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Modulation of Neuronal Nitric Oxide Release by Soluble Guanylyl Cyclase in Guinea Pig Colon
Biochemical and Biophysical Research Communications 280, 1130 –1134 (2001) doi:10.1006/bbrc.2001.4241, available online at http://www.idealibrary.com on
Modulation of Neuronal Nitric Oxide Release by Soluble Guanylyl Cyclase in Guinea Pig Colon K. Halle´n,* ,† C. Olgart,† L. E. Gustafsson,† and N. P. Wiklund* ,† *Department of Urology, Karolinska Hospital, 171 76 Stockholm, Sweden; and †Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Received December 28, 2000
Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3ⴕ,5ⴕ-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexuscontaining smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO 2ⴚ. In the presence of 8-BrcGMP, YC-1 and zaprinast (all at 10 ⴚ4 M) the NO/NO 2ⴚrelease increased to 152 ⴞ 16% (P < 0.05), 164 ⴞ 37% (P < 0.05) and 290 ⴞ 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10 ⴚ5 M) decreased the evoked release of NO/NO 2ⴚ to 49 ⴞ 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones. © 2001 Academic Press Key Words: nitric oxide (NO); nitrite (NO 2ⴚ); autonomic neurotransmission; cyclic guanosine 3ⴕ,5ⴕmonophosphate (cGMP); soluble guanylyl cyclase (sGC); phosphodiesterase (PDE); YC-1; zaprinast; ODQ.
Nitric oxide (NO) has been identified as a neurotransmitter in both the peripheral and central nervous systems (1–3). It accounts for many autonomic responses in the cardiovascular system, as well as in the gastrointestinal and urogenital tracts, such as regulation of blood flow and blood pressure (4) inhibition of gastrointestinal motility (5) and relaxation of the ureAbbreviations used: NO, nitric oxide; NO2⫺, nitrite; NO 3⫺, nitrate; NOS, NO synthase; sGC, soluble guanylyl cyclase; cGMP, cyclic guanosine 3⬘,5⬘-monophosphate; PDE, phosphodiesterase; YC-1, 3-(5⬘hydroxymethyl-2⬘-furyl)-1-benzylindazole; ODQ, (1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one); DMSO, dimethyl sulfoxide; L-NAME, N nitro-L-arginine methyl ester; TTX, tetrodotoxin. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
thra during the micturition reflex (6). Although NO is involved in several important physiological functions, remarkably little is known about the regulation of neuronal release of NO in the peripheral autonomic nervous system. The majority of effects of NO are mediated via the soluble guanylyl cyclase (sGC)/cyclic guanosine 3⬘,5⬘-monophosphate (cGMP) pathway. However, NO may also exert direct effects on ion channels in smooth muscle cells (7). During stimulation of nitrergic neurones, NO is formed, released and will diffuse over the synaptic cleft, pass the cell membrane of surrounding cells and interact with the heme moiety of sGC (8). This leads to an increased formation of cGMP, which will affect cell activity via cGMP-dependent protein kinases. cGMP is degraded in the cells by phosphodiesterases (PDE), in particular PDE V (9). NO itself is rapidly oxidised to nitrite (NO 2⫺) and in the presence of heme-containing proteins (e.g., haemoglobin) it will be further oxidised to nitrate (NO 3⫺) (10). The sGC/cGMP pathway is present in many autonomic neurones as confirmed by immunohistochemistry (11). Several studies have been aimed at the sGC/ cGMP pathway in neurones but the research has been hampered by the lack of specific pharmacological tools. The introduction of ODQ, a selective sGC inhibitor (12), and YC-1, a sGC stimulator (13), has opened up possibilities to elucidate guanylyl cyclase mediated effects more selectively. As yet, it is unclear whether there might be an autoregulatory feedback mechanism via the sGC/cGMP pathway for the neuronal release of NO. The aim of the present study was therefore to investigate if sGC modulates nerve-induced NO release from peripheral autonomic neurones in the guinea-pig gastrointestinal tract. MATERIALS AND METHODS Tissue preparation. Male Dunkin-Hartley guinea pigs (350 –500 g) were anaesthetised by carbon dioxide and exsanguinated. The mesenteric artery was cannulated and perfused with 0.9% saline. The distal part of the colon was removed and a 25 cm long strip of the
