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Questioning use of l -arginine to augment synthesis of nitric oxide in pharmacological studies
Abstracts
following food intake and digestion. It is understood that sensory perception by the abdominal vagus initiates and sustains the coordinated processes of gastrointestinal motility, circulation, absorption, exocrine and endocrine secretion, immunity, and satiation. In this study, we examined effects of luminal amino acids on the afferent nerve activities of the rat abdominal vagus. Male Sprague–Dawley rats were used for this experiment. Under urethane anesthesia, the nerve bundle of the left gastric or celiac branch was split, and vagal filaments were dissected from the main nerve trunk, and placed on a silver hook recording electrode. Recordings were all made from the peripheral cut end of the vagal nerve. Blood pressure and body temperature were continuously monitored during experiments. Intraduodenal application of each amino acid all modulated the vagal afferent activities of the rat celiac branch. For instance, the celiac afferents were activated by glutamic acid, tryptophan, and lysine, and suppressed by glycine. However, the afferent fibers of the rat gastric vagus increased their firing rate solely with intragastric application of glutamic acid. Thus, glutamate was a unique amino acid that could be sensed by the both abdominal vagal afferents. Moreover, intravenous application of 5-HT3 antagonist, granisetron strongly attenuated the glutamate-induced gastric afferent activation but failed to inhibit the glutamate-induced celiac afferent activation. This potently indicates possible existence of some sensing systems for glutamate in the rat upper gastrointestinal mucosa, and that luminal glutamate-sensing mechanism by the vagus might be quite differed between the stomach and intestine. Assuming there is a universal co-existence of free glutamate with dietary protein, the glutamate sensing by the abdominal vagus might have a new physiological role in the gastric and intestinal phases of protein digestion via the vagal reflex. doi:10.1016/j.autneu.2007.06.152
I-P-039 Modulatory mechanism of the striated muscle motility by a local neural reflex in the rat esophagus Takahiko Shiina a, Yasutake Shimizu a, Ammar Boudaka a , Juergen Woerl b, Tadashi Takewaki a a Laboratory of Veterinary Physiology, Department of Basic Veterinary Science, The United Graduate School, Gifu University, Gifu, Japan b Institute of Anatomy, University of Erlangen–Nuremberg, Germany The external muscle layers of the mammalian esophagus contain striated muscle fibers. These striated muscle fibers are innervated exclusively by excitatory vagal efferents and the peristalsis in the striated esophageal muscle is modulated via vago-vagal reflexes. By contrast, the presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the mammalian esophagus has long been known.
93
However, the role of the intrinsic nervous system in the peristalsis of the striated muscle portion of the esophagus has remained enigmatic, and has been neglected in terms of peristaltic control. Hence, this study investigated the roles of the intrinsic neurons regulating the motility of the striated muscle in the rat esophagus. An isolated segment of rat esophagus was placed in an organ bath and the contractile responses were recorded using a force transducer. Since primary afferent nerve fibers closely innervate myenteric neurons in the esophagus, the primary afferent neurons were stimulated using capsaicin to activate the intrinsic neurons. Electrical stimulation of the vagus nerve trunk evoked twitch contractions of the striated muscles in the rat esophagus. Capsaicin inhibited the vagally evoked twitch contractions in a concentration-dependent manner. However, capsaicin did not significantly affect the vagally evoked twitch contractions of rat esophageal preparations whose capsaicinsensitive sensory neurons had been destroyed and the electrical field stimulation-induced non-neurogenic contractions of the striated muscle. In addition, capsaicin suppressed the acetylcholine release from the rat esophageal segment evoked by vagus nerve stimulation. These results indicate that the rat esophagus has a local neural reflex that regulates striated muscle motility by inhibiting