Neurogenic pathways in remote ischemic preconditioning induced cardioprotection: Evidences and possible mechanisms

Remote ischemic preconditioning (RIPC) is an intrinsic phenomenon whereby 3~4 consecutive ischemia-reperfusion cycles to a remote tissue (noncardiac) increases the tolerance of the myocardium to sustained ischemiareperfusion induced injury. Remote ischemic preconditioning induces the local release of chemical mediators which activate the sensory nerve endings to convey signals to the brain. The latter consequently stimulates the efferent nerve endings innervating the myocardium to induce cardioprotection. Indeed, RIPC-induced cardioprotective effects are reliant on the presence of intact neuronal pathways, which has been confirmed using nerve resection of nerves including femoral nerve, vagus nerve, and sciatic nerve. The involvement of neurogenic signaling has been further substantiated using various pharmacological modulators including hexamethonium and trimetaphan. The present review focuses on the potential involvement of neurogenic pathways in mediating remote ischemic preconditioning-induced cardioprotection.

Role of Nicotinic Acetylcholine Receptor alpha3 and alpha7 Subunits in Detrusor Overactivity Induced by Partial Bladder Outlet Obstruction in Rats / 대한배뇨장애요실금학회지

PURPOSE: To investigate the role of alpha3 and alpha7 nicotinic acetylcholine receptor subunits (nAChRs) in the bladder, using a rat model with detrusor overactivity induced by partial bladder outlet obstruction (BOO). METHODS: Forty Sprague-Dawley rats were used: 10 were sham-operated (control group) and 30 were observed for 3 weeks after partial BOO. BOO-induced rats were further divided into 3 groups: Two groups of 10 rats each received intravesicular infusions with hexamethonium (HM group; n=10) or methyllycaconitine (MLC group; n=10), which are antagonists for alpha3 and alpha7 nAChRs, respectively. The remaining BOO-induced rats received only saline infusion (BOO group; n=10). Based on the contraction interval measurements using cystometrogram, the contraction pressure and nonvoiding bladder contractions were compared between the control and the three BOO-induced groups. Immunofluorescent staining and Western blotting were used to analyze alpha3 and alpha7 nAChRs levels. RESULTS: The contraction interval of the MLC group was higher than that of the BOO group (P<0.05). Nonvoiding bladder contraction almost disappeared in the HM and MLC groups. Contraction pressure increased in the BOO group (P<0.05) compared with the control group and decreased in the HM and MLC groups compared with the BOO group (P<0.05). Immunofluorescence staining showed that the alpha3 nAChR signals increased in the urothelium, and the alpha7 nAChR signals increased in the urothelium and detrusor muscle of the BOO group compared with the control group. Western blot analysis showed that both alpha3 and alpha7 nAChR levels increased in the BOO group (P<0.05). CONCLUSIONS: Alpha3 and alpha7 nAChRs are associated with detrusor overactivity induced by BOO. Furthermore, nAChR antagonists could help in clinically improving detrusor overactivity.

Nicotine in High Concentration Causes Contraction of Isolated Strips of Rabbit Corpus Cavernosum

It is well known that cigarette smoke can cause erectile dysfunction by affecting the penile vascular system. However, the exact effects of nicotine on the corpus cavernosum remains poorly understood. Nicotine has been reported to cause relaxation of the corpus cavernosum; it has also been reported to cause both contraction and relaxation. Therefore, high concentrations of nicotine were studied in strips from the rabbit corpus cavernosum to better understand its effects. The proximal penile corpus cavernosal strips from male rabbits weighing approximately 4 kg were used in organ bath studies. Nicotine in high concentrations (10(-5)~10(-4) M) produced dose-dependent contractions of the corpus cavernosal strips. The incubation with 10(-5) M hexamethonium (nicotinic receptor antagonist) significantly inhibited the magnitude of the nicotine associated contractions. The nicotine-induced contractions were not only significantly inhibited by pretreatment with 10(-5) M indomethacin (nonspecific cyclooxygenase inhibitor) and with 10(-6) M NS-398 (selective cyclooxygenase inhibitor), but also with 10(-6) M Y-27632 (Rho kinase inhibitor). Ozagrel (thromboxane A2 synthase inhibitor) and SQ-29548 (highly selective TP receptor antagonist) pretreatments significantly reduced the nicotine-induced contractile amplitude of the strips. High concentrations of nicotine caused contraction of isolated rabbit corpus cavernosal strips. This contraction appeared to be mediated by activation of nicotinic receptors. Rho-kinase and cyclooxygenase pathways, especially cyclooxygenase-2 and thromboxane A2, might play a pivotal role in the mechanism associated with nicotine-induced contraction of the rabbit corpus cavernosum.