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longitudinal muscle layer with the underlying myenteric plexus was isolated according to Rang (14), folded to a 5 cm long preparation and tied between thin threads. Measurements of NO/NO 2⫺. The tissue was mounted in a glass chamber at a load of 10 mN with continuous superfusion (1 ml min ⫺1) of Tyrode’s solution (Na ⫹ 161 mM, K ⫹ 2.8 mM, Ca 2⫹ 1.8 mM, Mg 2⫹ 0.5 mM, Cl ⫺ 144 mM, HCO 3⫺ 24 mM, H 2PO 3⫺ 0.4 mM, glucose 5.5 mM), heated to 37°C and continuously aerated with 6.5% CO 2 in O 2. The NO-substrate L-arginine (10 ⫺5 M) was added to the Tyrode’s solution and the tissue was left for 180 min in order to equilibrate before drugs were added. Transmural electrical stimulation (50 V biphasic, 32 Hz, pulse duration 1 ms) was applied for 1 min at 45 min intervals with needle shaped silver electrodes using a Grass S88 stimulator. The electrodes were placed approximately 4.5 cm apart. The muscle activity of the tissue was recorded isometrically by a Grass transducer (FT03) and displayed with a Grass model 7P1 Polygraph (Grass Instruments, Quincy, MA, USA). The tissue superfusate was collected prior to and during electrical stimulation. Aliquots of 1 ml were injected into a reaction chamber containing 100 ml 1% sodium iodide in deoxygenated and concentrated hot acetic acid, where NO 2⫺ is reduced to NO. NO was carried further by a stream of N 2 into a reaction chamber and reacted with ozone under vacuum to give rise to photons, which were counted in a photomultiplier. The system was cautiously calibrated by injecting aliquots of NaNO 2 solution and using peak heights for construction of standard curves for calculation of unknown samples (15, 16). The detection limit as 1–2 pmol NaNO 2 per ml sample. The concentrations of 8-Br-cGMP (10 ⫺4 M), YC-1 (10 ⫺4 M) and zaprinast (10 ⫺4 M) were chosen on the basis of their maximal inhibitory effect on nervestimulation-evoked contractions in organbath experiments (data not shown). The concentration of ODQ (10 ⫺5 M) was chosen in accordance to the maximal enhancing effect on nerve-stimulation-evoked contractions in organbath experiments (data not shown). Drugs. L-Arginine, 8-Br-cGMP, zaprinast and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO), YC-1 was from
TABLE 1 ⫺ 2
Release of NO/NO from Guinea Pig Colon upon Electrical Stimulation [pmol/min]
Basal release Evoked release
Basal release Evoked release
Basal release Evoked release
Basal release Evoked release
Control
8-Br-cGMP 10 ⫺4 M
65 ⫾ 10 27 ⫾ 3
65 ⫾ 8 40 ⫾ 5
Control
YC-1 10 ⫺4 M
57 ⫾ 8 17 ⫾ 3
73 ⫾ 13 25 ⫾ 5
Control
Zaprinast 10 ⫺4 M
48 ⫾ 9 14 ⫾ 5
48 ⫾ 8 28 ⫾ 6
Control
ODQ 10 ⫺5 M
65 ⫾ 9 23 ⫾ 6
67 ⫾ 8 11 ⫾ 3
n n.s. *
5 5
TABLE 2
Contractile Responses to Nerve Stimulation in Guinea Pig Colon [mN] Control
8-Br-cGMP 10 ⫺4 M
50 ⫾ 5
40 ⫾ 4
Control
YC-1 10 ⫺4 M
56 ⫾ 8
29 ⫾ 6
Control
Zaprinast 10 ⫺4 M
68 ⫾ 9
65 ⫾ 9
Control
ODQ 10 ⫺5 M
49 ⫾ 14
60 ⫾ 19
n *
5 n
*
7 n
n.s.
6 n
*
6
Note. Muscular contraction of the plexus-containing longitudinal muscle of guinea pig colon upon electrical stimulation (50 V, 32 Hz, pulse duration 1 ms). Statistically significant increases and decreases of the contractile response by 8-Br-cGMP (10 ⫺4 M), YC-1 (10 ⫺4 M), zaprinast (10 ⫺4 M) and ODQ (10 ⫺5 M) are denoted by * (P ⬍ 0.05). Results are shown as mN, mean ⫾ SEM; n denotes number of experiments.
Alexis Corporation (San Diego, CA) and ODQ was from Tocris Cookson Ltd. (Bristol, UK). ODQ, YC-1 and zaprinast were dissolved in DMSO. All other solutions were prepared from ultra filtrated water (18.2 M⍀ resistance after passage through ␣Q-filter, Millipore (Bedford, MA)). DMSO diluted 1:500 or 1:1000 in the tissue superfusate did not release any NO 2⫺. Drugs diluted 1:1000 in Tyrode’s solution did not contain any detectable NO 2⫺ (data not shown). Statistical analysis. Experimental data are expressed as means ⫾ SEM. Statistical significance was tested according to Student’s t test for paired observations, except where normality failed, then Wilcoxon’s tests was used. n indicates the number of experiments.
