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Histochemical localization of cGMP in canine proximal colon
ABSTRACTS
October 1992
OF PAPERS
1375
HISTCICHEMICAL L.OCALIZATION OF cGMP IN CANINE PROXIMAL COLON. C.W.R. Shuttleworth, C. Xue, SM. Ward and K.M. Sanders. Dept. Physiology, University of Nevada, Reno NV. Nitric oxide (NO) may be an inhibitory neurotransmitter in gastrointestinal smooth muscles. NO activates soluble guanylate cvclases. and it is wssible that cGMP mediates the inhibitory effects of NO. Immunohistochemical techniques were used to determine whether cGMP levels increase in response to NO. Thin strips of muscle with mucosa removed were stimulated with NO (60 ~1 saturated solution by bolus), sodium nitropmsside (SNP) (lo-4 M, IO min). or electrical field stimulation (EFS: 5Hz.30-120 s). M&B 22948 (104M) was added to inhibit cGMp ph&phohiesterask activity, and atropine (1odM) was also present during EFS. After stimuli, muscles were fixed with paraformaldehyde (4%). Formaldehyde stabilized cGMP was localized with antiserum C-5 (1:150; deVente and Steinbusch. Acta histochem. 92, 13-38, 1992) and conventional immunocytochemical techniques for visualization at the light or EM level. None of the stimuli resulted in demonstrable increases in cGMP-like immunoreactivity (cGMP-LI) in smooth muscle cells of the circular or longitudinal muscle layers. Both NO and SNP (but not EFS) caused striking increases in cGMP-LI in endothelial cells and vascular smooth muscle cells. cGMP-LI also increased in a subpopulation of nerve cell bodies in myenteric and submucosal ganglia following either EFS or NO stimulation. NO also increased cGMP-LI in the interstitial cells at the submucosal surface of the circular muscle layer that are thought to be electrical pacemakers. These results suggest: i) changes in cGMP in colonic muscle cells are small in response to NO, or ii) effects of NO on muscle cells are mediated via a cGMP’-independent mechanism. In contrast, NO increased cGMP-LI in interstitial cells, and it is possible that a portion of the inhibitory effects of NO could be mediated through the electrical coupling between these cells and smooth muscle cells. The role of the cGMP response in enteric neurons is unknown, but it is possible that NO has pre-junctional effects, regulating the release of other neurotransmitters. (Supported by DK 41315)
NO NANC NERVES MODULATE MIGRATING MOTOR COMPLEXES (MMC’S) AND POSTPRANDIAL STATE. SK. Sama, M.F. Otterson, R.P. Ryan and V.E. Cowles, Dcpts. Surg. & Physiol., Mcd Coil of WI Milwaukee, WI 53226 and Pablo&i VAMC, Milwaukee, WI 53295. We investigated the role of nitric oxide containing non-adrenergic non-cholinergic (NANC) nerves in the regulation of MMCs in the small intestine and cyclic motor activity (CMA) in the stomach in 4 conscious dogs. Each dog was ins!mmentcd with 8 strain gauge transducers on the small intestine and 1 to 2 on the annum. After control recordings, L-NAME was infused i.v. for 4 h @ 10 mg/kg/h or 15 mg&/h starting at 5 min after the end of MMC in the duodenum. The recording was continued for another 4 h. Additional 8 h recordings were made for the next 4 days. L-arginine (150 mg/kg for 8 h), L-NAME (15 mg/kgfh for 4 h) + L-arginine (150 m&g for 8 h), or Angiotensin (10 ng/kg/min) were infused. For postprsndisl experiments, a 650 kCal meal was given 1 h after the start of L-NAME (15 mg&/h for 4 h) infusion. A catheter was implanted in the carotid artery to monitor blood pressure. RESULTS: The infusion of L-NAME at 10 mg/kglh or 15 mg/kgfh for 4 h first induced a premature MMC cycle and then disrupted MMC cycling for the remainder of the day. In several experiments the small intestine exhibited amyogenesia. The gasnic sntmm exhibited a series of continuous contractions with no CMA’s. The MMc’s in the small intestine rcturncd on the second day? but the cyclic motor activity in the stomach remained disrupted. L-argmine alone had no significant effect on MMC cycling, but when given with L-NAME it inhibited the disruptive effect of L-NAME on MMC cycling and gastric CMA. The increase in arterial blood pressure due to Angiotensin II and L-NAME was of the same order but Angiotensin II did not affect the MMC cycling or gastric CMA. During control, a 650 kCa1 solid meal disrupted MMC cycling for 350 f 20 min. Infusion of L-NAME 1 h before the meal significandy decreased the period of MMC disruption to 175 f 29 min. CONCLUSION: 1) NO NANC nerves play a significant role in the disruption of MMC cycling by a meal. 2) NO NANC nerves also play a significant role in the generation of gastric cyclic motor activity, probably by producing the inhibition of contractions to produce phase I activity. These effects are not secondary to increase in arterial blood pressure. Intravenous L-arginine inhibits the in vivo motor effects of LNAMB. Supported in part by DK32346 and VA Research Service
