Characterizing the hippocampal theta's response to carbachol; using a complete septo-hippocampal preparation / Caracterización de la respuesta theta del hipocampo al carbacol en una preparación septo-hipocámpica completa

Abstract: The present study describes the pharmacological analysis of the effects of carbachol, a cholinergic agonist, on hippocampal theta activity. Knowing that this activity is critically related to cognitive function and altered in patients with neurodegeneration, pharmacological efforts aiming to directly modulate hippocampal theta activity becomes of central importance. In a recently developed complete septo-hippocampal preparation, carbachol elicited significant theta power enhancement with 1 μM. Concentrations under 1 μM and over 2 μM carbachol caused significant reduction in the power of hippocampal theta activity. Carbachol effects were completely blocked with the cholinergic antagonist scopolamine. At the experimental level, it is the first time the direct action of a cholinergic agonist is evaluated in the septo-hippocampal pathway completely isolated. However, carbachol as a cholinergic agonist is a drug with a certain level of nonspecific response. That is why to correct this experimental limitation, we used scopolamine (cholinergic antagonist) which allowed us to corroborate the effects on the cholinergic pathway. In summary, electrophysiological assays demonstrated an effective concentration range of carbachol specifically modulating hippocampal theta activity.

Resumen: El presente estudio describe el análisis farmacológico de los efectos de carbacol, un agonista colinérgico, sobre la actividad theta del hipocampo. Sabiendo que esta actividad está críticamente relacionada con la función cognitiva y alterada en pacientes con neurodegeneración, los esfuerzos farmacológicos destinados a modular directamente la actividad theta del hipocampo se vuelven de gran importancia. En una preparación completa que contiene la región septal media conectada al hipocampo, desarrollada recientemente, 1 μM de carbacol provocó un incremento significativo a nivel de potencia en la actividad theta del hipocampo. Las concentraciones menores de1 μM y mayores a 2 μM causaron una reducción significativa en la potencia de la actividad theta. Los efectos del carbacol fueron completamente bloqueados con la escopolamina, antagonista colinérgico. A nivel experimental, es la primera vez que se evalúa la acción directa de un agonista colinérgico en la vía septo-hipocámpica completamente aislada. Sin embargo, el carbacol como agonista colinérgico es un fármaco que presenta cierto nivel de respuesta inespecífica. Es por eso que para corregir esta limitante experimental, se utilizó escopolamina (antagonista colinérgico) lo que nos permitió corroborar los efectos sobre la vía colinérgica. En resumen, nuestros estudios electrofisiológicos demostraron un intervalo de concentración eficaz del carbacol que modula específicamente la actividad theta del hipocampo.

Effectiveness and safety evaluation of low-dose carbachol application during implantable collamer lens implantation for myopic eyes / 中华实验眼科杂志

Background Carbachol solution (0.01％) is an agonist of M cholinoceptor and skeletal muscle N cholinoceptor,and it is used to play miotic effect and open peripheral iridectomic hole during the surgery of implantable collamer lens (ICL) implantation in order to lower intraocular pressure (IOP).However,the anterior chamber injection of 0.01％ carbachol solution often causes relevant complications,while whether lower dose of carbachol solution is effective and safe is unclear.Objective This study was to compare the effectiveness and safety between 0.01％ carbachol solution and 0.005％ carbachol solution after anterior chamber injection during the ICL implantation.Methods One hundred and fifty-two eyes of 76 cataract patients were included in Daping Hospital of Third Military Medical University from September 2014 to September 2015.ICL implantation and periphery iridectomy were carried out on both eyes of the patients and the 0.01％ carbachol solution was injected into the anterior chamber during the surgery of the right eyes and 0.01％ carbachol solution was used in the left eyes.The operation duration and IOP at postoperative 2 hours and systemic choline-like reaction were compared between 0.01％ carbachol solution group and 0.005％ carbachol solution group.Results The mean operation duration was (11.86± 2.39) minutes and (11.22 ± 1.85) minutes in the 0.01％ carbachol group and 0.005％ carbachol group,without significant difference between two groups (t =1.851,P =0.066).IOP was (15.76 ± 2.18) mmHg and (13.58 ±2.24)mmHg in the 0.01％ carbachol group before and after surgery,and those in the 0.005％ carbachol group was (15.70±2.35)mmHg and (13.12±2.17)mmHg,there was no significant difference in the IOP between the two concentrations of carbachol (Fsroup =0.986,P=O.322).The IOP at postoperative 2 hours was lower than that before operation,the difference was statistically significant(Ftime =97.339,P =0.000).There was no interaction between drug concentration and time (Fcorrelation =0.772,P =0.381).The incidences of complications,such as dizziness,nausea and vomiting,were lower in the 0.005％ carbachol group than those in the 0.01％ carbachol group (x2 =13.01,5.16,4.03,all at P＜0.05).Conclusions Carbachol solution (0.005％) can play intraoperative miosis effect and maintain effective operation duration in ICL implantation.In addition,the application of 0.005％ carbachol solution is quite safe in both intraoperation and postoperation.