RESULTS
n * *
7 7 n
n.s. *
6 6 n
n.s. *
6 6
Note. NO/NO 2⫺ release from plexus-containing longitudinal muscle of guinea pig colon upon electrical stimulation (50 V, 32 Hz, pulse duration 1 ms). Statistically significant increases and decreases of NO/NO 2⫺ release by 8-Br-cGMP (10 ⫺4 M), YC-1 (10 ⫺4 M), zaprinast (10 ⫺4 M) and ODQ (10 ⫺5 M) are denoted by * (P ⬍ 0.05). Results are shown as pmol/min, mean ⫾ SEM; n denotes number of experiments.
Electrical stimulation (50 V, 32 Hz, 1 ms) of the plexus-containing smooth muscle preparation evoked a smooth muscle contraction and a marked increase of NO/NO 2⫺ in the tissue superfusate as described previously (15, 16) and the stimulation-evoked release of NO/NO 2⫺ was reproducible over several hours (15, 16). Thus, the NO/NO 2⫺ release during the third stimulation amounted to 105 ⫾ 11% (n ⫽ 23). Addition of YC-1 (10 ⫺4 M) to the superfusate increased the basal release of NO/NO 2⫺ by 24 ⫾ 8% (P ⬍ 0.05, n ⫽ 7), while 8-Br-cGMP (10 ⫺4 M), zaprinast (10 ⫺4 M) or ODQ (10 ⫺5 M) did not have any significant effect. However, the cGMP-analogue 8-Br-cGMP (10 ⫺4 M) increased the nerve stimulation-induced NO/NO 2⫺ release to 152 ⫾ 16% (P ⬍ 0.05, n ⫽ 5) and inhibited the nerve-induced contractions to 79 ⫾ 3% (P ⬍ 0.05, n ⫽ 5) of controls (Tables 1 and 2, Fig. 1). Further-
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FIG. 1. Effect of 8-Br-cGMP (10 ⫺4 M) on nerve-induced NO/NO 2⫺ release and contractile responses in plexus-containing guinea pig colon longitudinal muscle. Each bar represents the evoked release during a period of transmural nerve stimulation (50 V, 32 Hz, pulse duration 1 ms). 8-Br-cGMP (10 ⫺4 M) increased the nerve induced NO/NO 2⫺ release and inhibited the nerve-induced-contractions (n ⫽ 5, P ⬍ 0.05).
cells (11). Furthermore, the NO-induced inhibition of contractile responses to electrical stimulation in the gastrointestinal tract has been shown to be mediated by the sGC/cGMP pathway (21). Thus, the importance of NO as a transmitter within the gastrointestinal tract has been established but the regulation of NO release from autonomic neurones has not been fully understood. The recent introduction of pharmacological drugs that may selectively stimulate or inhibit sGC activity has opened up possibilities to study the involvement of the sGC/cGMP pathway in the regulation of neuronal NO release. NO released from neurones is rapidly oxidised to nitrite and nitrate which can be quantified by chemiluminescence as previously described (15, 16). We found that drugs that are known to cause accumulation of cGMP (8-Br-cGMP, YC-1 and zaprinast) enhanced the nerve-induced release of NO. In line with this finding, we could note that inhibition of sGC elicited a marked decrease in nerve-induced NO release. Thus, we conclude that the sGC/cGMP pathway is involved in the regulation of neuronal NO release. The sGC inhibitor ODQ increased the smooth muscle contraction in guinea-pig colon. This effect was likely caused either by decreased formation of cGMP in smooth muscle cells or decreased release of NO from the neurones. Conversely, the cGMP-elevating agents
more, stimulation of the sGC by YC-1 (10 ⫺4 M) and inhibition of PDE V by zaprinast (10 ⫺4 M) increased the nerve-induced NO/NO 2⫺ release to 164 ⫾ 37% (P ⬍ 0.05, n ⫽ 7) and to 290 ⫾ 67% (P ⬍ 0.05, n ⫽ 6) of controls, respectively (Table 1). The nerve-induced contractions were decreased to 51 ⫾ 5% (P ⬍ 0.05, n ⫽ 7) by YC-1 (10 ⫺4 M), while zaprinast (10 ⫺4 M) did not affect the contractions significantly (Table 2). Conversely, inhibition of the sGC by ODQ (10 ⫺5 M) decreased the nerve-stimulation-evoked release of NO/ NO 2⫺ to 49 ⫾ 7% (P ⬍ 0.05, n ⫽ 6) and enhanced the nerve-induced contractions to 121 ⫾ 6% (P ⬍ 0.05, n ⫽ 6) of controls (Tables 1 and 2, Fig. 2). DISCUSSION NO is an important neurotransmitter within the gastrointestinal tract. The effects are mainly inhibitory, such as relaxation of the cardia and reduction of the intestinal motility (17). The enzyme responsible for NO synthesis, the neuronal NO synthase (nNOS), has been found in the submucosa and in the myenteric plexus in the gastrointestinal wall, by immunocytochemistry and Western blot (18 –20). The majority of the NO effects in the gut are mediated via the sGC/cGMP pathway. NO has been shown to cause accumulation of cGMP-like immunoreactivity in colonic smooth muscle
FIG. 2. Effect of ODQ (10 ⫺5 M) on nerve-induced NO/NO 2⫺ release and contractile responses in plexus-containing guinea pig colon longitudinal muscle. Each bar represents the evoked release during a period of transmural nerve stimulation (50 V, 32 Hz, pulse duration 1 ms). ODQ (10 ⫺5 M) decreased the nerve-induced NO/NO 2⫺ release and enhanced the nerve-induced contractions (n ⫽ 6, P ⬍ 0.05).