Further casbane-type diterpenes from the soft coral Sinularia depressa / 中国天然药物

AIM@#To study the minor diterpenes from the soft coral Sinularia depressa@*METHOD@#The chemical constituents were isolated and purified by various chromatographic techniques, and the chemical structures, including absolute configuration, were established on the basis of detailed analysis of spectroscopic data and by literature comparison with the data of related known compounds.@*RESULTS@#A new casbane-type diterpene, 2-epi-10-hydroxydepressin (1), was isolated and identified.@*CONCLUSION@#Compound 1 is a new casbane-type diterpene.

Autonomic Nerve Regulation of Colonic Peristalsis in Guinea Pigs

BACKGROUND/AIMS: Colonic peristalsis is mainly regulated via intrinsic neurons in guinea pigs. However, autonomic regulation of colonic motility is poorly understood. We explored a guinea pig model for the study of extrinsic nerve effects on the distal colon. METHODS: Guinea pigs were sacrificed, their distal colons isolated, preserving pelvic nerves (PN) and inferior mesenteric ganglia (IMG), and placed in a tissue bath. Fecal pellet propagation was conducted during PN and IMG stimulation at 10 Hz, 0.5 ms and 5 V. Distal colon was connected to a closed circuit system, and colonic motor responses were measured during PN and IMG stimulation. RESULTS: PN stimulation increased pellet velocity to 24.6 +/- 0.7 mm/sec (n = 20), while IMG stimulation decreased it to 2.0 +/- 0.2 mm/sec (n = 12), compared to controls (13.0 +/- 0.7 mm/sec, P < 0.01). In closed circuit experiments, PN stimulation increased the intraluminal pressure, which was abolished by atropine (10(-6) M) and hexamethonium (10(-4) M). PN stimulation reduced the incidence of non-coordinated contractions induced by NG-nitro-L-arginine methyl ester (L-NAME; 10(-4) M). IMG stimulation attenuated intraluminal pressure increase, which was partially reversed by alpha-2 adrenoceptor antagonist (yohimbine; 10(-6) M). CONCLUSIONS: PN and IMG input determine speed of pellet progression and peristaltic reflex of the guinea pig distal colon. The stimulatory effects of PN involve nicotinic, muscarinic and nitrergic pathways. The inhibitory effects of IMG stimulation involve alpha-2 adrenoceptors.

The pressor effect of angiotensin-(1-7) in the rat rostral ventrolateral medulla involves multiple peripheral mechanisms

