Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons / 神经科学通报·英文版

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.

Increased Tone of the Human Colon Muscle by Bisacodyl In Vitro

BACKGROUND/AIMS: Although bisacodyl is a widely administered laxative, its underlying mechanism of action remains generally unknown. This study focuses on investigating the effects of bisacodyl on the human colon muscle contraction, and elucidating its mechanism of action. METHODS: Sigmoid colon muscle strips (20 longitudinal and 18 circular muscles) were obtained from 20 subjects who underwent colectomy for colon cancer. Isometric force measurements were calculated in response to electrical field stimulation (EFS, 0.3 milliseconds in trains of 10 Hz for 20 seconds, 150 V). Peak and nadir (tone) during and after EFS, were measured in a controlled state, and after sequential addition of bisacodyl (1 μM), atropine (1 μM), N-nitro-L-arginine (L-NNA, 100 μM), MRS2500 (1 μM), and tetrodotoxin (TTX, 1 μM) to the organ bath. RESULTS: Transient phasic contractions were observed during EFS, and after cessation of EFS. In the longitudinal muscles, nadir during EFS, and tone after EFS, significantly increased after addition of bisacodyl, and persisted after sequential addition of atropine, L-NNA, MRS2500, and TTX, indicating a direct action of bisacodyl on the smooth muscle. In the second experiment, pretreatment of TTX abolished EFS-induced phasic contractions. Although no phasic contraction was produced after perfusion of bisacodyl, tone was increased, thereby supporting evidence of a direct mechanism of action of bisacodyl on the colon smooth muscle. CONCLUSIONS: Bisacodyl increases the tone of longitudinal muscle in the human sigmoid colon through a direct action on the smooth muscle. Further study is warranted to investigate the neural mechanism of action of bisacodyl.

Botulinum toxin type A enhances the inhibitory spontaneous postsynaptic currents on the substantia gelatinosa neurons of the subnucleus caudalis in immature mice

Botulinum toxin type A (BoNT/A) has been used therapeutically for various conditions including dystonia, cerebral palsy, wrinkle, hyperhidrosis and pain control. The substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc) receive orofacial nociceptive information from primary afferents and transmit the information to higher brain center. Although many studies have shown the analgesic effects of BoNT/A, the effects of BoNT/A at the central nervous system and the action mechanism are not well understood. Therefore, the effects of BoNT/A on the spontaneous postsynaptic currents (sPSCs) in the SG neurons were investigated. In whole cell voltage clamp mode, the frequency of sPSCs was increased in 18 (37.5%) neurons, decreased in 5 (10.4%) neurons and not affected in 25 (52.1%) of 48 neurons tested by BoNT/A (3 nM). Similar proportions of frequency variation of sPSCs were observed in 1 and 10 nM BoNT/A and no significant differences were observed in the relative mean frequencies of sPSCs among 1–10 nM BoNT/A. BoNT/A-induced frequency increase of sPSCs was not affected by pretreated tetrodotoxin (0.5 µM). In addition, the frequency of sIPSCs in the presence of CNQX (10 µM) and AP5 (20 µM) was increased in 10 (53%) neurons, decreased in 1 (5%) neuron and not affected in 8 (42%) of 19 neurons tested by BoNT/A (3 nM). These results demonstrate that BoNT/A increases the frequency of sIPSCs on SG neurons of the Vc at least partly and can provide an evidence for rapid action of BoNT/A at the central nervous system.

Histamine Excites Rat GABAergic Ventral Pallidum Neurons via Co-activation of H1 and H2 Receptors / 神经科学通报·英文版

The ventral pallidum (VP) is a crucial component of the limbic loop of the basal ganglia and participates in the regulation of reward, motivation, and emotion. Although the VP receives afferent inputs from the central histaminergic system, little is known about the effect of histamine on the VP and the underlying receptor mechanism. Here, we showed that histamine, a hypothalamic-derived neuromodulator, directly depolarized and excited the GABAergic VP neurons which comprise a major cell type in the VP and are responsible for encoding cues of incentive salience and reward hedonics. Both postsynaptic histamine H1 and H2 receptors were found to be expressed in the GABAergic VP neurons and co-mediate the excitatory effect of histamine. These results suggested that the central histaminergic system may actively participate in VP-mediated motivational and emotional behaviors via direct modulation of the GABAergic VP neurons. Our findings also have implications for the role of histamine and the central histaminergic system in psychiatric disorders.