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8-Br-cGMP and YC-1 used in this study caused decreased contraction of the smooth muscle in guinea pig colon and increases in nerve-induced NO release. Zaprinast however, did not affect the contraction of the smooth muscle and the reason for this is unclear at present. It might be that zaprinast may enhance the release or the effect of a contractile factor since NO in a cGMP-dependent way can contract certain intestinal smooth muscle (22). Furthermore studies are required to determine to what extent the effects of cGMP are mediated by changes in formation of cGMP in smooth muscle cells or alterations in neuronal NO release. There are reports stating that YC-1 and ODQ lack specificity for the sGC (23, 24). However, we have used three different pharmacological agents, all of which act by increasing the cGMP levels in the effector cells. They all use different mechanisms, but still the consequences are similar: facilitation of NO release. This suggests that the observed changes in neuroeffector responses are indeed due to alterations in the sGC/ cGMP pathway. In fact, application of the sGC inhibitor ODQ resulted in the opposite effect, further supporting a role for cGMP in this respect. It is unclear whether the sGC/cGMP modulation of neuronal NO release would occur in the nitrergic neurone or in some other cell type. One possible mechanism would be that cGMP itself upregulates the formation of NO within the nitrergic neurone. However, this is unlikely since NOS does not co-localise with cGMP in either myenteric or submucosal neurones in the canine proximal colon (11). Furthermore, there is ample evidence showing that NO inhibits its own synthesis by negative feedback on the NOS isoforms nNOS (25), iNOS (26), and eNOS (27). This speaks against the possibility that an increased NO release from the peripheral autonomic neurones could be caused by NO mediated NOS stimulation. It is more likely that this modulation occurs in a cell located close to the nitrergic neurone. cGMP upregulation has been shown in smooth muscle cells, neurones and interneurones, and it is possible that the cGMP upregulation would lead to release of a factor that may facilitate further NO release. This hypothesis has to be further explored. CONCLUSION In conclusion, by using various drugs that affect the sGC/cGMP pathway in guinea pig colon we have demonstrated that intervention with this pathway modulates the nerve-stimulation-induced NO release. Taken together, these data strongly suggest that NO exerts a positive feedback on its own release from peripheral autonomic neurones via the sGC/cGMP pathway. ACKNOWLEDGMENT This work was supported by Gunvor och Josef Ane´rs stiftelse, Stiftelsen Johanna Hagstrand och Sigfrid Linne´rs minne, Swedish
Society for Medical Research, the Swedish MRC Grants 11199 and 7919, and the Karolinska Institute.
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17. Brookes, S. J. (1993) Neuronal nitric oxide in the gut. J. Gastroenterol. Hepatol. 8, 590 – 603. 18. Llewellyn-Smith, I. J., Song, Z. M., Costa, M., Bredt, D. S., and Snyder, S. H. (1992) Ultrastructural localization of nitric oxide synthase immunoreactivity in guinea-pig enteric neurons. Brain Res. 577, 337–342. 19. Nichols, K., Krantis, A., and Staines, W. (1992) Histochemical localization of nitric oxide-synthesizing neurons and vascular sites in the guinea-pig intestine. Neuroscience 51, 791–799. 20. Sheng, H., Schmidt, H. H., Nakane, M., Mitchell, J. A., Pollock, J. S., et al. (1992) Characterization and localization of nitric oxide synthase in non-adrenergic non-cholinergic nerves from bovine retractor penis muscles. Br. J. Pharmacol. 106, 768 –773. 21. Olgart, C., Hallen, K., Wiklund, N. P., Iversen, H. H., and Gustafsson, L. E. (1998). Blockade of nitrergic neuroeffector transmission in guinea-pig colon by a selective inhibitor of soluble guanylyl cyclase. Acta Physiol. Scand. 162, 89 –95. 22. Olgart, C., Wiklund, N. P., and Gustafsson, L. E. (1997) Blockade of nitric oxide-evoked smooth muscle contractions by an inhibitor of guanylyl cyclase. Neuroreport 8, 3355–3358.