EFFECT OF CHRONIC ADMINISTRATION OF No-NITRO-LARGININE (LNNA) ON THE OPOSSUM ESOPHAGUS AND LOWER ESOPHAGEAL SPHINCTER (LES) RESEMBLES ACHALASIA. J.F. Helm, R.D. Layman, M.D. Eckert. Medical College of Wisconsin, Milwaukee, WI. Achalasia is characterized by loss of esophageal peristalsis and a hypertensive LES that fails to relax with deglutition. Because inhibitory innervation is impaired in achalasia while cholinergic excitatory nerves are believed to be intact, we studied the effect-of chronic inhibitory nerve blockade bv LNNA, lOa mol/ke. iv per dav, on swallow-initiated peristalsis and LES relaxation in 6 opossums A perfused manometric catheter assembly was used to record intraluminal pressures from the smooth-muscle esophagus, while LES pressure-was monitored by a sleeve device. Acutely, the first dose of LNNA comnletelv inhibited LES relaxation without affecting resting LES pressure significantly. Although LNNA alone did not abolish primary peristalsis, the velocity of peristalsis increased from 2.4 to 6.2 cm/s (~~0.05) due to a shortening of the latency between swallow initiation and onset of contraction in the distal smooth-muscle esophagus. Given after LNNA, atropine 50 ug/kg iv abolished peristalsis. Chronic administration of LNNA for 4-6 weeks not only maintained persistent inhibition of LES relaxation for 24 hours between doses, but also increased resting LES pressure from 33 to 59 mmHg (p ~0.05). With chronic LNNA treatment, peristalsis was lost and swallows initiated only simultaneous “common cavity” pressure rises of identical waveform in the distal esophagus. Bddy weight was maintained during the studv. We conclude: 1) Although acute blockade of inhibitory nerves abolishes LES relaxation, Swallowing continues to initiate peristaltic contractions, albeit with an increased velocity of propagation. These peristaltic contractions are mediated by cholinergic excitatory nerves. 2) Chronic blockade of inhibitory nerves results in loss of peristalsis and a hvnertensive LES that fails to relax, findings which resemble achalasia. 3) Loss of cholinergically mediated peristalsis with chronic inhibitory nerve impairment may be an adaptive response to obstruct&r by a hypertensive LES that fails to relax with deglutition.
AMPLIFICATION AND PROPAGATION OF NITRIC OXIDE RELEASE BY INTERSTITIAL CELLS IN CANINE COLON. N.G. Publicover, E.M. Hammond and KM. Sanders. Dept. of Physiology, Univ. of Nevada School of Medicine, Reno, NV 89557. Enzymatically dispersed interstitial cells (1C.s)and smooth muscle cells (SMCs) were used to investigate mechanisms that regulate nitric oxide (NO) transmission. Cells isolated from the canine colon were loaded with the Ca2+-sensitive dye, flu03. A videobased imaging system was used to monitor relative changes in [Ca2’]i in pairs of cells (IC-SMC and SMC-SMC) located in close proximity to each other. Exogenously applied NO (10” M) caused an increase in [Ca2+]. in ICs and a decrease in [Ca2’]. in SMCs. In ICs, removal of Catt from the extracellular medium’(no added Ca2’ and 5 mM EGTA) did not block the increase in [Ca2’]i caused by NO, suggesting that transmembrane flux of Ca2’ was not responsible for the NO response. Incubation of ICs in ryanodine (10M5M for 30 min) complet;? blocked the NO response, suggesting-that the source of Ca was from intracellular stores. When [Caztli was elevated in an IC by micropressure ejection of Bav K 8644 in the vicinitv of the IC. lCa2+1: decreased in nearbv SMCs, demonstrating the ielease of a diffusibie substance from the IC. This transmission was blocked by L-NAME, L-NMMA, or oxyhemoglobin indicating that the diffusible substance may be NO. When [Ca2’], was elevated in SMCs by micropressure ejection of Bay K 8644, no effects were observed in adjacent ICs or SMCs. In ICs, the ability of NO to increase [Ca2’]i which, in turn, causes release of NO provides the components for positive-feedback amplification of NO signalling. Furthermore, the ability of NO to trigger this positive-feedback in adjacent ICs provides a mechanism for active propagation of the NO response. Results suggest that NO release from the network of ICs in the colon might be an important mechanism to amplify and propagate NO signals from enteric inhibitory nerves during normal activity, or from cells that release NO during inflammatory responses. (Supported by NIH grant DK 41315.)