Effect of pure carbachol or combined with dietary fiber on intestinal mucosal barrier of rats after diffuse brain injury / 中国中西医结合急救杂志

Objective To study the protective effect of pure carbachol or combined with dietary fiber on intestinal mucosal barrier of rats after diffuse brain injury (DBI).Methods An adult male Wistar rat model of DBI was reproduced by gravitational shock method. The rats injured and survived after resuscitation were divided into three groups: model group (n = 40), carbachol group (n = 40) and carbachol combined with dietary fiber group (combined group,n = 32). In addition, a control group was established by simply an incision performed on the scalp, and the rats could drink freely (n = 5). In the experimental groups, 2 hours after resuscitation the rats began to receive gavage, 6 hours once, the liquid amount 15 mL/kg should be assured in every 6 hours, and if insufficient, normal saline was supplemented. In model group, normal saline 90 mL/kg was given, in carbachol group, carbachol 300μg/kg was administered and in combined group, carbachol 300μg/kg combined with dietary fiber 60 mL/kg was supplied. At 3 (combined group being excluded), 6, 12, 24 and 48 hours after resuscitation, the rats were anesthetized to collect samples and detect the plasma levels of D-lactate and activity of diamine oxidase (DAO) respectively, and the changes of villus height of small intestine were examined by a light microscope.Results The plasma D-lactate levels and the activities of DAO at any time point in the experimental groups were significant higher than those in control group (allP < 0.01). Along with the prolongation of time, the levels of plasma D-lactate and DAO activities in carbachol and carbachol plus diatary fiber groups were gradually lower than those of the model group, and at 48 hours after injury they reached their valley values [D-lactate (ng/L): 6.32±0.79, 7.46±1.67 vs. 17.65±1.53, DAO activity (kU/L): 0.76±0.01, 0.86±0.01 vs. 2.23±0.15]. Under light microscopy, compared with control group, the villus height of small intestinal mucosa at any time point in any experimental group was gradually lowered, and reached the valley values at 12 hours, then gradually increased , and peaked at 48 hours, the villus height in carbachol group and combined group was higher than that in model group (μm: 265.36±10.20, 261.54±10.38 vs. 247.51±9.39, bothP < 0.05).Conclusion When only carbachol is administered into the rat intestine early after diffuse brain injury in rats, beginning from 6 hours after injury, the protective effect of intestinal mucosal barrier is shown, representing decrease of plasma D-lactate level and DAO activity, amelioration of intestinal mucosal damage and protection of intestinal mucosal barrier; under the same above situation, the carbachol combined with dietary fiber was applied, showing the similar above carbachol protective effects.

The protection of carbachol on intestinal barrier function in patients with trauma / 中国中西医结合急救杂志

