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Nitric oxide-mediated relaxation and NADPH diaphorase activity in the human colon: Functional and histochemical differences between circular and longitudinal smooth muscle
A712
AGA ABSTRACTS
• NITRIC OXIDE-MEDIATED RELAXATION AND NADPH DIAPHORASE ACTIVITY IN THE HUMAN COLON : FUNCTIONAL AND HISTOCHEMICAL DIFFERENCES BETWEEN CIRCULAR AND LONGITUDINAL SMOOTH MUSCLE. S. Yamato M. Matsukawa, K. Matsueda, R. Shoda, J. Miwa, R. Suzuki, E. Shimojyo, N. Umeda,* S. Morita, O. Kobori** and S. Saitoh***. *Division of Gastroenterology, **Division of Surgery, and ***Division of Pathology, International Medical Center of Japan. Tokyo, Japan. Background: Coordinated contraction and relaxation of the circular and longitudinal muscle of the gut has been suggested to be essential to propel the intraluminal contents. However, few studies have been conducted in the human colon. The aim of this study was to examine whether a response to electrical field stimulation(EFS) differs in the circular and longitudinal muscle of the human colon, and if so, whether the difference has any correlation to the distribution of nitric oxide(NO) and acetylcholine(Ach)related nerves. Methods: (1)Human colonic tissue was obtained from the specimen of partial colecmmy for colonic cancer. After removing the mucosa, muscle strips of the circular and longitudinal dimension were suspended in organ bath and isometric tension was measured by force transducer. Total 48 strips from 8 patients (iigmoid 6, transverse 2) were examined. Basal tension of lg was applied to muscle strips and EFS Was applied(0.5ms, 15v, 1-10Hz, 60sec train). The effects of L-NAME and atropine on EFS-induced relaxation or contraction were studied. (2)NADPH diaphorase(NADPHD) and Ach esterase(AchE) activity were measured by histochemical staining. Results: (1)In resting state, circular muscle, hut not longitudinal muscle, showed phasic contractions. In the circular muscle, EFS abolished phasic contractions and induced frequency-dependent relaxation during stimulation ("on" relaxation) followed by contraction ("off" contraction). "On" relaxation was dose-dependently inhibited by LNAME, and L-arginine reversed the inhibition. In the longitudinal muscle, EFS induced contraction during stimulatinnCon" contraction) and "off" contraction was occasioualy observed. "On" contraction was completely abolished by atropine(ll~M)i Tetrodotoxin(l~tM) abolished EFS-induced relaxation or contraction in both muscles. SNP relaxed and CCh contracted both muscles. (2)NADPHD activity was observed !n the nerve fibers mainly in the circular muscle and scarcely in the longitudinal muscle. AchE was found in both but more in the longitudinal muscle. Conclusion: In the human colon, EFS induced NO-mediated relaxation in the circular muscle and Ach-mediated contraction in the longitudinal muscle. Such phenomena were suggested to be due to different distribution of NO and Ach-related nerve fibers in each muscle layer.