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October 1992
OF PAPERS
1375
HISTCICHEMICAL L.OCALIZATION OF cGMP IN CANINE PROXIMAL COLON. C.W.R. Shuttleworth, C. Xue, SM. Ward and K.M. Sanders. Dept. Physiology, University of Nevada, Reno NV. Nitric oxide (NO) may be an inhibitory neurotransmitter in gastrointestinal smooth muscles. NO activates soluble guanylate cvclases. and it is wssible that cGMP mediates the inhibitory effects of NO. Immunohistochemical techniques were used to determine whether cGMP levels increase in response to NO. Thin strips of muscle with mucosa removed were stimulated with NO (60 ~1 saturated solution by bolus), sodium nitropmsside (SNP) (lo-4 M, IO min). or electrical field stimulation (EFS: 5Hz.30-120 s). M&B 22948 (104M) was added to inhibit cGMp ph&phohiesterask activity, and atropine (1odM) was also present during EFS. After stimuli, muscles were fixed with paraformaldehyde (4%). Formaldehyde stabilized cGMP was localized with antiserum C-5 (1:150; deVente and Steinbusch. Acta histochem. 92, 13-38, 1992) and conventional immunocytochemical techniques for visualization at the light or EM level. None of the stimuli resulted in demonstrable increases in cGMP-like immunoreactivity (cGMP-LI) in smooth muscle cells of the circular or longitudinal muscle layers. Both NO and SNP (but not EFS) caused striking increases in cGMP-LI in endothelial cells and vascular smooth muscle cells. cGMP-LI also increased in a subpopulation of nerve cell bodies in myenteric and submucosal ganglia following either EFS or NO stimulation. NO also increased cGMP-LI in the interstitial cells at the submucosal surface of the circular muscle layer that are thought to be electrical pacemakers. These results suggest: i) changes in cGMP in colonic muscle cells are small in response to NO, or ii) effects of NO on muscle cells are mediated via a cGMP’-independent mechanism. In contrast, NO increased cGMP-LI in interstitial cells, and it is possible that a portion of the inhibitory effects of NO could be mediated through the electrical coupling between these cells and smooth muscle cells. The role of the cGMP response in enteric neurons is unknown, but it is possible that NO has pre-junctional effects, regulating the release of other neurotransmitters. (Supported by DK 41315)
NO NANC NERVES MODULATE MIGRATING MOTOR COMPLEXES (MMC’S) AND POSTPRANDIAL STATE. SK. Sama, M.F. Otterson, R.P. Ryan and V.E. Cowles, Dcpts. Surg. & Physiol., Mcd Coil of WI Milwaukee, WI 53226 and Pablo&i VAMC, Milwaukee, WI 53295. We investigated the role of nitric oxide containing non-adrenergic non-cholinergic (NANC) nerves in the regulation of MMCs in the small intestine and cyclic motor activity (CMA) in the stomach in 4 conscious dogs. Each dog was ins!mmentcd with 8 strain gauge transducers on the small intestine and 1 to 2 on the annum. After control recordings, L-NAME was infused i.v. for 4 h @ 10 mg/kg/h or 15 mg&/h starting at 5 min after the end of MMC in the duodenum. The recording was continued for another 4 h. Additional 8 h recordings were made for the next 4 days. L-arginine (150 mg/kg for 8 h), L-NAME (15 mg/kgfh for 4 h) + L-arginine (150 m&g for 8 h), or Angiotensin (10 ng/kg/min) were infused. For postprsndisl experiments, a 650 kCal meal was given 1 h after the start of L-NAME (15 mg&/h for 4 h) infusion. A catheter was implanted in the carotid artery to monitor blood pressure. RESULTS: The infusion of L-NAME at 10 mg/kglh or 15 mg/kgfh for 4 h first induced a premature MMC cycle and then disrupted MMC cycling for the remainder of the day. In several experiments the small intestine exhibited amyogenesia. The gasnic sntmm exhibited a series of continuous contractions with no CMA’s. The MMc’s in the small intestine rcturncd on the second day? but the cyclic motor activity in the stomach remained disrupted. L-argmine alone had no significant effect on MMC cycling, but when given with L-NAME it inhibited the disruptive effect of L-NAME on MMC cycling and gastric CMA. The increase in arterial blood pressure due to Angiotensin II and L-NAME was of the same order but Angiotensin II did not affect the MMC cycling or gastric CMA. During control, a 650 kCa1 solid meal disrupted MMC cycling for 350 f 20 min. Infusion of L-NAME 1 h before the meal significandy decreased the period of MMC disruption to 175 f 29 min. CONCLUSION: 1) NO NANC nerves play a significant role in the disruption of MMC cycling by a meal. 2) NO NANC nerves also play a significant role in the generation of gastric cyclic motor activity, probably by producing the inhibition of contractions to produce phase I activity. These effects are not secondary to increase in arterial blood pressure. Intravenous L-arginine inhibits the in vivo motor effects of LNAMB. Supported in part by DK32346 and VA Research Service