acetylcholine release from vagal motor neurons and that capsaicin can activate this inhibitory neural reflex via capsaicin-sensitive afferent neurons. Next, pharmacological analyses were used to identify possible neurotransmitters involved in the local neural reflex. A nitric oxide synthase inhibitor blocked the inhibitory effect of capsaicin on the motility of esophageal striated muscle, and exogenous application of a nitric oxide donor mimicked the inhibitory effect of capsaicin. Furthermore, a selective tachykinin NK1 receptor antagonist significantly blocked the inhibitory effect of capsaicin on the motility of esophageal striated muscles, and a tachykinin receptor agonist inhibited the twitch contractions evoked by vagal stimulation. These data suggest that nitric oxide and tachykinins are crucial neurotransmitters in the local neural reflex. The local reflex arc might be involved in coordinating esophageal peristalsis in the striated muscle portion. doi:10.1016/j.autneu.2007.06.153
I-P-040 Questioning use of L-arginine to augment synthesis of nitric oxide in pharmacological studies William T. Talman, Julie E. Langesek Laboratory of Neurobiology, Department of Neurology, University of Iowa and Veterans Affairs Medical Center, United States L-arginine (L-Arg), the critical substrate in enzymatic synthesis of nitric oxide (NO.), is often used in pharmacological studies to assess effects of synthesis and release of
94
Abstracts
NO.; D-Arg, the inactive isomer, is often used as a control. We have similarly used L-Arg, but we have found that the free amino acid L-Arg and L-Arg HCl differed in the cardiovascular effects they elicited when injected into the nucleus tractus solitarii (NTS). We conjectured that L-Arg may elicit effects through mechanisms (e.g. volume or osmolarity) independent of synthesis of NO.. If that were the case, then we predicted that L-Arg administered bilaterally into the NTS would lead to hypertension with or without an acute depressor effect and that D-Arg and L-Arg would elicit similar effects. In anesthetized adult male Sprague Dawley rats instrumented for recording arterial blood pressure (AP) and heart rate (HR) we assessed changes in each variable after unilateral microinjection (50 nl) of L-Arg (free amino acid; Sigma, St. Louis, MO), D-Arg (free amino acid; Sigma), LArg HCl (Sigma) or D-Arg HCl (Sigma) at doses of 1, 10, and 50 nmol. In some animals we injected L-Arg HCl or D-Arg HCl (10 or 50 nmol) bilaterally into NTS. Dose-related depressor and bradycardiac responses followed injection of either free L-Arg or free D-Arg unilaterally into the NTS. There were no significant differences in responses elicited by one isomer of the free amino acid compared with the other. In contrast, D-Arg HCl elicited significantly lower MAP and HR responses than did L-Arg HCl except that a dose of 1 nmol produced the same HR responses with each isomer. While responses for MAP and HR were dose-related for free D-Arg and for D-Arg HCl, depressor and bradycardiac responses were greater after injection of the free amino acid than after injection of D-Arg HCl at all doses. Bilateral microinjection of free L-Arg into the NTS at a dose of 10 nmol/50 nl led to a mild but significant elevation of MAP with no significant change in HR. By comparison, bilateral injection of L-Arg HCl (10 nmol/50 nl) did not change MAP or HR whereas bilateral injection of 10 and 50 nmol doses of D-Arg HCl both increased MAP with little change in HR. Considerable caution must be used in concluding that responses to L-Arg result from synthesis of NO.. The preparation (free or HCl) of the amino acid should always be specified.
Background
doi:10.1016/j.autneu.2007.06.154
1) Coronary angiography in the acute and chronic phases found no significant stenosis in all of the patients. Left ventricular (LV) wall motion returned to normal by 17.6 ±6.4 days. LV ejection fraction was 45.7 ± 8.8% in the acute phase, while it was 69.8 ± 6.8% ( p b 0.001) after the improvement of wall motion. 2) Between 0 M and 3 M, SDNN and SDANN improved significantly from 88.8 ± 35.5 to 109.5 ±33.4 ms ( p = 0.01) and from 79.9 ± 34.7 to 99.3 ± 40.3 ms ( p = 0.03), respectively. However, rMSSD and pNN50 showed no changes. 3) No significant changes were observed between 0 M and 3 M in LF, HF, and TF values.