Korean red ginseng excitation of paraventricular nucleus neurons via non-N-methyl-D-aspartate glutamate receptor activation in mice

It has been reported that Korean red ginseng (KRG), a valuable and important traditional medicine, has varied effects on the central nervous system, suggesting its activities are complicated. The paraventricular nucleus (PVN) neurons of the hypothalamus has a critical role in stress responses and hormone secretions. Although the action mechanisms of KRG on various cells and systems have been reported, the direct membrane effects of KRG on PVN neurons have not been fully described. In this study, the direct membrane effects of KRG on PVN neuronal activity were investigated by using a perforated patch-clamp in ICR mice. In gramicidin perforated patch-clamp mode, KRG extract (KRGE) induced repeatable depolarization followed by hyperpolarization of PVN neurons. The KRGE-induced responses were concentration-dependent and persisted in the presence of tetrodotoxin, a voltage sensitive Na+ channel blocker. The KRGE-induced responses were suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (10 µM), a non-N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, but not by picrotoxin, a type A gamma-aminobutyric acid receptor antagonist. The results indicate that KRG activates non-NMDA glutamate receptors of PVN neurons in mice, suggesting that KRG may be a candidate for use in regulation of stress responses by controlling autonomic nervous system and hormone secretion.

Action of Mitochondrial Substrates on Neuronal Excitability in Rat Substantia Gelatinosa Neurons

Recent studies indicate that mitochondria are an important source of reactive oxygen species (ROS) in the spinal dorsal horn. In our previous study, application of malate, a mitochondrial electron transport complex I substrate, induced a membrane depolarization, which was inhibited by pretreatment with ROS scavengers. In the present study, we used patch clamp recording in the substantia geletinosa (SG) neurons of spinal slices, to investigate the cellular mechanism of mitochondrial ROS on neuronal excitability. DNQX (an AMPA receptor antagonist) and AP5 (an NMDA receptor antagonist) decreased the malate-induced depolarization. In an external calcium free solution and addition of tetrodotoxin (TTX) for blockade of synaptic transmission, the malateinduced depolarization remained unchanged. In the presence of DNQX, AP5 and AP3 (a group I metabotropic glutamate receptor (mGluR) antagonist), glutamate depolarized the membrane potential, which was suppressed by PBN. However, oligomycin (a mitochondrial ATP synthase inhibitor) or PPADS (a P2 receptor inhibitor) did not affect the substrates-induced depolarization. These results suggest that mitochondrial substrate-induced ROS in SG neuron directly acts on the postsynaptic neuron, therefore increasing the ion influx via glutamate receptors.