EFFECT OF CHRONIC ADMINISTRATION OF No-NITRO-LARGININE (LNNA) ON THE OPOSSUM ESOPHAGUS AND LOWER ESOPHAGEAL SPHINCTER (LES) RESEMBLES ACHALASIA. J.F. Helm, R.D. Layman, M.D. Eckert. Medical College of Wisconsin, Milwaukee, WI. Achalasia is characterized by loss of esophageal peristalsis and a hypertensive LES that fails to relax with deglutition. Because inhibitory innervation is impaired in achalasia while cholinergic excitatory nerves are believed to be intact, we studied the effect-of chronic inhibitory nerve blockade bv LNNA, lOa mol/ke. iv per dav, on swallow-initiated peristalsis and LES relaxation in 6 opossums A perfused manometric catheter assembly was used to record intraluminal pressures from the smooth-muscle esophagus, while LES pressure-was monitored by a sleeve device. Acutely, the first dose of LNNA comnletelv inhibited LES relaxation without affecting resting LES pressure significantly. Although LNNA alone did not abolish primary peristalsis, the velocity of peristalsis increased from 2.4 to 6.2 cm/s (~~0.05) due to a shortening of the latency between swallow initiation and onset of contraction in the distal smooth-muscle esophagus. Given after LNNA, atropine 50 ug/kg iv abolished peristalsis. Chronic administration of LNNA for 4-6 weeks not only maintained persistent inhibition of LES relaxation for 24 hours between doses, but also increased resting LES pressure from 33 to 59 mmHg (p ~0.05). With chronic LNNA treatment, peristalsis was lost and swallows initiated only simultaneous “common cavity” pressure rises of identical waveform in the distal esophagus. Bddy weight was maintained during the studv. We conclude: 1) Although acute blockade of inhibitory nerves abolishes LES relaxation, Swallowing continues to initiate peristaltic contractions, albeit with an increased velocity of propagation. These peristaltic contractions are mediated by cholinergic excitatory nerves. 2) Chronic blockade of inhibitory nerves results in loss of peristalsis and a hvnertensive LES that fails to relax, findings which resemble achalasia. 3) Loss of cholinergically mediated peristalsis with chronic inhibitory nerve impairment may be an adaptive response to obstruct&r by a hypertensive LES that fails to relax with deglutition.
AMPLIFICATION AND PROPAGATION OF NITRIC OXIDE RELEASE BY INTERSTITIAL CELLS IN CANINE COLON. N.G. Publicover, E.M. Hammond and KM. Sanders. Dept. of Physiology, Univ. of Nevada School of Medicine, Reno, NV 89557. Enzymatically dispersed interstitial cells (1C.s)and smooth muscle cells (SMCs) were used to investigate mechanisms that regulate nitric oxide (NO) transmission. Cells isolated from the canine colon were loaded with the Ca2+-sensitive dye, flu03. A videobased imaging system was used to monitor relative changes in [Ca2’]i in pairs of cells (IC-SMC and SMC-SMC) located in close proximity to each other. Exogenously applied NO (10” M) caused an increase in [Ca2+]. in ICs and a decrease in [Ca2’]. in SMCs. In ICs, removal of Catt from the extracellular medium’(no added Ca2’ and 5 mM EGTA) did not block the increase in [Ca2’]i caused by NO, suggesting that transmembrane flux of Ca2’ was not responsible for the NO response. Incubation of ICs in ryanodine (10M5M for 30 min) complet;? blocked the NO response, suggesting-that the source of Ca was from intracellular stores. When [Caztli was elevated in an IC by micropressure ejection of Bav K 8644 in the vicinitv of the IC. lCa2+1: decreased in nearbv SMCs, demonstrating the ielease of a diffusibie substance from the IC. This transmission was blocked by L-NAME, L-NMMA, or oxyhemoglobin indicating that the diffusible substance may be NO. When [Ca2’], was elevated in SMCs by micropressure ejection of Bay K 8644, no effects were observed in adjacent ICs or SMCs. In ICs, the ability of NO to increase [Ca2’]i which, in turn, causes release of NO provides the components for positive-feedback amplification of NO signalling. Furthermore, the ability of NO to trigger this positive-feedback in adjacent ICs provides a mechanism for active propagation of the NO response. Results suggest that NO release from the network of ICs in the colon might be an important mechanism to amplify and propagate NO signals from enteric inhibitory nerves during normal activity, or from cells that release NO during inflammatory responses. (Supported by NIH grant DK 41315.)
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