Objective To observe the protection of carbachol on intestinal barrier function in patients with trauma. Methods A prospective randomized controlled trial was conducted. Seventy patients after trauma with a definite diagnosis of multiple organ dysfunction syndrome(MODS)from Department of Critical Care Medicine in Hebei United University Affiliated Hospital were included. According to random number table,the patients were divided into a carbachol treatment group(37 cases)and a mosapride citrate treatment group(33 cases),and all the patients in the two groups were treated by antibacterial drugs,supportive agents for organ function,surgery, etc symptomatic treatment. Based on the conventional treatment,in the carbachol treatment group,carbachol was administered through a stomach tube at the dose of 0.2 mg/kg,twice a day,and the dose was doubled if no exhaust or defecation persisted for 3 days after treatment,while in the mosapride group,mosapride citrate was given at the dose of 5 mg once and thrice a day,the therapeutic course of both groups being 7 days. On the 1st,3rd,5th, 7th day after admission,peripheral venous fasting blood in early morning was collected,the activity of diamine oxidase(DAO),expression rates of CD11b+and CD18+in polymorphonuclear neutrophil(PMN),contents of tumour necrosis factor-α(TNF-α)and interleukin-10(IL-10) were detected,and the clinical curative effects were observed. Results Compared to the mosapride citrate treatment group,the total effective rate was significantly higher in the carbachol treatment group on the 7th day after treatment〔70.3%(26/37)vs. 45.5%(15/33),P<0.05〕. The activity of DAO,expression rates of CD11b+and CD18+in PMN,contents of TNF-αand IL-10 in the carbachol treatment group were decreased with the extension of time,and reached valley values on the 7th day,the differences were statistically significant in the comparisons with those in mosapride citrate treatment group at the same time point〔DAO(mg/L):3.21±0.52 vs. 3.93±0.51,CD11b+:(14.89±2.16)% vs.(28.92±1.59)%,CD18+:(53.67±2.44)% vs. (72.46±4.08)%, TNF-α(ng/L):111.44±16.42 vs. 129.73±18.74, IL-10(ng/L):67.71±38.83 vs. 121.45±40.23,all P<0.05〕. At the various time points,the above indexes had no obvious changes in mosapride citrate treatment group. Conclusion Carbachol can ameliorate the ischemic/reperfusion(I/R)injury in patients with intestinal barrier dysfunction after trauma,decrease the release of inflammatory cytokines in vivo,and promote peristalsis of intestinal tract,therefore carbachol has clinical value of protecting intestinal barrier function.

Selective mastoporan inhibition of muscarinic activation of bovine tracheal smooth muscle

Muscarinic activation of bovine tracheal smooth muscle (BTSM) leading to smooth muscle contraction involves the generation of two cGMP signals (20 and 60 s), being 20s peak associated with soluble (sGC) and the second (60s) to membrane-bound Natriuretic Peptide- receptor-Guanylylcy clases (NPR-GC). In this study, we showed that pre-incubation of isolated BTSM strips with mastoparan and superactive mastoparan (mastoparan 7) decreased significantly the muscarinic dependent contractile smooth muscle responses in dose-dependent and non-competitive manner. Moreover, mastoparan (50 nM) inhibited completely the BTSM-muscarinic contractile responses and affected dramatically the carbachol-dependent cGMP signals being the first cGMP signal inhibited in a 63 ± 5%, whereas the second signal disappeared. Mastoparan inhibition of muscarinic activation is specific since other spasmogens as serotonin and histamine fully contracted these BTSM strips under mastoparan treatment. Cyclic GMP levels were evaluated by exposing BTSM strips to activators of NO-sensitive sGC as Sodium Nitroprussiate (SNP) and Natriuretic Peptides as CNP-53 for membrane-bound NPR-GC. Thus, SNP and CNP increased in a binary way, in more than 20 fold cGMP levels at 30-40 s being both increments inhibited by mastoparan. Furthermore, the Gi/o-protein involvement on mastoparan inhibition of cGMP elevations induced by CNP and SNP is suggested by Pertussis toxin pre-treatment, which reversed mastoparan effects. These results indicate that muscarinic signal transduction cascades leading to airway smooth muscle contractions involved two different guanylyl cyclases being both regulated by mastoparan-sensitive G-proteins. ANP, Natriuretic Peptide type A; ASM, Airway Smooth Muscle; BTSM, Bovine Tracheal Smooth Muscle; CNP-53, Natriuretic Peptide type C-53; GPCR, G-Protein Coupled Receptor; Gq16, Heterotrimeric G protein subtype 16; Gi/o, Heterotrimeric G protein subtype...