Slow Wave Activity and Modulations in Mouse Jejunum Myenteric Plexus In Situ

BACKGROUND/AIMS: Myenteric plexus interstitial cells of Cajal (ICC-MY) are involved in the generation of gut pacemaker activity and neuronal communication. We performed patch clamp on ICC-MY in situ to observe the changes of pacemaker activity in response to neural modulations. METHODS: A fresh longitudinal muscle with myenteric plexus (LMMP) from mouse jejunum was prepared. ICC-MY and ganglion neurons embedded in the layer of longitudinal muscles were targeted by patch clamping in whole-cell configuration in a model of current or voltage clamp. Neurogenic modulators were applied to evaluate their effects on ICC pacemaker activity. RESULTS: In situ ICC-MY showed spontaneous and rhythmical voltage oscillations with a frequency of 27.2 ± 3.9 cycles/min, amplitude of 32.6 ± 6.3 mV, and resting membrane potential of −62.2 ± 2.8 mV. In situ neurons showed electrically evocable action potential in single or multiple spikes. Pacemaker activity was modulated by neuronal activators through receiving a neuronal input. Application of tetrodotoxin depolarized pacemaker potentials in a dose dependent manner, and decreased the amplitude at tetrodotoxin 0.3 μM for about 40 ± 10%; capsaicin (1 μM) ameliorated ICC-MY K+ current for about 49 ± 14.8%; and, nitric oxide hyperpolarized pacemaker potential and decreased the amplitude and frequency. CONCLUSIONS: The in situ preparation patch clamp study further demonstrates that the pacemaker activity is an intrinsic property of ICC. The neurogenic activators change and shape pacemaker potential and activity in situ. LMMP preparation in situ patch clamp provides an ideal platform to study the functional innervation of the ICC and the enteric neural system, thereby, for evaluating the neural regulation of pacemaker activity, especially in disorder models.

The Effect of Deoxycholic Acid on Secretion and Motility in the Rat and Guinea Pig Large Intestine

BACKGROUND/AIMS: Bile acid is an important luminal factor that affects gastrointestinal motility and secretion. We investigated the effect of bile acid on secretion in the proximal and distal rat colon and coordination of bowel movements in the guinea pig colon. METHODS: The short-circuit current from the mucosal strip of the proximal and distal rat colon was compared under control conditions after induction of secretion with deoxycholic acid (DCA) as well as after inhibition of secretion with indomethacin, 1,2-bis (o-aminophenoxy) ethane-N,N,N′,N′-tetra-acetic acid (an intracellular calcium chelator; BAPTA), and tetrodotoxin (TTX) using an Ussing chamber. Colonic pressure patterns were also evaluated in the extracted guinea pig colon during resting, DCA stimulation, and inhibition by TTX using a newly developed pressure-sensing artificial stool. RESULTS: The secretory response in the distal colon was proportionate to the concentration of DCA. Also, indomethacin, BAPTA, and TTX inhibited chloride secretion in response to DCA significantly (P < 0.05). However, these changes were not detected in the proximal colon. When we evaluated motility, we found that DCA induced an increase in luminal pressure at the proximal, middle, and distal sensors of an artificial stool simultaneously during the non-peristaltic period (P < 0.05). In contrast, during peristalsis, DCA induced an increase in luminal pressure at the proximal sensor and a decrease in pressure at the middle and distal sensors of the artificial stool (P < 0.05). CONCLUSIONS: DCA induced a clear segmental difference in electrogenic secretion. Also, DCA induced a more powerful peristaltic contraction only during the peristaltic period.

Tetrodotoxin poisoning caused by Goby fish consumption in southeast China: a retrospective case series analysis

OBJECTIVES: To investigate an unusual outbreak of tetrodotoxin poisoning in Leizhou, southeast China, a case series analysis was conducted to identify the source of illness. METHODS: A total of 22 individuals experienced symptoms of poisoning, including tongue numbness, dizziness, nausea and limb numbness and weakness. Two toxic species, Amoya caninus and Yongeichthys nebulosus, were morphologically identified from the batches of gobies consumed by the patients. Tetrodotoxin levels in the blood and Goby fish samples were detected using liquid chromatography-tandem mass spectrometry. RESULTS: The tetrodotoxin levels in the remaining cooked Goby fish were determined to be 2090.12 µg/kg. For Amoya caninus, the toxicity levels were 1858.29 µg/kg in the muscle and 1997.19 µg/kg in the viscera and for Yongeichthys nebulosus, they were 2783.00 µg/kg in the muscle and 2966.21 µg/kg in the viscera. CONCLUSION: This outbreak demonstrates an underestimation of the risk of Goby fish poisoning. Furthermore, the relationships among the toxic species, climates and marine algae present should be clarified in the future. .