OBJECTIVE: In the present study, the peripheral mechanism that mediates the pressor effect of angiotensin-(1-7) in the rostral ventrolateral medulla was investigated. METHOD: Angiotensin-(1-7) (25 pmol) was bilaterally microinjected in the rostral ventrolateral medulla near the ventral surface in urethane-anesthetized male Wistar rats that were untreated or treated (intravenously) with effective doses of selective autonomic receptor antagonists (atenolol, prazosin, methyl-atropine, and hexamethonium) or a vasopressin V1 receptor antagonist [d(CH2)5 -Tyr(Me)-AVP] given alone or in combination. RESULTS: Unexpectedly, the pressor response produced by angiotensin-(1-7) (16 ± 2 mmHg, n = 12), which was not associated with significant changes in heart rate, was not significantly altered by peripheral treatment with prazosin, the vasopressin V1 receptor antagonist, hexamethonium or methyl-atropine. Similar results were obtained in experiments that tested the association of prazosin and atenolol; methyl-atropine and the vasopressin V1 antagonist or methyl-atropine and prazosin. Peripheral treatment with the combination of prazosin, atenolol and the vasopressin V1 antagonist abolished the pressor effect of glutamate; however, this treatment produced only a small decrease in the pressor effect of angiotensin-(1-7) at the rostral ventrolateral medulla. The combination of hexamethonium with the vasopressin V1 receptor antagonist or the combination of prazosin, atenolol, the vasopressin V1 receptor antagonist and methyl-atropine was effective in blocking the effect of angiotensin-(1-7) at the rostral ventrolateral medulla. CONCLUSION: These results indicate that angiotensin-(1-7) triggers a complex pressor response at the rostral ventrolateral medulla that involves an increase in sympathetic tonus, release of vasopressin and possibly the inhibition of a vasodilatory mechanism.

Regional Difference in Colonic Motility Response to Electrical Field Stimulation in Guinea Pig

BACKGROUND/AIMS: In isolated guinea-pig colon, we investigated regional differences in peristalsis evoked by intrinsic electrical nerve stimulation. METHODS: Four colonic segments from mid and distal colon of Hartley guinea pigs, were mounted horizontally in an organ bath. Measurement of pellet propulsion time, intraluminal pressure, electrical field stimulation (EFS; 0.5 ms, 60 V, 10 Hz), and response of pharmacological antagonists, were performed to isolated segments of colon to determine the mechanisms underlying peristaltic reflexes evoked by focal electrical nerve stimuli. RESULTS: In fecal pellet propulsion study, the velocity of pellet propulsion was significantly faster in the distal colon and decreased gradually to the proximal part of the mid colon. Intraluminal pressure recording studies showed that luminal infusion initiated normal peristaltic contractions (PCs) in 82% trials of the distal colon, compared to that of mid colon. In response to EFS, the incidence of PCs was significantly increased in the distal colon in contrast, the incidence of non-peristaltic contractions (NPCs) was significantly higher in the middle-mid colon, distal-mid colon and distal colon, compared to that of proximal-mid colon. Addition of L-NAME into the bath increased the frequency of NPCs. EFS failed to cause any PCs or NPCs contractions in the presence of hexamethonium, atropine or tetrodotoxin. CONCLUSIONS: This study has revealed that electrical nerve stimulation of distal colon is the most likely region to elicit a peristaltic wave, compared with the mid or proximal colon. Our findings suggest that EFS-evoked PCs can be modulated by endogenous nitric oxide.

Autonomic and cardiovascular effects of pentobarbital anesthesia during trigeminal stimulation in cats / 国际口腔科学杂志·英文版

Stimulation of the trigeminal nerve can elicit various cardiovascular and autonomic responses; however, the effects of anesthesia with pentobarbital sodium on these responses are unclear. Pentobarbital sodium was infused intravenously at a nominal rate and the lingual nerve was electrically stimulated at each infusion rate. Increases in systolic blood pressure (SBP) and heart rate (HR) were evoked by lingual nerve stimulation at an infusion rate between 5 and 7 mg·kg(-1)·h(-1). This response was associated with an increase in the low-frequency band of SBP variability (SBP-LF). As the infusion rate increased to 10 mg·kg(-1)·h(-1) or more, decreases in SBP and HR were observed. This response was associated with the reduction of SBP-LF. In conclusion, lingual nerve stimulation has both sympathomimetic and sympathoinhibitory effects, depending on the depth of pentobarbital anesthesia. The reaction pattern seems to be closely related to the autonomic balance produced by pentobarbital anesthesia.