I-P-041 Cardiac autonomic nervous function in patients with reversible ventricular dysfunction takotsubo cardiomyopathy Yoshihiro J. Akashi a , Giuseppe Barbaro b , Tsuneharu Sakurai c, Kiyoshi Nakazawa a, Fumihiko Miyake a a Division of Cardiology, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Japan b Cardiology Unit, Department of Medical Pathophysiology, University “La Sapienza”, Rome, Italy c Division of Laboratory Medicine, St. Marianna University School of Medicine, Kawasaki, Japan
Transient apical ballooning, so-called “takotsubo cardiomyopathy (TC)” is a condition in which the symptoms resemble acute myocardial infarction, but there is no significant stenosis on coronary angiography. Several causes have been proposed for this disease, including neurogenic stunned myocardium caused by alternation of catecholamine dynamics. Although it is a characteristic of this disease that wall motion recovers dramatically, there have been no investigations of the changes of autonomic nervous function. Therefore, we compared cardiac autonomic function in the acute and chronic phases of TC. Methods The subjects were 10 consecutive patients (mean age: 70.1 ± 13.7 years) who underwent cardiac catheterization on the day of onset of symptoms and were diagnosed with TC. After the normalization of wall motion, a cardiac catheterization was repeated. To assess autonomic nervous function, Holter ECG monitoring was done within 3 days after the onset of symptoms (0 M) and 3 months after (3 M), and the standard deviation of the NN intervals over 24 h (SDNN), the 24-hour standard deviation of the mean value of the difference between the NN intervals for each 5-minutes segment (SDANN), the root-means-square of the successive differences of the NN intervals (rMSSD), and the percentage of differences between the successive NN intervals exceeding 50 ms (pNN50) were calculated by time-area analysis of heart rate variability over 24 h. In addition, the low frequency (LF: 0.04–0.15 Hz), high frequency (HF: 0.15– 0.40 Hz) and total frequency (TF: b 0.4 Hz) components of the power spectrum of heart rate variability were investigated. Ten age-matched healthy volunteers were recruited in our institutions. Results
Conclusions The results of this study support our previous hypothesis that TC might be due to neurogenic stunned myocardium
following food intake and digestion. It is understood that sensory perception by the abdominal vagus initiates and sustains the coordinated processes of gastrointestinal motility, circulation, absorption, exocrine and endocrine secretion, immunity, and satiation. In this study, we examined effects of luminal amino acids on the afferent nerve activities of the rat abdominal vagus. Male Sprague–Dawley rats were used for this experiment. Under urethane anesthesia, the nerve bundle of the left gastric or celiac branch was split, and vagal filaments were dissected from the main nerve trunk, and placed on a silver hook recording electrode. Recordings were all made from the peripheral cut end of the vagal nerve. Blood pressure and body temperature were continuously monitored during experiments. Intraduodenal application of each amino acid all modulated the vagal afferent activities of the rat celiac branch. For instance, the celiac afferents were activated by glutamic acid, tryptophan, and lysine, and suppressed by glycine. However, the afferent fibers of the rat gastric vagus increased their firing rate solely with intragastric application of glutamic acid. Thus, glutamate was a unique amino acid that could be sensed by the both abdominal vagal afferents. Moreover, intravenous application of 5-HT3 antagonist, granisetron strongly attenuated the glutamate-induced gastric afferent activation but failed to inhibit the glutamate-induced celiac afferent activation. This potently indicates possible existence of some sensing systems for glutamate in the rat upper gastrointestinal mucosa, and that luminal glutamate-sensing mechanism by the vagus might be quite differed between the stomach and intestine. Assuming there is a universal co-existence of free glutamate with dietary protein, the glutamate sensing by the abdominal vagus might have a new physiological role in the gastric and intestinal phases of protein digestion via the vagal reflex. doi:10.1016/j.autneu.2007.06.152
I-P-039 Modulatory mechanism of the striated muscle motility by a local neural reflex in the rat esophagus Takahiko Shiina a, Yasutake Shimizu a, Ammar Boudaka a , Juergen Woerl b, Tadashi Takewaki a a Laboratory of Veterinary Physiology, Department of Basic Veterinary Science, The United Graduate School, Gifu University, Gifu, Japan b Institute of Anatomy, University of Erlangen–Nuremberg, Germany The external muscle layers of the mammalian esophagus contain striated muscle fibers. These striated muscle fibers are innervated exclusively by excitatory vagal efferents and the peristalsis in the striated esophageal muscle is modulated via vago-vagal reflexes. By contrast, the presence of a distinct ganglionated myenteric plexus in the striated muscle portion of the mammalian esophagus has long been known.