Modulatory effects of taurine on jejunal contractility

Taurine (2-aminoethanesulfonic acid) is widely distributed in animal tissues and has diverse pharmacological effects. However, the role of taurine in modulating smooth muscle contractility is still controversial. We propose that taurine (5-80 mM) can exert bidirectional modulation on the contractility of isolated rat jejunal segments. Different low and high contractile states were induced in isolated jejunal segments of rats to observe the effects of taurine and the associated mechanisms. Taurine induced stimulatory effects on the contractility of isolated rat jejunal segments at 3 different low contractile states, and inhibitory effects at 3 different high contractile states. Bidirectional modulation was not observed in the presence of verapamil or tetrodotoxin, suggesting that taurine-induced bidirectional modulation is Ca2+ dependent and requires the presence of the enteric nervous system. The stimulatory effects of taurine on the contractility of isolated jejunal segments was blocked by atropine but not by diphenhydramine or by cimetidine, suggesting that muscarinic-linked activation was involved in the stimulatory effects when isolated jejunal segments were in a low contractile state. The inhibitory effects of taurine on the contractility of isolated jejunal segments were blocked by propranolol and L-NG-nitroarginine but not by phentolamine, suggesting that adrenergic β receptors and a nitric oxide relaxing mechanism were involved when isolated jejunal segments were in high contractile states. No bidirectional effects of taurine on myosin phosphorylation were observed. The contractile states of jejunal segments determine taurine-induced stimulatory or inhibitory effects, which are associated with muscarinic receptors and adrenergic β receptors, and a nitric oxide associated relaxing mechanism.

Poisoning after ingestion of pufferfish in Brazil: report of 11 cases

In this brief communication the authors report eleven cases of human poisoning caused by ingestion of pufferfish meat. Three patients (two children and one adult) were seriously affected. The circumstances that precipitated the poisoning are discussed as well as the clinical aspects observed. No deaths were registered and the patients did not present sequelae after the episode.

Characteristics of Diprophylline-Induced Bidirectional Modulation on Rat Jejunal Contractility

In this study, we propose that diprophylline exerts bidirectional modulation (BM) on the isolated rat jejunal segment depending on its contractile state. The results supported the hypothesis. Diprophylline (20 microM) exerted stimulatory effects on the contractility of jejunal segment in six low contractile states while inhibitory effects in six high contractile states, showing the characteristics of BM. Diprophylline-induced stimulatory effect was significantly blocked by atropine, indicating the correlation with cholinergic activation. Diprophylline-induced inhibitory effect was partially blocked by phentolamine, propranolol, and L-N-Nitro-Arginine respectively, indicating their correlation with sympathetic activation and nitric oxide-mediated relaxing mechanisms. Diprophylline-induced BM was abolished by tetrodotoxin or in a Ca2+ free condition or pretreated with tyrosine kinase inhibitor imatinib, suggesting that diprophylline-induced BM is Ca2+ dependent, and that it requires the presence of enteric nervous system as well as pacemaker activity of interstitial cells of Cajal. Diprophylline significantly increased the reduced MLCK expression and myosin extent in constipation-prominent rats and significantly decreased the increased MLCK expression and myosin extent in diarrhea-prominent rats, suggesting that the change of MLCK expression may also be involved in diprophylline-induced BM on rat jejunal contractility. In summary, diprophylline-exerted BM depends on the contractile states of the jejunal segments, requires the presence of Ca2+, enteric nervous system, pacemaker activity of interstitial cells of Cajal, and MLCK-correlated myosin phosphorylation. The results suggest the potential implication of diprophylline in relieving alternative hypo/hyper intestinal motility.