The Effect of Autonomic Nerve System on Neurotensin Induced Motility in Isolated, Vascularly Perfused Rat Colon

BACKGROUND/AIMS: Although neurotensin (NT) stimulates colon motility and the passage of intestinal contents, the associated mechanism of action remains unclear. The objective of this study was to investigate the effects of NT on colon motility using isolated rat colon. METHODS: Intraluminal pressure was measured at both the proximal and distal portions of the isolated colon. An isolated rat colon was perfused with Krebs solution via the superior mesenteric artery. After stabilization, NT was administered in concentrations of 14, 28, 138 and 276 pM. After pretreatment with phentolamine, propranolol, hexamethonium, atropine or tetrodotoxin, NT was administered at a concentration of 276 pM, and then the intraluminal pressure was monitored. RESULTS: NT significantly increased colon motility at concentrations of 14, 28, 138, and 276 in the proximal colon (25.1+/-6.5%, 175.4+/-117.0%, 240.8+/-115.1% and 252.3+/-110.6%, respectively) and in the distal colon (35.6+/-11.8%, 97.5+/-35.1%, 132.7+/-36.7% and 212.1+/-75.2%, respectively). The stimulant effect of NT was more potent in the proximal colon, in a concentration-dependent manner (P<0.05). The stimulant effect of NT was significantly inhibited by atropine at both the proximal and distal colon and by tetrodotoxin at the proximal colon, but not by tetrodotoxin at the distal colon and not by propranolol, phentolamine, or hexamethonium at both the proximal and distal colon. CONCLUSIONS: NT increased colon motility at both the proximal and distal portions of the rat colon. The effects were more prominent at the proximal portion. The results of this study suggest that the stimulant action of NT may be mediated by local cholinergic muscarinic receptors.

Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca2+ dependent exocitosis of vasoactive compounds. Using the Ca2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23). The increase in cytosolic Ca2+ induced by 10µM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 µM of hexamethonium. On the other hand, the increase in cytosolic Ca2+ could be depressed by nifedipine (0.01 -100 µM) or withdrawal of extracellular Ca2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca2+ channels, influx of external Ca2+ and liberation of vasoactive compounds.

The mechanism of action of neuropeptide Y in rat colonic motility / 대한내과학회지

BACKGROUND/AIMS: Neuropepetide Y (NPY) is involved in the regulation of several gut functions, but the neuronal action of NPY has not been fully investigated. This study was designed to investigate the effect and mechanism of action of NPY on motility in the proximal and distal rat colon. METHODS: Rat colon with an intact superior mesenteric artery was isolated. After a basal period, NPY was administered at concentrations of 14 pM, 70 pM, 140 pM, and 280 pM. Intraluminal pressures were monitored in the proximal and distal colon. The contractile response was expressed as a percent change of motility indices over the basal level. After a pre-infusion of atropine (AT), tetrodotoxin (TTX), propranolol, hexamethonium, and phentolamine, NPY was infused at a concentration of 140 pM, and pressures were monitored. RESULTS: NPY increased the colonic motility at concentrations of 14, 70, 140, and 280 pM in the proximal colon (28.5+/-28.2%, 48.4+/-34.3%, 122.9+/-97.3%, 68.2+/-28.1%, respectively) and in the distal colon (44.9+/-25.9%, 103.8+/-72.0%, 237.1+/-131.0%, 93.0+/-63.9%, respectively) in a dose-dependent manner. The enhancing effect of NPY (140 pM) on colonic motility was significantly suppressed by pretreatment with atropine, propranolol, and TTX. However, the effect of NPY was not inhibited by hexamethonium or phentolamine. CONCLUSION: NPY increases colonic motility. The enhancing effect of NPY on colonic motility may require cholinergic input via muscarinic receptors or adrenergic input via beta-receptors.