La activación muscarínica del músculo liso de las vías aéreasrelacionada a la contracción de dicho músculo liso esta asociada a la generación de dos señales de GMPc (20 y 60 s), siendo la señal de los 20s relacionado a la activación de la guanililciclasa soluble mientras que el pico de los 60s a la guanililciclasa unida membranas y sensible a péptidos natriuréticos (NPR-GC). En este trabajo, nosotros mostramos que la pre-incubación de fragmentos del músculo liso traqueal de bovino (BTSM) con mastoparan y su análogo superactivo (mastoparan 7), en una forma dosis dependiente, son capaces de disminuir de manera significativa la actividad contráctil dependiente de agentes muscarinicos. Adicionalmente, mastoparan (50 nM) inhibió completamente la respuesta contráctil muscarinica del BTSM y afectó dramáticamente los picos de GMPc asociados a la activación muscarinica siendola primera señal inhibida en un 63 ± 5%, mientras que la segunda señal desapareció completamente. Esta inhibición del mastoparan de la activación muscarínica es especifica ya que otros espamogenos como la serotonina y la histamina fueron capaces de inducir respuestas máximas en presencia del mastoparan y su análogos. Este efecto del mastoparan sobre los niveles del GMPc fue evaluado en presencia de otros agentes generadores de este segundo mensajero como son el nitroprusiato de sodio (SNP) que activa la guanililciclasa soluble sensible a NO y los péptidos natriureticos como el CNP-53 (CNP) activador de la NPR-GC asociada a membranas plasmáticas. Tanto, el SNP como el CNP aumentaronen mas de 50 veces los niveles de GMPc a los 30-40 s en forma bifasica, siendo estos incrementos inhibidos de manera significativa por el mastoparan. Ademas, se sugiere la participación de proteínas Gi/o en los efectos inhibitoriosdel mastoparan, porque la Toxina pertussis revertió los efectos inhibitorios. Estos resultados indican que la cascada de activación muscarinica que conduce...

Antidipsogenic effects of central adenosine-5'-triphosphate

Besides other physiological functions, adenosine-5'-triphosphate (ATP) is also a neurotransmitter that acts on purinergic receptors. In spite of the presence of purinergic receptors in forebrain areas involved with fluid-electrolyte balance, the effect of ATP on water intake has not been investigated. Therefore, we studied the effects of intracerebroventricular (icv) injections of ATP (100, 200 and 300 nmol/µL) alone or combined with DPCPX or PPADS (P1 and P2 purinergic antagonists, respectively, 25 nmol/µL) on water intake induced by water deprivation. In addition, the effect of icv ATP was also tested on water intake induced by intragastric load of 12 percent NaCl (2 mL/rat), acute treatment with the diuretic/natriuretic furosemide (20 mg/kg), icv angiotensin II (50 ng/µL) or icv carbachol (a cholinergic agonist, 4 nmol/µL), on sodium depletion-induced 1.8 percent NaCl intake, and on food intake induced by food deprivation. Male Holtzman rats (280-320 g, N = 7-11) had cannulas implanted into the lateral ventricle. Icv ATP (300 nmol/µL) reduced water intake induced by water deprivation (13.1 ± 1.9 vs saline: 19.0 ± 1.4 mL/2 h; P < 0.05), an effect blocked by pre-treatment with PPADS, but not DPCPX. Icv ATP also reduced water intake induced by NaCl intragastric load (5.6 ± 0.9 vs saline: 10.3 ± 1.4 mL/2 h; P < 0.05), acute furosemide treatment (0.5 ± 0.2 vs saline: 2.3 ± 0.6 mL/15 min; P < 0.05), and icv angiotensin II (2.2 ± 0.8 vs saline: 10.4 ± 2.0 mL/2 h; P < 0.05), without changing icv carbachol-induced water intake, sodium depletion-induced 1.8 percent NaCl intake and food deprivation-induced food intake. These data suggest that central ATP, acting on purinergic P2 receptors, reduces water intake induced by intracellular and extracellular dehydration.