• D I S P A R A T E A C T I O N S OF P 2 - P U R I N O C E P T O R A G O N I S T S O N MYENTERIC CHOLINERGIC NEURONS. W.M. Yau, L.A. Reese and M.L. Youther. School of Medicine, Southern Illinois University, Carbondale, IL. A recently recognized signal transduction m e c h a n i s m from activation of P2-purinoceptor is a closing of K ÷ channel on m y e n t e r i c cholinergic neurons. To further elucidate the involvement of K ÷ channel with P2-purinoceptor, we have e x a m i n e d and c o m p a r e d two different P2 agonists, 2-chloro-ATP (CATP) and ~, ~ - m e t h y l e n e - A T P (~ATP). This study u s e d the efflux of [3H]ACh from longitudinal m u s c l e myenteric plexus strips o f guinea pig small intestine to test their actions. RESULTS: i) CATP has an inhibitory action while ~ATP caused a dose-dependent s t i m u l a t i o n of ACh in the range from 10 -6 t o 10 -4 M. T h e inhibitory action of CATP on electrical field stimulation of ACh release was dose related (10 -9 to 10 -6 M) 2) The inhibitory action of CATP was not b l o c k e d by the P2 antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2',4'd i s u l p h o n i c acid). 3) The (xATP-evoked A C h was significantly inhibited by K+-channel openers pinacidil and diazoxide. 4) The inhibitory action of CATP was b l o c k e d in a d o s e - d e p e n d e n t m a n n e r by 4-aminopyridine, a K÷-channel blocker. ~5) The inhibitory action of CATP p e r s i s t e d in the p r e s e n c e of S - ( 4 - N i t r o b e n z y l ) - 6 - t h i o i n o s i n e , an adenosine uptake inhibitor. 6) The Ca2÷-activated K ÷ channel b l o c k e r apamin was unable to alter the excitatory action of OUATP. Our data suggest that K ÷ channel activity is intimately related to P2 receptor activation. Of the two agonist tested, (XATP b e h a v e d as a stimulant of cholinergic neuron by a closure of K+-channel. CATP has the opposite effect by inhibiting c h o l i n e r g i c activity t h r o u g h an opening of K÷-channel. There is evidence to suggest that Ca2÷-activated K ÷ channel is not involved in ~ATP action. It is unlikely that the inhibitory action of CATP is related to adenosine m o i e t y or its metabolites. (Supported by NIH DK-26860)
GASTROENTEROLOGY, VOl. 108, No. 4
ANTRAL GASTRIN AND SOMATOSTATIN GENE EXPRESSION IS DECREASED BY INTRACISTERNAL INJECTION OF TRH ANALOG IN RATS. H.Yang, V. WU and Y.Tach6. CURE/Gastroenteric Biology Center, Dept. of Medicine, UCLA, Los Angeles, CA 90073. BACKGROUND: Thyrotropin-releasing hormone (TRH) acts centrally to stimulate gastric acid, histamine, serotonin and prostaglandin secretion through activation Of the vagus, However, plasma gastrin levels do not changed after central TRH administration. Also, intravenous infusion of gastrin antibody dose not influence the gastric acid response to intracisternal TRH (Digestion, 54: 65, 1993). PURPOSE: To explore the effect of central TRH induced vagal stimulation on antral gastrin and somatostatin gene expression in conscious rats. METHODS: TRH analog, RX 77368 (30 rig), was injected intracisternally in fasted rats (250-320 g) under light ether anesthesia and sacrificed 30. 60 and 90 min after injection. The antrum was removed for RNA extraction and Northern blot analysis. Twenty micrograms of total antral RNA was separated on a 1.2% formaldehyde gel and then transferred by electroblotting to a nylon membrane. The membrane was hybridized with gastrin and somatostatin cRNA probes separately. RESULTS: Intracisternal injection of normal saline did not influence antral gastrin and somatostatin mRNA levels compared with non-injected rats. RX 77368 30 ng injected intracisternally significantly decreased antral gastrin mRNA signals by 50% (n=5) and by 70% (n=4) at 30 and 60 min after the injection respectively but showed no significant difference in the two groups 90 min after the injection. Antral somatostatin mRNA levels also decreased by 40% (n=5) at 30 min after the injection but showed no difference in the two groups 60 and 90 min after the injection. CONCLUSION: Intracisternal injection of TRH analog, which activates the vagus nerve, did not stimulate antral gastrin and somatostatin gene expression, instead it decreases antral gastrin and somatostatin gene expression. (Supported by NIH grant DK 30110)