Neuromuscular Blocking Actions of Hexamethonium, Rocuronium and (-)Vesamicol / 대한마취과학회지

BACKGROUND: Hexamethonium (HM) and Rocuronium (R) are nAChR antagonists. However, there is some controversy as to whether R has a selective presynaptic effect. (-)Vesamicol (V) inhibits the transport of acetylcholine into the vesicles. This study compared the neuromuscular blockade of HM, R and V. METHODS: Hemidiaphragm-phrenic nerve preparations (male Sprague-Dawley rats [150-250 g]) were bathed in a Krebs solution maintained at 32oC and aerated with a mixture of 95% O2 and 5% CO2. Isometric forces were generated in response to 0.1 Hz, and 1.9-second 50 Hz with supramaximal stimulation (0.2 ms, rectangular) of the phrenic nerve. HM, R and V were added sequentially to achieve an 80-90% decrease in the ST. The ECs for ST, PTT and TF were calculated using a probit model. The antagonism indices of calcium (5 mM) and neostigmine (N) (250 nM) were assessed at the 85+/-5% level. RESULTS: The potency of ST, PTT and TF were respectively, 5.92, 3.56 and 1.99 mM for HM, 10.81, 5.27 and 4.4 1micronM for R, and 19.4, 15.2 and 13.3micronM for V. The neuromuscular blockades of R were reversed by N but not by calcium. Those of V were not reversed by either of them. Calcium and N inhibited the decrease in ST and TF by HM, respectively. CONCLUSIONS: The mechanism for how HM and R affect the neuromuscular blockade are different. V might not affect the release of acetylcholine.

The Effect of Alcohol in the Nicotinic Acetylcholine Receptor / 대한정신약물학회지

OBJECTIVE: It has been well known that alcohol can modulate several ligand-gated ion channel and voltage-gated ion channels. But the roles of alcohol in the autonomic neurons still remain unclear. In this study, thus we characterized the neuronal acetylcholine receptor (nnAChRs) and investigated the modulation of nnAChRs by ethanol (EtOH). METHODS: We used whole-cells which were acutely dissociated male rat major pelvic ganglion (MPG) neurons, and used gramicidin perforated patch clamp techniques. RESULTS: MPG neurons can be classified on the basis of the response of the soma membrane to depolarizing current pulses ; either tonic or phasic neurons. Sympathetic neurons expressing T-type Ca(2+) channels showed tonic firing pattern, while parasympathetic neurons lacking T-type Ca(2+) channels phasic firing to depolarizing current pulses. When hyperpolarizing currents were injected, sympathetic neurons produced post-anodal rebound spikes, while parasympathetic neurons were silent. Under current clamp mode, Acetylcholine (ACh) evoked significant membrane depolarization and produced subsequently marked membrane hyperporization. Under whole-cell mode, application of ACh-induced inward currents held at holding potentials below 0 mV and reversal potential was close to 0 mV, an equilibrium potential of nonselective cation channel. The ACh-activated current was blocked by methyllycaconitine (MLA ; 10 micrometer), hexamethonium (100 micrometer) and alpha-bungarotoxin (alpha-BuTx ; 100 nM), nAChRs antagonists. EtOH (40 mM) potentiated ACh-induced depolarization and hyperpolarization. EtOH also increased both alpha-BuTx-sensitive and -insensitive ACh-activated currents. Futhermore, EtOH potentiated 5-HT-activated current but had a little effect on GABA-activated current. CONCLUSION: These results suggest that EtOH modulates nnAChRs and 5-HT receptors in MPG neurons.