The changes of HMGB-1 in patients with sepsis and the effect of carbachol on prevention of HMGB-1 / 中国综合临床

Objective To observe the changes of HMGB-1 of burned patient with sepsis and to investigate the effect of carbaehol on production of HMGB-1 from human monocytes stimulated by lipopolysaccharide(LPS) and its mechanism. Methods The peripheral blood samples of burned patients with sepsis and healthy donors were taken for isolation of monoeyte. The subjects were divided into six groups: the menocytes group were added only with 1640 culture medium,LPS group were added only with LPS;nicotine group and carbachol group were with carbachol first or nicotine for 5 rain and then with LPS. The levels of HMGB-1 were tested by EIJSA. α-bungarotoxin + carba-chol group were added with atropine or α-bungarotoxin for 5 min and then were given carbachol and 5 min later were stimulated with LPS. 48 h-incubation later supemate were collected for the detection of HMGB-1 by ELISA. Results HMGB-1 levels of burned patients with sepsis were ( 12.94±6.54)μ/L,which were much higher than that of healthy donors( [ (2.01±0.03 )μ/L), P <0.01 ]. When monoeytes were stimulated by LPS alone, concentrations of HMGB-1 were (9.39±1.37 ) μ/L, which were obviously higher than that of controls [( 1.48±0.69 ) μ/L), P < 0.01]. Mter pretreated by carbaehol or nicotine,concentrations of HMGB-1 were (3.52±1.64)μ/L and (4.01± 1.56) μ/L respectively, which were obviously decreased compared with that of LPS stimulation alone group ( P < 0.01). When pretreatedby atropine before addition of carbachol, there was no significant changes in concentrations of HMGB-1 [ (3.87±2.01 )μ/L]. When pretreated with α-Bungaretoxin before carbachol adminitration, inhibitive effect of carbachol on production of HMGB-1 were blocked [(8.97±1.97 )μ/L ]. Conclusion The release of HMGB-1 in burned patients with sepsis is increased. Carbachol could obviously reduce the production of HMGB-1, which may affect through activating α 7 subunit of cholinergic N receptor.

Effect of carbachol on the expression of la on monocyte and lymphocyte apoptosis rate of the burned rats with delayed resuscitation / 中国医师杂志

Objective To explore the changing trend of Ia on monocyte, lymphocyte apoptosis rate, TNF-α and IL-6 in abdominal aorta of burned rats with delayed resuscitation and the influence of application of carbachol on them. Methods Adult male Wistar rats were randomly divided into normal control group(n =8), scald group(n =48) and scald with carbachol treatment group(n =48). In latter two groups, rats were inflicted with 30% total body surface area (TBSA) full-thickness scald and delayed fluid resuscitation. All scald rats were sacrificed at the 6th hours or 1st, 2nd, 3rd, 7th, 14th day after scald, with 8 rats at each time point. Expression of Ia antigen on monocyte and lymphocyte apoptosis rate were determined by direct immunofluorescence on a flow cytometer, and TNF-α and IL-6 was measured by ELISA. Results Expression of la on monocyte was obviously lower than that of controls. The lowest levels were recorded on the 6th hours and 1st day after scald. Subsequently, Ia was elevated gradually, but still lower than that of normal rats(P <0. 01). After administration of carbachol, Ia expression was obviously promoted, compared with the simple scald group (P <0. 01). Lymphocyte apoptosis rate, TNF-α and IL-6 was higher than that of controls(P <0. 01). After administration of cavachol, , lymphocyte apoptosis rate and TNF-α and IL-6 was obviously down-regulated on the 6th hours, 1st day, 2nd day and 3rd day after scald injury, compared with the simple scald group (P < 0. 01 or 0. 05). Conclusion After severe burn with delayed fluid resuscitation, there is a low la expression, high lymphocyte apoptosis rate and increased releasing of proinflammatory cytokine. Immune function was suppressed. Carbacho] could improve the immune function of scald rats with delayed fluid resuscitation.