• E X C I T A T O R Y V A L L I N O I D S E N S O R Y R E C E P T O R INPUT TO THE MYENTERIC CHOLINERGIC NEURONS. W.M. Yau, L.A. Reese and M.L. Youther. School of Medicine, Southern Illinois University, Carbondale, IL. R e s i n i f e r a t o x i n (RTX) represents a specific but ultrapotent class of sensorotoxin w h i c h activates a subpopulation of sensory receptors. It has been recently recognized for its interaction with v a l l i n o i d (capsacinoid) receptors; however, RTX is 100-10,000 fold more potent than capsaicin in responses common to both. This study c h a r a c t e r i z e d the R T X - s e n s i t i v e sensory p a t h w a y which involved in the excitation of myenteric c h o l i n e r g i c neurons in guinea pig small intestine. Longitudinal m u s c l e m y e n t e r i c plexus strips were u s e d for r a d i o c h e m i c a l efflux of [3H]ACh. RESULTS: i) RTX caused a dosedependent stimulation of A C h in the range from 10 -1° to 10 -7 M; this stimulation was blocked by tetrodotoxin. 2) To rule out a p o s s i b l e s e n s o r y input by way of 5-HT neurons, a highly specific 5HT~ antagonist (SC-53606A) was employed. SC-53606A was ineffective in decreasing the R T X - e v o k e d A C h release. 3) Suppression of endogenous opioid receptors by naloxone (NX) has led to a significant rise in ACh output with RTX. Of all the endogenous opioid pePtides tested, the K receptor agonist dynorphin A appeared to be responsible for this inhibition. 4) RTX was unable to elicit further A C h release during supramaximal electrical field stimulation using stimulus p a r a m e t e r s known to excite cholinergic neurons, further supporting a RTX action on sensory afferent to the cholinergic. N X was ineffective in altering this field-evoked A C h response. 5) Response to RTX was unaltered when cAMP-dependent p r o t e i n kinase A activity was b l o c k e d by Rp-cAMPS. Our data suggest the p r e s e n c e of a discrete, RX-sensitive, intrinsic excitatory afferent input to the m y e n t e r i c cholinergic which is tonically under the influence of opioid suppression. (Supportedby NIH DK-26860)
AGA ABSTRACTS
• NITRIC OXIDE-MEDIATED RELAXATION AND NADPH DIAPHORASE ACTIVITY IN THE HUMAN COLON : FUNCTIONAL AND HISTOCHEMICAL DIFFERENCES BETWEEN CIRCULAR AND LONGITUDINAL SMOOTH MUSCLE. S. Yamato M. Matsukawa, K. Matsueda, R. Shoda, J. Miwa, R. Suzuki, E. Shimojyo, N. Umeda,* S. Morita, O. Kobori** and S. Saitoh***. *Division of Gastroenterology, **Division of Surgery, and ***Division of Pathology, International Medical Center of Japan. Tokyo, Japan. Background: Coordinated contraction and relaxation of the circular and longitudinal muscle of the gut has been suggested to be essential to propel the intraluminal contents. However, few studies have been conducted in the human colon. The aim of this study was to examine whether a response to electrical field stimulation(EFS) differs in the circular and longitudinal muscle of the human colon, and if so, whether the difference has any correlation to the distribution of nitric oxide(NO) and acetylcholine(Ach)related nerves. Methods: (1)Human colonic tissue was obtained from the specimen of partial colecmmy for colonic cancer. After removing the mucosa, muscle strips of the circular and longitudinal dimension were suspended in organ bath and isometric tension was measured by force transducer. Total 48 strips from 8 patients (iigmoid 6, transverse 2) were examined. Basal tension of lg was applied to muscle strips and EFS Was applied(0.5ms, 15v, 1-10Hz, 60sec train). The effects of L-NAME and atropine on EFS-induced relaxation or contraction were studied. (2)NADPH diaphorase(NADPHD) and Ach esterase(AchE) activity were measured by histochemical staining. Results: (1)In resting state, circular muscle, hut not longitudinal muscle, showed phasic contractions. In the circular muscle, EFS abolished phasic contractions and induced frequency-dependent relaxation during stimulation ("on" relaxation) followed by contraction ("off" contraction). "On" relaxation was dose-dependently inhibited by LNAME, and L-arginine reversed the inhibition. In the longitudinal muscle, EFS induced contraction during stimulatinnCon" contraction) and "off" contraction was occasioualy observed. "On" contraction was completely abolished by atropine(ll~M)i Tetrodotoxin(l~tM) abolished EFS-induced relaxation or contraction in both muscles. SNP relaxed and CCh contracted both muscles. (2)NADPHD activity was observed !n the nerve fibers mainly in the circular muscle and scarcely in the longitudinal muscle. AchE was found in both but more in the longitudinal muscle. Conclusion: In the human colon, EFS induced NO-mediated relaxation in the circular muscle and Ach-mediated contraction in the longitudinal muscle. Such phenomena were suggested to be due to different distribution of NO and Ach-related nerve fibers in each muscle layer.