The Clinical Features and Prognostic Factors in Adults with Acute Etrodotoxin Poisoning Caused by Ingesting Puffer Fish

PURPOSE: We conducted this study in order to determine clinical features and prognostic factors in adults with acute tetrodotoxin (TTX) poisoning caused by ingestion of puffer fish. METHODS: In this retrospective study, 107 patients were diagnosed with TTX poisoning. The subjects were divided into two groups according to duration of treatment; Group I, patients were discharged within 48 hours (n=76, 71.0%), Group II patients were discharged after more than 48 hours (n=31, 29.0%). Group II was subsequently divided into two subgroups [IIa (n=12, 11.2%), IIb (n=19, 17.8%)] according to the need for mechanical ventilation support. RESULTS: In multivariable logistic regression analysis, the predictors of the need for treatment over 48 hours were dizziness (odds ratio [OR], 4.72; 95% confidence intervals [CI], 1.59-12.83), time interval between onset of symptom and ingestion (OR, 0.56; 95% CI, 0.16-0.97), PaCO2<35 mmHg (OR, 8.37; 95% CI, 2.37-23.59). In addition, predictors of the need for mechanical ventilation were a time interval between onset of symptoms and ingestion (OR, 0.54; 95% CI, 0.11-0.96) and PaCO2<35 mmHg (OR, 5.65; 95% CI, 1.96-18.66). CONCLUSION: Overall, dizziness, time interval between onset of symptoms and ingestion, DeltaDBP and PaCO2<35 mmHg predict the need for treatment over 48 hours, time interval between onset of symptoms and ingestion and PaCO2<35 mmHg predict the need for mechanical ventilation support after acute TTX poisoning.

Nitrergic Pathway Is the Major Mechanism for the Effect of DA-9701 on the Rat Gastric Fundus Relaxation

BACKGROUND/AIMS: DA-9701 significantly improved gastric accommodation by increasing the postprandial gastric volume. In this study, we investigated how DA-9701 affects the rat gastric fundus relaxation. METHODS: Gastric fundus muscle strips (9 longitudinal and 7 circular muscles) were obtained from rats. Electrical field stimulation (EFS) was performed at various frequencies (1, 5, 10 and 20 Hz) and train durations (1, 5, 10 and 20 seconds) to select optimal condition for experiments. Isometric force measurements were performed in response to EFS. Peak and nadir were observed during the first 1 minute after initiation of EFS in control state and after sequential addition of atropine (1 microM), DA-9701 (0.5, 5, 25 and 50 microg), N-nitro-L-arginine (L-NNA, 100 microM), MRS2500 (1 microM) and tetrodotoxin (TTX, 1 microM) to the organ bath. RESULTS: The optimal frequency and duration of EFS to evoke nerve-mediated relaxation was determined as 5 Hz for 10 seconds. Addition of L-NNA in the presence of atropine and DA-9701 (50 microg) decreased nadir by inhibiting relaxation from -0.054 +/- 0.021 g to -0.022 +/- 0.015 g (P = 0.026) in longitudinal muscles. However, subsequent application of MRS2500 in the presence of atropine, DA-9701 (50 microg) and L-NNA did not affect nadir. In circular muscles, subsequent addition of L-NNA and MRS2500 in the presence of atropine and DA-9701 (50 microg) did not show significant change of nadir. CONCLUSIONS: Our data suggest that the effect of DA-9701 on the rat gastric fundus relaxation is mainly mediated by nitrergic rather than purinergic pathway.

A Case of Hemoperitoneum due to Spontaneous Rupture of Visceral Artery Aneurysm after Ingestion of Puffer Fish / 대한구급학회지

Ingestion of puffer fish can cause intoxication, which produces a wide range of symptoms due to the presence of neurotoxins, such as tetrodotoxin, in puffer fish. Abdominal pain is just one of the symptoms that should be treated with symptomatic and supportive therapy. This study reports a case of a 56-year-old male patient with abdominal pain, who was admitted to the emergency room with a diagnosis of puffer fish poisoning. In this case, the abdominal pain did not improve, but rather, the symptoms worsened. Finally, the cause of the abdominal pain was found to be hemoperitoneum due to active bleeding at the greater omentum, as observed on abdominal computed tomography; the source of bleeding was the right colic artery branch. Embolization was performed successfully, and the post-intervention course was uneventful. The patient was discharged without any complications within 13 days after admission.