23. Friebe, A., Mullershausen, F., Smolenski, A., Walter, U., Schultz, G., et al. (1998) YC-1 potentiates nitric oxide- and carbon monoxide-induced cyclic GMP effects in human platelets. Mol. Pharmacol. 54, 962–967. 24. Feelisch, M., Kotsonis, P., Siebe, J., Clement, B., and Schmidt, H. H. (1999) The soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3,-a] quinoxalin-1-one is a nonselective heme protein inhibitor of nitric oxide synthase and other cytochrome P-450 enzymes involved in nitric oxide donor bioactivation. Mol. Pharmacol. 56, 243–253. 25. Rogers, N. E., and Ignarro, L. J. (1992) Constitutive nitric oxide synthase from cerebellum is reversibly inhibited by nitric oxide formed from L-arginine. Biochem. Biophys. Res. Commun. 189, 242–249. 26. Assreuy, J., Cunha, F. Q., Liew, F. Y., and Moncada, S. (1993) Feedback inhibition of nitric oxide synthase activity by nitric oxide. Br. J. Pharmacol. 108, 833– 837. 27. Buga, G. M., Griscavage, J. M., Rogers, N. E., and Ignarro, L. J. (1993) Negative feedback regulation of endothelial cell function by nitric oxide. Circ. Res. 73, 808 – 812.
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Modulation of Neuronal Nitric Oxide Release by Soluble Guanylyl Cyclase in Guinea Pig Colon K. Halle´n,* ,† C. Olgart,† L. E. Gustafsson,† and N. P. Wiklund* ,† *Department of Urology, Karolinska Hospital, 171 76 Stockholm, Sweden; and †Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
Received December 28, 2000
Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3ⴕ,5ⴕ-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexuscontaining smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO 2ⴚ. In the presence of 8-BrcGMP, YC-1 and zaprinast (all at 10 ⴚ4 M) the NO/NO 2ⴚrelease increased to 152 ⴞ 16% (P < 0.05), 164 ⴞ 37% (P < 0.05) and 290 ⴞ 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10 ⴚ5 M) decreased the evoked release of NO/NO 2ⴚ to 49 ⴞ 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones. © 2001 Academic Press Key Words: nitric oxide (NO); nitrite (NO 2ⴚ); autonomic neurotransmission; cyclic guanosine 3ⴕ,5ⴕmonophosphate (cGMP); soluble guanylyl cyclase (sGC); phosphodiesterase (PDE); YC-1; zaprinast; ODQ.
Nitric oxide (NO) has been identified as a neurotransmitter in both the peripheral and central nervous systems (1–3). It accounts for many autonomic responses in the cardiovascular system, as well as in the gastrointestinal and urogenital tracts, such as regulation of blood flow and blood pressure (4) inhibition of gastrointestinal motility (5) and relaxation of the ureAbbreviations used: NO, nitric oxide; NO2⫺, nitrite; NO 3⫺, nitrate; NOS, NO synthase; sGC, soluble guanylyl cyclase; cGMP, cyclic guanosine 3⬘,5⬘-monophosphate; PDE, phosphodiesterase; YC-1, 3-(5⬘hydroxymethyl-2⬘-furyl)-1-benzylindazole; ODQ, (1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one); DMSO, dimethyl sulfoxide; L-NAME, N nitro-L-arginine methyl ester; TTX, tetrodotoxin. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
thra during the micturition reflex (6). Although NO is involved in several important physiological functions, remarkably little is known about the regulation of neuronal release of NO in the peripheral autonomic nervous system. The majority of effects of NO are mediated via the soluble guanylyl cyclase (sGC)/cyclic guanosine 3⬘,5⬘-monophosphate (cGMP) pathway. However, NO may also exert direct effects on ion channels in smooth muscle cells (7). During stimulation of nitrergic neurones, NO is formed, released and will diffuse over the synaptic cleft, pass the cell membrane of surrounding cells and interact with the heme moiety of sGC (8). This leads to an increased formation of cGMP, which will affect cell activity via cGMP-dependent protein kinases. cGMP is degraded in the cells by phosphodiesterases (PDE), in particular PDE V (9). NO itself is rapidly oxidised to nitrite (NO 2⫺) and in the presence of heme-containing proteins (e.g., haemoglobin) it will be further oxidised to