• D I S P A R A T E A C T I O N S OF P 2 - P U R I N O C E P T O R A G O N I S T S O N MYENTERIC CHOLINERGIC NEURONS. W.M. Yau, L.A. Reese and M.L. Youther. School of Medicine, Southern Illinois University, Carbondale, IL. A recently recognized signal transduction m e c h a n i s m from activation of P2-purinoceptor is a closing of K ÷ channel on m y e n t e r i c cholinergic neurons. To further elucidate the involvement of K ÷ channel with P2-purinoceptor, we have e x a m i n e d and c o m p a r e d two different P2 agonists, 2-chloro-ATP (CATP) and ~, ~ - m e t h y l e n e - A T P (~ATP). This study u s e d the efflux of [3H]ACh from longitudinal m u s c l e myenteric plexus strips o f guinea pig small intestine to test their actions. RESULTS: i) CATP has an inhibitory action while ~ATP caused a dose-dependent s t i m u l a t i o n of ACh in the range from 10 -6 t o 10 -4 M. T h e inhibitory action of CATP on electrical field stimulation of ACh release was dose related (10 -9 to 10 -6 M) 2) The inhibitory action of CATP was not b l o c k e d by the P2 antagonist PPADS (pyridoxal-phosphate-6-azophenyl-2',4'd i s u l p h o n i c acid). 3) The (xATP-evoked A C h was significantly inhibited by K+-channel openers pinacidil and diazoxide. 4) The inhibitory action of CATP was b l o c k e d in a d o s e - d e p e n d e n t m a n n e r by 4-aminopyridine, a K÷-channel blocker. ~5) The inhibitory action of CATP p e r s i s t e d in the p r e s e n c e of S - ( 4 - N i t r o b e n z y l ) - 6 - t h i o i n o s i n e , an adenosine uptake inhibitor. 6) The Ca2÷-activated K ÷ channel b l o c k e r apamin was unable to alter the excitatory action of OUATP. Our data suggest that K ÷ channel activity is intimately related to P2 receptor activation. Of the two agonist tested, (XATP b e h a v e d as a stimulant of cholinergic neuron by a closure of K+-channel. CATP has the opposite effect by inhibiting c h o l i n e r g i c activity t h r o u g h an opening of K÷-channel. There is evidence to suggest that Ca2÷-activated K ÷ channel is not involved in ~ATP action. It is unlikely that the inhibitory action of CATP is related to adenosine m o i e t y or its metabolites. (Supported by NIH DK-26860)
GASTROENTEROLOGY, VOl. 108, No. 4
ANTRAL GASTRIN AND SOMATOSTATIN GENE EXPRESSION IS DECREASED BY INTRACISTERNAL INJECTION OF TRH ANALOG IN RATS. H.Yang, V. WU and Y.Tach6. CURE/Gastroenteric Biology Center, Dept. of Medicine, UCLA, Los Angeles, CA 90073. BACKGROUND: Thyrotropin-releasing hormone (TRH) acts centrally to stimulate gastric acid, histamine, serotonin and prostaglandin secretion through activation Of the vagus, However, plasma gastrin levels do not changed after central TRH administration. Also, intravenous infusion of gastrin antibody dose not influence the gastric acid response to intracisternal TRH (Digestion, 54: 65, 1993). PURPOSE: To explore the effect of central TRH induced vagal stimulation on antral gastrin and somatostatin gene expression in conscious rats. METHODS: TRH analog, RX 77368 (30 rig), was injected intracisternally in fasted rats (250-320 g) under light ether anesthesia and sacrificed 30. 