Carbachol alleviates intestinal inflammation during enteral fluid resuscitation of rats with burn shock / 中华急诊医学杂志

Objective To investigate the effect of carbachol on local gut inflammation during entetal resuscitation of rats with bum shock. Method Thirty-eight Wistar rats were subjected to 35%TBSA full thickness scald injury, and enteral fluid was infused into animal intestines via duodenal stomas 30 minutes post bum. The animals were randomly divided into four groups: no resuscitation (Control, n = 8), enteral resuscitation using either a glucose electrolyte solution (GES, n = 10) or GES plus carbachol (60 μg·kg-1,GES/CAR, n = 10), or carbachol alone (CAR, n = 10) .The volumeof GES infusion was based on the Parkland formula (4 ml· 1% TB-SA-1·Kg-1) - All animals were sacrificed 4 hours post bum, and specimens of jejunal tissue were collected to determine the levels of tumor necrosis factor (TNF)-α, nitric oxide (NO), nitric oxide synthase (NOS) and myeloperoxidase (MPO). Serum assays for plasma diamine oxidase (DAO) activities were also performed. Results There were no statistical differences in the intestinal levels of NOS, NO, TNF-α and MPO, and plasma OAO activities, between the GES group and the control group. Compared to the GES group, the GES/CAR group showed significantly lowered levels of intestinal NOS (1.276 ±0.391 vs. 1.818 ±0.436, P＜0.05), NO (0.925 ±0.402 vs. 1.561 ±0.190, P ＜ 0.05, TNF-α (0.87±0.13 vs. 1.94±0.47, P ＜0.01) and MPO (0.465 ±0.092 vs. 0.832±0.214, P＜0.05),and reduction in plasma DAO activites (0.732±0.192 vs. 1.381 ±0.564, P ＜0.05). The CAR group also showed significantly lowered levels of intestinal NOS, NO, TNF-α and MPO and reduced plasma DAO activites, compared to the GES group. Conclusions Theses results suggest that carbachol significantly inhibits the release of proinflammatory mediator and attenuates local inflammation in gut during enteral fluid resuscitation of rats in rats with bum shock. We postulate that carbachol may exert its and-inflammatory effects via the cholinergic anti-inflammatory pathway.

Effects of carbachol on intestinal inflammation and mucosal blood flow in rats with gut ischemia-reperfusion / 中国医师杂志

Objective To investigate the effects of carbachol on intestinal inflammation and mucosal blood flow after gut ischemia-repedusion(I/R) in rat. Method A jejunal sac was formed in Wistar rats. The superior mesenteric artery (SMA) was occluded for 45 mi-nutes followed by 240 minutes of reperfusion. Animals were random divided into three groups: sham operation, L/R + saline injection (I/R + NS) and I/R + carbachol injection (0.1mg/kg, I/R + Ca). Immediately after occluded of SAM blood flow, either 0.1mg/kg of carba-chol or same account of 0.9% saline was injected into the jejunal sac. The pathological injury was observed with HE staining. The activity of DAO and content of TNF-α in intestinal mucosa tissue were determined. Mucosal blood flow was measured by laser Doppler. All measure-ments were done at 0 min, 30 min, 60 min, 120 min, and 240 min after reperfusion. Result In I/R group the activity of DAO in intestinal mucosa and mucosal blood flow deceased, meanwhile the content of TNF-α gut tissue was dramatically increased than those in sham operation (P<0.01). Severe pathological changes were observed in intestinal mucosa. After injection of carbachol, the activity of DAO and mucosal blood flow increased (P<0.01), but the content of TNF-α in intestinal mucosa were dramatically decreased (P<0.01), compared with those in I/R group. Conclusion Administration of carbachol protects intestinal ischemia-reperfusion injury by attenuating intestinal mucosa inflammation and increasing gut mueosal blood flow.

Relationship between Carbachol Hyperstimulation-Induced Pancreatic Acinar Cellular Injury and Trypsinogen or NF-κB Activation in Rats in vitro / 华中科技大学学报（医学）（英德文版）

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulationinduced pancreatic acinar cellular injury and trypsinogen activation or NF-κB activation in rats was studied in vitro. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc), and NF-κB inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The cellular injury was evaluated by measuring the leakage of LDH from pancreatic acinar cells. The results showed that as compared with control group, 10-3 mol/L carbachol induced a significant increase of the intracellular trypsin activity and the leakage of LDH from pancreatic acinar cells. Pretreatment with 2 mmol/L pefabloc could significantly decrease the activity of trypsin and the leakage of LDH from pancreatic acinar cells (P ＜0.01) following the treatment with a high concentration of carbachol (10-3 mol/L) in vitro. The addition of 10-2 mol/L PDTC didn't result in a significant decrease in the activity of trypsin and the leakage of LDH from pancreatic acinar cells treated with a high concentration of carbachol (10-3 mol/L) in vitro (P＞0.05). It was concluded that intracellular trypsinogen activation is likely involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro. NF-κB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