93
However, the role of the intrinsic nervous system in the peristalsis of the striated muscle portion of the esophagus has remained enigmatic, and has been neglected in terms of peristaltic control. Hence, this study investigated the roles of the intrinsic neurons regulating the motility of the striated muscle in the rat esophagus. An isolated segment of rat esophagus was placed in an organ bath and the contractile responses were recorded using a force transducer. Since primary afferent nerve fibers closely innervate myenteric neurons in the esophagus, the primary afferent neurons were stimulated using capsaicin to activate the intrinsic neurons. Electrical stimulation of the vagus nerve trunk evoked twitch contractions of the striated muscles in the rat esophagus. Capsaicin inhibited the vagally evoked twitch contractions in a concentration-dependent manner. However, capsaicin did not significantly affect the vagally evoked twitch contractions of rat esophageal preparations whose capsaicinsensitive sensory neurons had been destroyed and the electrical field stimulation-induced non-neurogenic contractions of the striated muscle. In addition, capsaicin suppressed the acetylcholine release from the rat esophageal segment evoked by vagus nerve stimulation. These results indicate that the rat esophagus has a local neural reflex that regulates striated muscle motility by inhibiting acetylcholine release from vagal motor neurons and that capsaicin can activate this inhibitory neural reflex via capsaicin-sensitive afferent neurons. Next, pharmacological analyses were used to identify possible neurotransmitters involved in the local neural reflex. A nitric oxide synthase inhibitor blocked the inhibitory effect of capsaicin on the motility of esophageal striated muscle, and exogenous application of a nitric oxide donor mimicked the inhibitory effect of capsaicin. Furthermore, a selective tachykinin NK1 receptor antagonist significantly blocked the inhibitory effect of capsaicin on the motility of esophageal striated muscles, and a tachykinin receptor agonist inhibited the twitch contractions evoked by vagal stimulation. These data suggest that nitric oxide and tachykinins are crucial neurotransmitters in the local neural reflex. The local reflex arc might be involved in coordinating esophageal peristalsis in the striated muscle portion. doi:10.1016/j.autneu.2007.06.153
I-P-040 Questioning use of L-arginine to augment synthesis of nitric oxide in pharmacological studies William T. Talman, Julie E. Langesek Laboratory of Neurobiology, Department of Neurology, University of Iowa and Veterans Affairs Medical Center, United States L-arginine (L-Arg), the critical substrate in enzymatic synthesis of nitric oxide (NO.), is often used in pharmacological studies to assess effects of synthesis and release of
94
Abstracts
NO.; D-Arg, the inactive isomer, is often used as a control. We have similarly used L-Arg, but we have found that the free amino acid L-Arg and L-Arg HCl differed in the cardiovascular effects they elicited when injected into the nucleus tractus solitarii (NTS). We conjectured that L-Arg may elicit effects through mechanisms (e.g. volume or osmolarity) independent of synthesis of NO.. If that were the case, then we predicted that L-Arg administered bilaterally into the NTS would lead to hypertension with or without an acute depressor effect and that D-Arg and L-Arg would elicit similar effects. In anesthetized adult male Sprague Dawley rats instrumented for recording arterial blood pressure (AP) and heart rate (HR) we assessed changes in each variable after unilateral microinjection (50 nl) of L-Arg (free amino acid; Sigma, St. Louis, MO), D-Arg (free amino acid; Sigma), LArg HCl (Sigma) or D-Arg HCl (Sigma) at doses of 1, 10, and 50 nmol. In some animals we injected L-Arg HCl or D-Arg HCl (10 or 50 nmol) bilaterally into NTS. Dose-related depressor and bradycardiac responses followed injection of either free L-Arg or free D-Arg unilaterally into the NTS. There were no significant differences in responses elicited by one isomer of the free amino acid compared with the other. In contrast, D-Arg HCl elicited significantly lower MAP and HR responses than did L-Arg HCl except that a dose of 1 nmol produced the same HR responses with each isomer. While responses for MAP and HR were dose-related for free D-Arg and for D-Arg HCl, depressor and bradycardiac responses were greater after injection of the free amino acid than after injection of D-Arg HCl at all doses. Bilateral microinjection of free L-Arg into the NTS at a dose of 10 nmol/50 nl led to a mild but significant elevation of MAP with no significant change in HR. By comparison, bilateral injection of L-Arg HCl (10 nmol/50 nl) did not change MAP or HR whereas bilateral injection of 10 and 50 nmol doses of D-Arg HCl both increased MAP with little change in HR. Considerable caution must be used in concluding that responses to L-Arg result from synthesis of NO.. The preparation (free or HCl) of the amino acid should always be specified.