nitrate (NO 3⫺) (10). The sGC/cGMP pathway is present in many autonomic neurones as confirmed by immunohistochemistry (11). Several studies have been aimed at the sGC/ cGMP pathway in neurones but the research has been hampered by the lack of specific pharmacological tools. The introduction of ODQ, a selective sGC inhibitor (12), and YC-1, a sGC stimulator (13), has opened up possibilities to elucidate guanylyl cyclase mediated effects more selectively. As yet, it is unclear whether there might be an autoregulatory feedback mechanism via the sGC/cGMP pathway for the neuronal release of NO. The aim of the present study was therefore to investigate if sGC modulates nerve-induced NO release from peripheral autonomic neurones in the guinea-pig gastrointestinal tract. MATERIALS AND METHODS Tissue preparation. Male Dunkin-Hartley guinea pigs (350 –500 g) were anaesthetised by carbon dioxide and exsanguinated. The mesenteric artery was cannulated and perfused with 0.9% saline. The distal part of the colon was removed and a 25 cm long strip of the
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longitudinal muscle layer with the underlying myenteric plexus was isolated according to Rang (14), folded to a 5 cm long preparation and tied between thin threads. Measurements of NO/NO 2⫺. The tissue was mounted in a glass chamber at a load of 10 mN with continuous superfusion (1 ml min ⫺1) of Tyrode’s solution (Na ⫹ 161 mM, K ⫹ 2.8 mM, Ca 2⫹ 1.8 mM, Mg 2⫹ 0.5 mM, Cl ⫺ 144 mM, HCO 3⫺ 24 mM, H 2PO 3⫺ 0.4 mM, glucose 5.5 mM), heated to 37°C and continuously aerated with 6.5% CO 2 in O 2. The NO-substrate L-arginine (10 ⫺5 M) was added to the Tyrode’s solution and the tissue was left for 180 min in order to equilibrate before drugs were added. Transmural electrical stimulation (50 V biphasic, 32 Hz, pulse duration 1 ms) was applied for 1 min at 45 min intervals with needle shaped silver electrodes using a Grass S88 stimulator. The electrodes were placed approximately 4.5 cm apart. The muscle activity of the tissue was recorded isometrically by a Grass transducer (FT03) and displayed with a Grass model 7P1 Polygraph (Grass Instruments, Quincy, MA, USA). The tissue superfusate was collected prior to and during electrical stimulation. Aliquots of 1 ml were injected into a reaction chamber containing 100 ml 1% sodium iodide in deoxygenated and concentrated hot acetic acid, where NO 2⫺ is reduced to NO. NO was carried further by a stream of N 2 into a reaction chamber and reacted with ozone under vacuum to give rise to photons, which were counted in a photomultiplier. The system was cautiously calibrated by injecting aliquots of NaNO 2 solution and using peak heights for construction of standard curves for calculation of unknown samples (15, 16). The detection limit as 1–2 pmol NaNO 2 per ml sample. The concentrations of 8-Br-cGMP (10 ⫺4 M), YC-1 (10 ⫺4 M) and zaprinast (10 ⫺4 M) were chosen on the basis of their maximal inhibitory effect on nervestimulation-evoked contractions in organbath experiments (data not shown). The concentration of ODQ (10 ⫺5 M) was chosen in accordance to the maximal enhancing effect on nerve-stimulation-evoked contractions in organbath experiments (data not shown). Drugs. L-Arginine, 8-Br-cGMP, zaprinast and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO), YC-1 was from
TABLE 1 ⫺ 2
Release of NO/NO from Guinea Pig Colon upon Electrical Stimulation [pmol/min]
Basal release Evoked release
Basal release Evoked release
Basal release Evoked release
Basal release Evoked release
Control
8-Br-cGMP 10 ⫺4 M
65 ⫾ 10 27 ⫾ 3
65 ⫾ 8 40 ⫾ 5
Control
YC-1 10 ⫺4 M
57 ⫾ 8 17 ⫾ 3
73 ⫾ 13 25 ⫾ 5
Control
Zaprinast 10 ⫺4 M
48 ⫾ 9 14 ⫾ 5
48 ⫾ 8 28 ⫾ 6
Control
ODQ 10 ⫺5 M
65 ⫾ 9 23 ⫾ 6
67 ⫾ 8 11 ⫾ 3
n n.s. *
5 5
TABLE 2
Contractile Responses to Nerve Stimulation in Guinea Pig Colon [mN] Control
8-Br-cGMP 10 ⫺4 M
50 ⫾ 5
40 ⫾ 4
Control
YC-1 10 ⫺4 M
56 ⫾ 8
29 ⫾ 6
Control
Zaprinast 10 ⫺4 M
68 ⫾ 9
65 ⫾ 9
Control
ODQ 10 ⫺5 M
49 ⫾ 14
60 ⫾ 19
n *
5 n
*
7 n
n.s.