Transient Diabetes Insipidus Associated with Tetrodotoxin Intoxication: A Case Report

Ingestion of puffer fish can result in severe and potentially lethal intoxication, referred to as tetrodotoxin intoxication. Tetrodotoxin is a potent neurotoxin well known for its ability to ability neuromuscular function. Tetrodotoxin is a specific and potent blocker of axonal sodium channel; it may block sodium channels in the axon of the neurons of the neurohypophysis, thereby inhibiting the release of vasopressin and causing diabetes insipidus neurotoxin. To our knowledge, previous report on diabetes insipidus causing tetrodotoxin is the only one case in Singapore. A married couple (69-year-old man and 57-year-old woman) ingested two green rough-backed puffer fish (Lagocephalus lunaris). They complained of paresthesia on perioral area and extremity and developed not only grade IV intoxication but also an increased urine output (4455 ml/day and 5035 ml/day), elevated serum sodium (157.4 mEq/L and 166.7 mEq/L) and elevated serum osmolality (324 mosmol/kg and 339 mosmol/kg), which suggested the development of diabetes insipidus. The administration of desmopressin nasal spray was successful in normalizing urine volume. Both were discharged on 20th and 18th hospital day, respectively, without any complications.

Determination of tetrodotoxin in seafood using graphitized carbon black clean-up with hilic ultra performance liquid chromatography-triple quadrupole mass spectrometry / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a rapid hilic ultra performance liquid chromatography (UPLC)-mass spectrum (MS)/MS method for determination of tetrodotoxin in seafood.</p><p><b>METHODS</b>The sample of muscle and liver of puffer fish and nassarius were extracted with aqueous solution containing 0.2% (V/V) acetic acid (the extract of liver must be purified through HLB cartridge), and then cleanup of extract was accomplished by solid-phase extraction with a graphitized carbon black cartridge. The analysis of tetrodotoxin was carried out on a chromatographic column (Acquity UPLC BEH Amide, 100 mm×2.1 mm×1.7 µm) with gradient elution of 95% (V/V) acetonitrile-H2O both containing 0.1% (V/V) formic acid and 2.0 mmol/L ammonium formate, and detected by positive electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode, and quantified by matrix-match standard solution.</p><p><b>RESULTS</b>The calibration curves were linear in the range of 30 - 10 000, 50 - 10 000 and 30 - 10 000 µg/kg of tetradotoxin in muscle and liver of puffer fish and in muscle of nassarius, respectively. The correlation coefficients were within 0.9963 - 0.9990. The limits of detection were 10, 20 and 10 µg/kg, and that of quantitation were 30, 50 and 30 µg/kg for muscle and liver of puffer fish and muscle of nassarius, respectively. The average recoveries were 81.5% - 93.1%, 82.3% - 106.0% and 83.5% - 95.2% for tetrodotoxin spiked in muscle and liver of puffer fish and in muscle of nassarius, respectively, with relative standard deviation (RSD) of 2.3% - 11%, 4.3% - 14.0% and 3.5% - 13.0% (n = 6).</p><p><b>CONCLUSION</b>The method was simple, accurate and sensitive, and could be successfully applied to the measurement of tetrodotoxin in puffer fish and nassarius.</p>

Postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pigs / 法医学杂志

OBJECTIVE@#To investigate the postmortem distribution of tetrodotoxin in tissues and body fluids of guinea pig, and to provide method and evidence for forensic identification and clinical diagnosis and treatment.@*METHODS@#Guinea pigs were intragastric administrated with 100, 50, 15 microg/kg tetrodotoxin, respectively. The poisoning symptoms were observed. The samples of heart, liver, spleen, lung, kidney, brain, stomach, intestines, bile, heart blood and urine were collected. The concentrations of tetrodotoxin in tissues and body fluids were measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS).@*RESULTS@#After administrated with tetrodotoxin, all guinea pigs came out poisoning signs including tachypnea, weary and dead finally. Tetrodotoxin concentrations in lung, stomach, intestines and urine were higher, followed by blood, heart and brain. The concentration in bile was the lowest.@*CONCLUSION@#Postmortem distribution of tetrodotoxin in guinea pig is uneven. The concentration in the lung, stomach, intestines, urine and heart blood are higher, those tissues could be used for diagnosis of tetrodotoxin poisoning.