Effect of carbachol on membrane potential of smooth muscle cells of gastric antrum of diabetic rats / 第三军医大学学报

Objective To study the effect of carbachol(CCH) on membrane potential of smooth muscle cells of gastric antrum of diabetic rats to verify the mechanism of gastric motility disturbance.Methods Fifty healthy Wistar rats of 2-month old,weighing 100-160 g,were divided into the control group(n=20) and diabetes mellitus group(n=30).Three months after the establishment of the rat model of diabetes by intraperitoneal injection of 60 mg/kg streptozotocin,gastric emptying time and gastric electrical activity were measured,and the resting membrane potentials of smooth muscle cells in antrum were measured by confocal laser scanning microscopy,then treated with carbachol of 10~(-9) mmol/L,10~(-8) mmol/L,10~(-7) mmol/L,the membrane potentials were measured.Results As compared with the normal rats,gastric emptying time in diabetic rats was significantly longer and abnormal gastric electric rhythm was significantly increased,the abnormal rhythm index(ARI) and the coefficient of variation(CV) of slow wave frequency in diabetic rats were significantly higher,but the resting membrane potentials remained unchanged and the sensitivity of CCH-induced membrane depolarization was increased.Conclusion The increase of sensitivity of CCH-induced membrane depolarization may be involved in the diabetes-induced gastric motor disorders.

Inhibitory Effects of Potassium Channel Blockers on Carbachol-induced Contraction in Rat Detrusor Muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 micrometer to 5 micrometer. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 micrometer thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potasssium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.

Effect of PKC antagonist-chelerythrine on mouse pancreatic secretion stimulated by cholecystokinin / 中国实用内科杂志

Objective To establish a mouse pancreatic exocrine secretion system,in order to study the effect of secretagogues,such as cholecystokinin (CCK) and carbachol (CCH),on the exocrine secretion of mouse pancreas,and use a protein kinase C (PKC) antagonist-chelerythrine(Che) to research the possible mechanism.Methods Prospective control study.Results CCK and CCH increased the secretion of amylase from the mouse pancreatic acini about two fold of that of the control (7.02%vs14.29%),and,CCK or CCH added Che,could increase the secretion of amylase to a level of about three fold(19.02%).Conclusion CCK or CCH stimulate the pancreatic secretion concerning the PKC cellular signal system;surprisingly,a PKC antagonist,chelerythrine,can reinforces their stimulation.

The gene expression profiles through activating endothelial target for acetylcholine / 中国药理学通报

Aim To investigate the effects of gene expression through activating endothelial target for acetylcholine (ETA).Methods Comparative studies were carried out to explore the distinct gene expression of carbachol and pilocarpine.Results Human coronary aortic endothelial cells were incubated with carbachol which activates ETA and muscarinic receptors and with pilocarpine which activates muscarinic receptors distinctly for 10 hours at the concentration of 100 ?mol?L -1.The endothelial gene expression was detected by BiostarH-14112S cDNA expression profiling microarrays containing 14 000 human unigenes. There were 801 differential genes totally (491 differentially expressed both by carbachol and by pilocarpine, 310 differentially expressed by carbachol exclusively). We found a significant increase in expression of 119 and a significant decrease in expression of 191 genes after treatment by carbachol preferentially. And these genes were not affected by pilocarpine. Further globally functional catagorization indicates that there are seven types of distinct expressed genes induced by carbachol selectively including membrane receptors and G proteins, ion channels, G protein coupled receptors, transcriptional factors, apoptosis-related genes, cell adhesive factors, thrombosis and atherosclerosis-related genes.Conclusion The differentially expressed genes evoked by carbachol exclusively are closely associated with ETA and its effectors.