6 n
*
6
Note. Muscular contraction of the plexus-containing longitudinal muscle of guinea pig colon upon electrical stimulation (50 V, 32 Hz, pulse duration 1 ms). Statistically significant increases and decreases of the contractile response by 8-Br-cGMP (10 ⫺4 M), YC-1 (10 ⫺4 M), zaprinast (10 ⫺4 M) and ODQ (10 ⫺5 M) are denoted by * (P ⬍ 0.05). Results are shown as mN, mean ⫾ SEM; n denotes number of experiments.
Alexis Corporation (San Diego, CA) and ODQ was from Tocris Cookson Ltd. (Bristol, UK). ODQ, YC-1 and zaprinast were dissolved in DMSO. All other solutions were prepared from ultra filtrated water (18.2 M⍀ resistance after passage through ␣Q-filter, Millipore (Bedford, MA)). DMSO diluted 1:500 or 1:1000 in the tissue superfusate did not release any NO 2⫺. Drugs diluted 1:1000 in Tyrode’s solution did not contain any detectable NO 2⫺ (data not shown). Statistical analysis. Experimental data are expressed as means ⫾ SEM. Statistical significance was tested according to Student’s t test for paired observations, except where normality failed, then Wilcoxon’s tests was used. n indicates the number of experiments.
RESULTS
n * *
7 7 n
n.s. *
6 6 n
n.s. *
6 6
Note. NO/NO 2⫺ release from plexus-containing longitudinal muscle of guinea pig colon upon electrical stimulation (50 V, 32 Hz, pulse duration 1 ms). Statistically significant increases and decreases of NO/NO 2⫺ release by 8-Br-cGMP (10 ⫺4 M), YC-1 (10 ⫺4 M), zaprinast (10 ⫺4 M) and ODQ (10 ⫺5 M) are denoted by * (P ⬍ 0.05). Results are shown as pmol/min, mean ⫾ SEM; n denotes number of experiments.
Electrical stimulation (50 V, 32 Hz, 1 ms) of the plexus-containing smooth muscle preparation evoked a smooth muscle contraction and a marked increase of NO/NO 2⫺ in the tissue superfusate as described previously (15, 16) and the stimulation-evoked release of NO/NO 2⫺ was reproducible over several hours (15, 16). Thus, the NO/NO 2⫺ release during the third stimulation amounted to 105 ⫾ 11% (n ⫽ 23). Addition of YC-1 (10 ⫺4 M) to the superfusate increased the basal release of NO/NO 2⫺ by 24 ⫾ 8% (P ⬍ 0.05, n ⫽ 7), while 8-Br-cGMP (10 ⫺4 M), zaprinast (10 ⫺4 M) or ODQ (10 ⫺5 M) did not have any significant effect. However, the cGMP-analogue 8-Br-cGMP (10 ⫺4 M) increased the nerve stimulation-induced NO/NO 2⫺ release to 152 ⫾ 16% (P ⬍ 0.05, n ⫽ 5) and inhibited the nerve-induced contractions to 79 ⫾ 3% (P ⬍ 0.05, n ⫽ 5) of controls (Tables 1 and 2, Fig. 1). Further-
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FIG. 1. Effect of 8-Br-cGMP (10 ⫺4 M) on nerve-induced NO/NO 2⫺ release and contractile responses in plexus-containing guinea pig colon longitudinal muscle. Each bar represents the evoked release during a period of transmural nerve stimulation (50 V, 32 Hz, pulse duration 1 ms). 8-Br-cGMP (10 ⫺4 M) increased the nerve induced NO/NO 2⫺ release and inhibited the nerve-induced-contractions (n ⫽ 5, P ⬍ 0.05).