Blood flow measurements in rats using four color microspheres during blockade of different vasopressor systems

The use of colored microspheres to adequately evaluate blood flow changes under different circumstances in the same rat has been validated with a maximum of three different colors due to methodological limitations. The aim of the present study was to validate the use of four different colors measuring four repeated blood flow changes in the same rat to assess the role of vasopressor systems in controlling arterial pressure (AP). Red (150,000), white (200,000), yellow (150,000), and blue (200,000) colored microspheres were infused into the left ventricle of 6 male Wistar rats 1) at rest and 2) after vasopressin (aAVP, 10 æg/kg, iv), 3) renin-angiotensin (losartan, 10 mg/kg, iv), and 4) sympathetic system blockade (hexamethonium, 20 mg/kg, iv) to determine blood flow changes. AP was recorded and processed with a data acquisition system (1-kHz sampling frequency). Blood flow changes were quantified by spectrophotometry absorption peaks for colored microsphere components in the tissues evaluated. Administration of aAVP and losartan slightly reduced the AP (-5.7 ± 0.5 and -7.8 ± 1.2 mmHg, respectively), while hexamethonium induced a 52 ± 3 mmHg fall in AP. The aAVP injection increased blood flow in lungs (78 percent), liver (117 percent) and skeletal muscle (>150 percent), while losartan administration enhanced blood flow in heart (126 percent), lungs (100 percent), kidneys (80 percent), and gastrocnemius (75 percent) and soleus (94 percent) muscles. Hexamethonium administration reduced only kidney blood flow (50 percent). In conclusion, four types of colored microspheres can be used to perform four repeated blood flow measurements in the same rat detecting small alterations such as changes in tissues with low blood flow.

Characteristics of Nicotinic Receptor Expressed in Human Retinoblastoma

PURPOSE: To identify the characteristics and physiological function of the nicotinic receptor expressed in human retinoblastoma cells. METHODS: We measured possible nicotinic signaling in WERI-Rb-1 cells using the Ca2+ imaging technique and the patch clamp method. RESULTS: 1) Nicotine-induced [Ca2+]i rise arose entirely through Ca2+ influx, which was completely abolished by hexamethonium (100 micro M). 2) Nicotine also induced remarkable depolarization from -56.6 +/- 3.7 mV to -29.6 +/- 3.6 mV (n=4) under current clamp mode, but it failed to directly activate the T-type Ca2+ channel expressed in retinoblastoma cells. CONCLUSIONS: Nicotinic activation can increase the intracellular calcium level through calcium influx in the undifferentiated retinoblastoma cells, which may play important roles in cell proliferation, differentiation, and cell death.

Efficacy and Tolerability of Mirtazapine and Sertraline in Treatment of Patients with Posttraumatic Stress Disorder with Depression: A Randomized Open Label Trial / 신경정신의학

OBJECTIVES: The aim of this study was to investigate the potential use of mirtazapine in Korean veterans diagnosed with PTSD, by comparing it with sertraline, a drug approved for use in PTSD in the USA. METHODS: Efficacy was eveluated by Clinician Adninistered PTSD Scale (CAPS-2), the Hamilton Rating Scale for Depressin (HAMD-17) and the Clinical Global lmpresaion Scale (CGI), at baseline and week 1, 2, 6. Response was defined as a > or =30% decrease in CAPS-2 total score, a > or =50% decrease in total HAMD-17 score, and s CGI-I score<3. RESULTS: 51 patients on mirtazapine (measn age/duration of illness:59.1/33.5 yrs) and 49 on sertratine (mean age/duration of illness:60.6/35.6 years) completed the study. Mean daily dosage was 34.1 mg for mirtazapine and 101.5 mg for sertraline. On CAPS-2 total score more patients responded in the mirtazapine group at weak 1 (13 vs. 2%) and week 2 (51vs. 31%). At week 6 this difference was statistically significant (88 vs. 69%, p=0.039), CAPS-2 total score. HAMD-17 total score and CGI-I score decreased significantly in both groups, with no significant differences between groups on all time points. Main side effects for the rnirtazapine group:dry mouth (19.8%) and constipation (19.6%), somnolence (15.7%), weight gain (1.96%). For the sertraline group:indigestion (14.3%), palpitation (6.1%) agitation (2.0%), epigastric soreness (2.0%), insomnia (2.0%), sexual dysfunction (2.0%). CONCLUSION: Mirtazapine appeared to be an effective and well-tolerated treatment for PTSD in Korean veterans.