60 and 90 min after injection. The antrum was removed for RNA extraction and Northern blot analysis. Twenty micrograms of total antral RNA was separated on a 1.2% formaldehyde gel and then transferred by electroblotting to a nylon membrane. The membrane was hybridized with gastrin and somatostatin cRNA probes separately. RESULTS: Intracisternal injection of normal saline did not influence antral gastrin and somatostatin mRNA levels compared with non-injected rats. RX 77368 30 ng injected intracisternally significantly decreased antral gastrin mRNA signals by 50% (n=5) and by 70% (n=4) at 30 and 60 min after the injection respectively but showed no significant difference in the two groups 90 min after the injection. Antral somatostatin mRNA levels also decreased by 40% (n=5) at 30 min after the injection but showed no difference in the two groups 60 and 90 min after the injection. CONCLUSION: Intracisternal injection of TRH analog, which activates the vagus nerve, did not stimulate antral gastrin and somatostatin gene expression, instead it decreases antral gastrin and somatostatin gene expression. (Supported by NIH grant DK 30110)
• E X C I T A T O R Y V A L L I N O I D S E N S O R Y R E C E P T O R INPUT TO THE MYENTERIC CHOLINERGIC NEURONS. W.M. Yau, L.A. Reese and M.L. Youther. School of Medicine, Southern Illinois University, Carbondale, IL. R e s i n i f e r a t o x i n (RTX) represents a specific but ultrapotent class of sensorotoxin w h i c h activates a subpopulation of sensory receptors. It has been recently recognized for its interaction with v a l l i n o i d (capsacinoid) receptors; however, RTX is 100-10,000 fold more potent than capsaicin in responses common to both. This study c h a r a c t e r i z e d the R T X - s e n s i t i v e sensory p a t h w a y which involved in the excitation of myenteric c h o l i n e r g i c neurons in guinea pig small intestine. Longitudinal m u s c l e m y e n t e r i c plexus strips were u s e d for r a d i o c h e m i c a l efflux of [3H]ACh. RESULTS: i) RTX caused a dosedependent stimulation of A C h in the range from 10 -1° to 10 -7 M; this stimulation was blocked by tetrodotoxin. 2) To rule out a p o s s i b l e s e n s o r y input by way of 5-HT neurons, a highly specific 5HT~ antagonist (SC-53606A) was employed. SC-53606A was ineffective in decreasing the R T X - e v o k e d A C h release. 3) Suppression of endogenous opioid receptors by naloxone (NX) has led to a significant rise in ACh output with RTX. Of all the endogenous opioid pePtides tested, the K receptor agonist dynorphin A appeared to be responsible for this inhibition. 4) RTX was unable to elicit further A C h release during supramaximal electrical field stimulation using stimulus p a r a m e t e r s known to excite cholinergic neurons, further supporting a RTX action on sensory afferent to the cholinergic. N X was ineffective in altering this field-evoked A C h response. 5) Response to RTX was unaltered when cAMP-dependent p r o t e i n kinase A activity was b l o c k e d by Rp-cAMPS. Our data suggest the p r e s e n c e of a discrete, RX-sensitive, intrinsic excitatory afferent input to the m y e n t e r i c cholinergic which is tonically under the influence of opioid suppression. (Supportedby NIH DK-26860)