cells (11). Furthermore, the NO-induced inhibition of contractile responses to electrical stimulation in the gastrointestinal tract has been shown to be mediated by the sGC/cGMP pathway (21). Thus, the importance of NO as a transmitter within the gastrointestinal tract has been established but the regulation of NO release from autonomic neurones has not been fully understood. The recent introduction of pharmacological drugs that may selectively stimulate or inhibit sGC activity has opened up possibilities to study the involvement of the sGC/cGMP pathway in the regulation of neuronal NO release. NO released from neurones is rapidly oxidised to nitrite and nitrate which can be quantified by chemiluminescence as previously described (15, 16). We found that drugs that are known to cause accumulation of cGMP (8-Br-cGMP, YC-1 and zaprinast) enhanced the nerve-induced release of NO. In line with this finding, we could note that inhibition of sGC elicited a marked decrease in nerve-induced NO release. Thus, we conclude that the sGC/cGMP pathway is involved in the regulation of neuronal NO release. The sGC inhibitor ODQ increased the smooth muscle contraction in guinea-pig colon. This effect was likely caused either by decreased formation of cGMP in smooth muscle cells or decreased release of NO from the neurones. Conversely, the cGMP-elevating agents
more, stimulation of the sGC by YC-1 (10 ⫺4 M) and inhibition of PDE V by zaprinast (10 ⫺4 M) increased the nerve-induced NO/NO 2⫺ release to 164 ⫾ 37% (P ⬍ 0.05, n ⫽ 7) and to 290 ⫾ 67% (P ⬍ 0.05, n ⫽ 6) of controls, respectively (Table 1). The nerve-induced contractions were decreased to 51 ⫾ 5% (P ⬍ 0.05, n ⫽ 7) by YC-1 (10 ⫺4 M), while zaprinast (10 ⫺4 M) did not affect the contractions significantly (Table 2). Conversely, inhibition of the sGC by ODQ (10 ⫺5 M) decreased the nerve-stimulation-evoked release of NO/ NO 2⫺ to 49 ⫾ 7% (P ⬍ 0.05, n ⫽ 6) and enhanced the nerve-induced contractions to 121 ⫾ 6% (P ⬍ 0.05, n ⫽ 6) of controls (Tables 1 and 2, Fig. 2). DISCUSSION NO is an important neurotransmitter within the gastrointestinal tract. The effects are mainly inhibitory, such as relaxation of the cardia and reduction of the intestinal motility (17). The enzyme responsible for NO synthesis, the neuronal NO synthase (nNOS), has been found in the submucosa and in the myenteric plexus in the gastrointestinal wall, by immunocytochemistry and Western blot (18 –20). The majority of the NO effects in the gut are mediated via the sGC/cGMP pathway. NO has been shown to cause accumulation of cGMP-like immunoreactivity in colonic smooth muscle
FIG. 2. Effect of ODQ (10 ⫺5 M) on nerve-induced NO/NO 2⫺ release and contractile responses in plexus-containing guinea pig colon longitudinal muscle. Each bar represents the evoked release during a period of transmural nerve stimulation (50 V, 32 Hz, pulse duration 1 ms). ODQ (10 ⫺5 M) decreased the nerve-induced NO/NO 2⫺ release and enhanced the nerve-induced contractions (n ⫽ 6, P ⬍ 0.05).
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8-Br-cGMP and YC-1 used in this study caused decreased contraction of the smooth muscle in guinea pig colon and increases in nerve-induced NO release. Zaprinast however, did not affect the contraction of the smooth muscle and the reason for this is unclear at present. It might be that zaprinast may enhance the release or the effect of a contractile factor since NO in a cGMP-dependent way can contract certain intestinal smooth muscle (22). Furthermore studies are required to determine to what extent the effects of cGMP are mediated by changes in formation of cGMP in smooth muscle cells or alterations in neuronal NO release. There are reports stating that YC-1 and ODQ lack specificity for the sGC (23, 24). However, we have used three different pharmacological agents, all of which act by increasing the cGMP levels in the effector cells. They all use different mechanisms, but still the consequences are similar: facilitation of NO release. This suggests that the observed changes in neuroeffector responses are indeed due to alterations in the sGC/ cGMP pathway. In fact, application of the sGC inhibitor ODQ resulted in the opposite effect, further supporting a role for cGMP in this respect. It is unclear whether the sGC/cGMP modulation of neuronal NO release would occur in the nitrergic neurone or in some other cell type. One possible mechanism would be that cGMP itself upregulates the formation of NO within the nitrergic neurone. However, this is unlikely since NOS does not co-localise with cGMP in either myenteric or submucosal neurones in the canine proximal colon (11). Furthermore, there is ample evidence showing that NO inhibits its own synthesis by negative feedback on the NOS isoforms nNOS (25), iNOS (26), and eNOS (27). This speaks against the possibility that an increased NO release from the peripheral autonomic neurones could be caused by NO mediated NOS stimulation. It is more likely that this modulation occurs in a cell located close to the nitrergic neurone. cGMP upregulation has been shown in smooth muscle cells, neurones and interneurones, and it is possible that the cGMP upregulation would lead to release of a factor that may facilitate further NO release. This hypothesis has to be further explored. CONCLUSION In conclusion, by using various drugs that affect the sGC/cGMP pathway in guinea pig colon we have demonstrated that intervention with this pathway modulates the nerve-stimulation-induced NO release. Taken together, these data strongly suggest that NO exerts a positive feedback on its own release from peripheral autonomic neurones via the sGC/cGMP pathway. ACKNOWLEDGMENT This work was supported by Gunvor och Josef Ane´rs stiftelse, Stiftelsen Johanna Hagstrand och Sigfrid Linne´rs minne, Swedish
Society for Medical Research, the Swedish MRC Grants 11199 and 7919, and the Karolinska Institute.
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