Background
doi:10.1016/j.autneu.2007.06.154
1) Coronary angiography in the acute and chronic phases found no significant stenosis in all of the patients. Left ventricular (LV) wall motion returned to normal by 17.6 ±6.4 days. LV ejection fraction was 45.7 ± 8.8% in the acute phase, while it was 69.8 ± 6.8% ( p b 0.001) after the improvement of wall motion. 2) Between 0 M and 3 M, SDNN and SDANN improved significantly from 88.8 ± 35.5 to 109.5 ±33.4 ms ( p = 0.01) and from 79.9 ± 34.7 to 99.3 ± 40.3 ms ( p = 0.03), respectively. However, rMSSD and pNN50 showed no changes. 3) No significant changes were observed between 0 M and 3 M in LF, HF, and TF values.
I-P-041 Cardiac autonomic nervous function in patients with reversible ventricular dysfunction takotsubo cardiomyopathy Yoshihiro J. Akashi a , Giuseppe Barbaro b , Tsuneharu Sakurai c, Kiyoshi Nakazawa a, Fumihiko Miyake a a Division of Cardiology, Department of Internal Medicine, St. Marianna University School of Medicine, Kawasaki, Japan b Cardiology Unit, Department of Medical Pathophysiology, University “La Sapienza”, Rome, Italy c Division of Laboratory Medicine, St. Marianna University School of Medicine, Kawasaki, Japan
Transient apical ballooning, so-called “takotsubo cardiomyopathy (TC)” is a condition in which the symptoms resemble acute myocardial infarction, but there is no significant stenosis on coronary angiography. Several causes have been proposed for this disease, including neurogenic stunned myocardium caused by alternation of catecholamine dynamics. Although it is a characteristic of this disease that wall motion recovers dramatically, there have been no investigations of the changes of autonomic nervous function. Therefore, we compared cardiac autonomic function in the acute and chronic phases of TC. Methods The subjects were 10 consecutive patients (mean age: 70.1 ± 13.7 years) who underwent cardiac catheterization on the day of onset of symptoms and were diagnosed with TC. After the normalization of wall motion, a cardiac catheterization was repeated. To assess autonomic nervous function, Holter ECG monitoring was done within 3 days after the onset of symptoms (0 M) and 3 months after (3 M), and the standard deviation of the NN intervals over 24 h (SDNN), the 24-hour standard deviation of the mean value of the difference between the NN intervals for each 5-minutes segment (SDANN), the root-means-square of the successive differences of the NN intervals (rMSSD), and the percentage of differences between the successive NN intervals exceeding 50 ms (pNN50) were calculated by time-area analysis of heart rate variability over 24 h. In addition, the low frequency (LF: 0.04–0.15 Hz), high frequency (HF: 0.15– 0.40 Hz) and total frequency (TF: b 0.4 Hz) components of the power spectrum of heart rate variability were investigated. Ten age-matched healthy volunteers were recruited in our institutions. Results
Conclusions The results of this study support our previous hypothesis that TC might be due to neurogenic stunned myocardium