Methionine Enhances the Contractile Activity of Human Colon Circular Smooth Muscle In Vitro

Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 microM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 microM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and Nw-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.

Involvement of veratridine-induced increase of reverse Na(+)/Ca(2+) exchange current in intracellular Ca(2+) overload and extension of action potential duration in rabbit ventricular myocytes / 生理学报

The objectives of this study were to investigate the effects of veratridine (VER) on persistent sodium current (I(Na.P)), Na(+)/Ca(2+) exchange current (I(NCX)), calcium transients and the action potential (AP) in rabbit ventricular myocytes, and to explore the mechanism in intracellular calcium overload and myocardial contraction enhancement by using whole-cell patch clamp recording technique, visual motion edge detection system, intracellular calcium measurement system and multi-channel physiological signal acquisition and processing system. The results showed that I(Na.P) and reverse I(NCX) in ventricular myocytes were obviously increased after giving 10, 20 μmol/L VER, with the current density of I(Na.P) increasing from (-0.22 ± 0.12) to (-0.61 ± 0.13) and (-2.15 ± 0.14) pA/pF (P < 0.01, n = 10) at -20 mV, and that of reverse I(NCX) increasing from (1.62 ± 0.12) to (2.19 ± 0.09) and (2.58 ± 0.11) pA/pF (P < 0.05, n = 10) at +50 mV. After adding 4 μmol/L tetrodotoxin (TTX), current density of I(Na.P) and reverse I(NCX) returned to (-0.07 ± 0.14) and (1.69 ± 0.15) pA/pF (P < 0.05, n = 10). Another specific blocker of I(Na.P), ranolazine (RAN), could obviously inhibit VER-increased I(Na.P) and reverse I(NCX). After giving 2.5 μmol/L VER, the maximal contraction rate of ventricular myocytes increased from (-0.91 ± 0.29) to (-1.53 ± 0.29) μm/s (P < 0.01, n = 7), the amplitude of contraction increased from (0.10 ± 0.04) to (0.16 ± 0.04) μm (P < 0.05, n = 7), and the baseline of calcium transients (diastolic calcium concentration) increased from (1.21 ± 0.08) to (1.37 ± 0.12) (P < 0.05, n = 7). After adding 2 μmol/L TTX, the maximal contraction rate and amplitude of ventricular myocytes decreased to (-0.86 ± 0.24) μm/s and (0.09 ± 0.03) μm (P < 0.01, n = 7) respectively. And the baseline of calcium transients reduced to (1.17 ± 0.09) (P < 0.05, n = 7). VER (20 μmol/L) could extend action potential duration at 50% repolarization (APD(50)) and at 90% repolarization (APD(90)) in ventricular myocytes from (123.18 ± 23.70) to (271.90 ± 32.81) and from (146.94 ± 24.15) to (429.79 ± 32.04) ms (P < 0.01, n = 6) respectively. Early afterdepolarizations (EADs) appeared in 3 out of the 6 cases. After adding 4 μmol/L TTX, APD(50) and APD(90) were reduced to (99.07 ± 22.81) and (163.84 ± 26.06) ms (P < 0.01, n = 6) respectively, and EADs disappeared accordingly in 3 cases. It could be suggested that: (1) As a specific agonist of the I(Na.P), VER could result in I(Na.P) increase and intracellular Na(+) overload, and subsequently intracellular Ca(2+) overload with the increase of reverse I(NCX). (2) The VER-increased I(Na.P) could further extend the action potential duration (APD) and induce EADs. (3) TTX could restrain the abnormal VER-induced changes of the above-mentioned indexes, indicating that these abnormal changes were caused by the increase of I(Na.P). Based on this study, it is concluded that as the I(Na.P) agonist, VER can enhance reverse I(NCX) by increasing I(Na.P), leading to intracellular Ca(2+) overload and APD abnormal extension.