAS relaxation was considerably shortened, leading to the earlier recovery of the basal IAS tone. The effects of L-glutamine on L-citrulline–induced decrease in the recovered IAS tone and on NANC nerve–mediated IAS relaxation were similar to those in porcine cerebral arteries.18 The experiments suggest that recovery of the basal IAS tone during the continuous EFS occurred primarily because of the depletion of the NO precursor L-arginine. In this experimental protocol, the recovered IAS tone responds with relaxation to the exogenous administration of both L-citrulline and L-arginine. Furthermore, the de/ 5e1b$$0027
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crease in IAS tone by L-citrulline was blocked by Lglutamine, whereas the decrease in the IAS tone by Larginine remained unaffected by the putative blocker of citrulline uptake. The data suggest that L-citrulline had to be taken and converted to L-arginine to be effective in causing the IAS relaxation in the L-arginine–depleted state. The data for L-glutamine confirm the site and specificity of action of L-citrulline because the relaxant effects of L-arginine that bypasses the uptake step were not modified by L-glutamine. According to the L-citrulline–arginine cycle, NO and L-citrulline are formed stoichiometrically (1:1) from Larginine by NOS; L-citrulline considered as a byproduct is converted to argininosuccinic acid by AS, which in turn recycles into L-arginine by the interaction of AL. After the schema, in L-arginine–deficient preparation (created by the continuous EFS when the IAS tone after the initial relaxation returns toward the prestimulus level), all three amino acids should restore the IAS relaxation if the recovery during the continuous EFS is caused by depletion of L-arginine. However, only L-citrulline and L-arginine, but not L-argininosuccinic acid, were successful in doing so in the current experimental setup. The lack of significant decrease in the IAS tension by Largininosuccinic acid in these experiments may be explained on the basis of the requirement for a specific transporter by this amino acid for its uptake into the myenteric neuronal cytosol. This study also suggests the presence of L-citrulline recycling enzymatic machinery (NOS, AS, and AL) in the IAS. The presence of NOS in the IAS19,20 and other regions of the gastrointestinal tract has been reviewed previously.21 – 25 Although the presence of AS and AL has been shown in a number of tissues,26 there is relatively little information on the presence of AS and AL in the peripheral nervous system of the gut.16 The latter studies showed the presence of AS and AL immunoreactivities in the varicose nerve fibers within the circular and longitudinal muscle layers of the canine proximal colon. Furthermore, such immunoreactivities were found to be colocalized with NOS. In our studies, although the presence of AS and AL immunoreactivities were not examined, the reversal of L-NNA–suppressed IAS relaxation by Lcitrulline, the restoration of decrease in the IAS tension by L-citrulline, and the blockade of the effect by L-glutamine strongly suggest the existence of the enzymatic machinery in the IAS. Furthermore, our recent studies showing the presence of AS and AL at the transcriptional level27 provide additional support to the concept. It has been frequently observed that NANC nerve – mediated IAS relaxation in in vivo2,4,28 and in vitro3,17 WBS-Gastro
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studies in response to short-term rectoanal inhibitory reflex by rectal balloon distention, sacral nerve stimulation and local intramural stimulation of the myenteric neurons, and EFS can sustain for several hours without any fade. It is possible that interstimulus intervals in these studies were such that the L-arginine stored in the appropriate compartments of the neurons may have been sufficient to cope with the demand or were just enough to allow for the continuous replenishment of L-arginine for the synthesis and release of the inhibitory neurotransmitter. However, in the present studies using continuous EFS, it was obvious that the IAS relaxation could be maintained for some time. However, the IAS tone began to recover in the presence of continuous EFS, and depending on the frequency of EFS, it took 15 – 30 minutes for the maximal recovery of 70% – 80%. We conclude that L-citrulline recycling may be largely responsible for the maintenance of the IAS relaxation in response to NANC nerve stimulation. Furthermore, the present studies in the IAS provide a novel L-arginine – deficient model to investigate the role of L-citrulline recycling in the nitrergic inhibitory neurotransmission in the enteric nervous system. Previous findings that certain analogues of L-arginine (that compete with endogenous L-arginine) block the NOS pathway responsible for the NANC nerve – mediated relaxation of human IAS that can be reversed by L-arginine29 suggest the importance of L-arginine – NOS pathway in anorectal reflex4 and in the pathophysiology of IAS relaxation.
10.
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15. 16.
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nate lyase: expression of the enzymes in rat tissues. J Biochem (Tokyo) 1995;117:952–957. 27. Rattan S, Mori M, Bandyopadhyay A, Chakder S. Evidence for Lcitrulline recycling being responsible for the maintenance of internal anal sphincter (IAS) relaxation by nonadrenergic noncholinergic (NANC) nerve stimulation (abstr). Gastroenterology 1996; 110:A742. 28. Culver PJ, Rattan S. Genesis of anal canal pressures in the opossum. Am J Physiol Gastrointest Liver Physiol 1986;251: G765–G771. 29. Burleigh DE. NG-Nitro-L-arginine reduces nonadrenergic, noncho-
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linergic relaxations of human gut. Gastroenterology 1992;102: 679–683.
Received July 22, 1996. Accepted December 10, 1996. Address requests for reprints to: Satish Rattan, D.V.M., 901 College, Thomas Jefferson University, 1025 Walnut Street, Philadelphia, Pennsylvania. Supported by National Institutes of Diabetes and Digestive and Kidney Diseases grant DK-35385 and an institutional grant from Thomas Jefferson University.
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