Difference in action sites between mecamylamine and hexamethonium on nicotinic receptors of sympathetic neurons / 生理学报

To compare the difference in action sites between mecamylamine (MEC) and hexamethonium (HEX) on nicotinic receptors of sympathetic neurons, we investigated the effects of MEC and HEX on the nicotine-induced currents in cultured superior cervical ganglion neurons by whole-cell patch clamp technique. The IC(50) of MEC and HEX for antagonizing the effect of 0.08 mmol/L nicotine was 0.0012 and 0.0095 mmol/L, respectively. Both MEC and HEX accelerated the desensitization of nicotinic receptors. Furthermore, by comparing their effects at holding potentials 30, 70 and 110 mV, it was indicated that their suppressing effect on the nicotine-induced currents was voltage-dependent. However, different from that of HEX, the inhibitory effect of MEC increased with administering the mixture of MEC and nicotine at intervals of 3 min, indicating a use-dependent effect of MEC. It is concluded that the action site of MEC on nicotinic receptors of sympathetic neurons is different from that of HEX.

The Regulation of AP-1 DNA Binding Activity by Long-term Nicotine Stimulation in Bovine Adrenal Medullary Chromaffin Cells: Role of Second Messengers

The signal pathways involved in the regulation of AP-1 DNA binding activity in long-term nicotine stimulated bovine adrenal medullary chromaffin (BAMC) cells have not been well characterized. To understand the involvement of second messengers in the regulation of AP-1 DNA binding activity, the present study was designed to define the time-course for inhibition of nicotine-induced responses by cholinergic antagonists, Ca2+ and calmodulin (CaM) antagonists, and calcium/calmodulin-dependent protein kinase (CaMK) II inhibitor using electrophoretic mobility shift assay. Nicotine (10microM) stimulation increased AP-1 DNA binding activity at 24 hr after treatment. Posttreatment with hexamethonium (1 mM) plus atropine (1microM) (HA), nimodipine (1microM), or calmidazolium (1microM) at 0.5, 3, and 6 hr after the nicotine treatment significantly inhibited the AP-1 DNA binding activity increased by long-term nicotine stimulation. However, posttreatment with HA, nimodipine, or calmidazolium at 9 or 12 hr after the nicotine treatment did not affect the nicotine-induced increase of AP-1 DNA binding activity. The pretreatment of BAMC cells with various concentrations of KN-62 inhibited the increase of AP-1 DNA binding activity induced by nicotine in a concentration-dependent manner. KN-62 (10microM) posttreatment beginning at 0.5, 3, or 6 hr after the nicotine treatment significantly inhibited the increase of AP-1 DNA binding activity. However, KN-62 posttreatment beginning at 9 or 12 hr after the nicotine treatment did not affect the increase of AP-1 DNA binding activity. This study suggested that stimulation (for at least 6 hr) of nicotinic receptors on BAMC cells was necessary for increase of AP-1 DNA binding activity, and activation of Ca2+, CaM, and CaMK II up to 6 hr at least seemed to be required for the increase of nicotine-induced AP-1 DNA binding activity.

Studies of Interaction between Hexamethonium and Lidocaine, alpha-Bungarotoxin or Decamethonium in Vitro / 대한마취과학회지

BACKGROUND: Interactions of neuromuscular blocking agents are antagonistic in a combination of depolarizing and nondepolarizing agents, additive in a combination of relative two compounds or synergistic in a combination of different two nondepolarizing agents. However, the interactions of neuromuscular blocking agents with a different site of action from each other have not been studied clearly. This study was designed to examine the interaction between hexamethonium and lidocaine, alpha-bungarotoxin or decamethonium with markedly different pre and postsynaptic sites of action. METHODS: Square wave, 0.1 Hz supramaximal stimuli or 2 Hz, 0.2 ms train of four (TOF) stimuli, was applied to the rat phrenic nerve-hemidiaphragm preparation, and the twitch height response was recorded mechanomyographically. The cumulative concentration effect and TOF ratio at each point of twitch depression after hexamethonium, lidocaine, alpha-bungarotoxin or decamethonium given were measured. The EC50 and EC95 of hexamethonium, lidocaine, alpha-bungarotoxin and decamethonium were calculated using an inhibitory sigmoid Emax model. In the experiment of each combination of two drugs, three points of the isobole for hexamethonium-lidocaine, hexamethonium-alpha-bungarotoxin and hexamethonium-decamethonium were established using ratios of 1 : 3, 1 : 1 and 3 : 1 of their EC50. Points on the line of theoretical additivity and 95% confidence intervals were calculated according to Tallarida et al. TOF ratios were observed at 75, 50 and 25% of the control twitch height value during each combination ratio of their EC50. RESULTS: Significant deviations of points on the isobole from the line of additivity to the left were found at all EC50 ratios of hexamethonium-lidocaine (P < 0.05 respectively), that to the right was found at all EC50 ratios of a hexamethonium-alpha-bungarotoxin and hexamethonium-decamethonium (P < 0.05 respectively). The magnitude of TOF fade depended upon the mixed ratios for their EC50. CONCLUSIONS: The interaction was found to be synergistic in the combination of hexamethonium- lidocaine, and antagonistic in the combination of hexamethonium-alpha-bungarotoxin and hexamethonium- decamethonium.

Neuromuscular Blocking Properties of beta-Bungarotoxin, Hexamethonium and Verapamil in the Rat Phrenic Nerve-Hemidiaphragm Preparation / 대한마취과학회지

BACKGROUND: beta-Bungarotoxin irreversibly changes the presynaptic membrane, hexamethonium acts on the presynaptic nicotinic receptor, and verapamil blocks the ion channels on the presynaptic membrane. The effect of these drugs on twitch height and train of four (TOF) ratio were investigated, as well as the reversal effects of neostigmine, pyridostigmine or 4-aminopyridine (4-AP) on the partial neuromuscular blockade induced by these drugs. METHODS: Square wave, 0.1 Hz supramaximal stimuli or 2 Hz, 0.2 ms train of four stimuli, was applied to the phrenic nerve-hemidiaphragm preparation of the rat, and the twitch height response was recorded mechanomyographically. The cumulative concentration effects and TOF ratios at each point of twitch depression after beta-bungarotoxin, hexamethonium or verapamil were measured. TOF ratios were observed at 75, 50 and 25% of the control twitch height value during observation of the concentration effect. The EC50 and EC95 of beta-bungarotoxin, hexamethonium or verapamil were calculated using an inhibitory sigmoid Emax model. The reversal effect of some doses of neostigmine, pyridostigmine or 4-aminopyridine to the partial neuromuscular block produced by EC50 of beta- bungarotoxin, hexamethonium or verapamil was determined. RESULTS: The EC50 and EC95 of beta-bungarotoxin, hexamethonium and verapamil were 0.0695 and 0.1160 microgram/ml, 1267.0 and 2033.5 microgram/ml and 29.45 and 37.99 microgram/ml respectively. TOF fade was marked with hexamethonium or verapamil but small with beta-bungarotoxin. Neostigmine or pyridostigmine did not reverse the partial neuromuscular block induced by beta-bungarotoxin, hexamethonium or verapamil. However, 4-AP produced a dose-dependent recovery of the twitch response (P < 0.05). CONCLUSIONS: beta-Bungarotoxin, hexamethonium and verapamil produced different degree of TOF fade, and this may be due to different sites of action of these drugs. 4-AP reversed effectively the partialneuromuscular block induced by beta-bungarotoxin, hexamethonium and verapamil, whereas, neostigmine and pyridostigmine did not.