Characterization of Intermediate Conductance K+ Channels in Submandibular Gland Acinar Cells

There are some evidences that K+ efflux evoked by muscarinic stimulation is not mainly mediated by large conductance K+ (BK) channels in salivary gland. In this experiment, we therefore characterised non BK channels in rat submandibular gland acinar cells and examined the possibility of agonist effect on this channel using a patch clamp technique. Two types of K+ channels were observed in these cells. BK channels were observed in 3 cells from total 6 cells and its average conductance was 152+/- 7 pS (n=3). The conductance of the another types of K+ channel was estimated as 71+/-7 pS (n=6). On the basis of the conductance of this channel, we defined this channel as intermediate conductance K+ (IK) channels, which were observed from all 6 cells we studied. When we increased Ca2+ concentration of the bath solution in inside-out mode, the IK channel activity was greatly increased, suggesting this channel is Ca2+ sensitive. We next examined the effect of carbachol (CCh) and isoproterenol on the activity of the IK channels. 10(-5) M isoproterenol significantly increased the open probability (Po) from 0.08+/-0.02 to 0.21+/-0.03 (n=4, P<0.05). Application of 10(-5) M CCh also increased Po from 0.048+/-0.03 to 0.55+/-0.33 (n=5, P<0.05) at the maximum channel activity. The degree of BK channel activation induced by the same concentration of CCh was lower than that of IK channels; Po value was 0.011+/-0.003 and 0.027+/-0.005 in control and during CCh stimulation (n=3), respectively. The result suggests that IK channels exist in salivary acinar cells and its channel activity is regulated by muscaricinic and beta- adrenergic agonist. We conclude that IK channels also play a putative role in secretion as well as the BK channels in rat submandibular gland acinar cells.

Effects of carbachol on gastric emptying and gastric blood flow in oral fluid resuscitation of burn shock in rats / 解放军医学杂志

Objective To observe the effects of carbachol(a cholinergic agent)on gastric emptying and gastric blood flow in resuscitation of early shock with oral administration of glucose-electrolyte solution(GES)in scalded rats.Methods Seventy-eight male Wistar rats were randomly assigned into five groups:shame scald(n=6),scald without fluid resuscitation(n=18),scald resuscitated with oral GES alone(n=18),scald resuscitated with oral carbachol alone(CAL,n=18),and scald resuscitated with oral carbachol plus GES(GES/CAL,n=18).About 35% TBSA scalded injury on back of rats was produced with boiling water(100℃,15s)in the latter four groups.GES(2ml/kg,which is equivalent to half volume of fluid infusion in first 24 hour according to Parkland' formula)was given twice in two-hour intervals by gavage beginning from 30min after the injury in GES and GES/CAL group.Carbachol(60?g/kg)was gavaged 30min after the injury in CAL and GES/CAL group.The gastric emptying rate was determined by phenol red emptying method and gastric blood flow was measured with laser Doppler at 2h,4h and 6h after the scald injury.Results The gastric emptying was obviously delayed and gastric blood flow was significantly reduced in scald animals compared with those in sham group(P

Mechanism of an increase in concentration of intracellular calcium by carbachol in human gastric smooth muscle cell / 대한내과학회지

BACKGROUND: The contraction of smooth muscle depends on an increase in the concentration of intracellular calcium ion and the source of this increase to various stimuli is different according to organs or species. Nevertheless, there have been only a few studies on human stomach smooth muscle. This study was designed to identify the source of the calcium utilized in the muscle contraction induced by carbachol, which is an important factor among various stimuli. METHODS: After the administration of carbachol with various conditions in cultured human stomach smooth muscle cells, fura-2-acetoxymethyl ester was used to measure the increase in the intracellular calcium concentration. RESULTS: (1) The carbachol-induced increase in the intracellular calcium concentration was not attenuated after removal of extracellular calcium. (2) Carbachol induced a small increase in the intracellular calcium concentration even after the depletion of intracellular calcium store. (3) Repeated histamine administration blocked the carbachol-induced increase in the intracellular calcium concentration in calcium-free extracellular solution. CONCLUSION: The main source of calcium utilized in human stomach smooth muscle contraction by carbachol is intracellular calcium store, particularly inositol triphosphate(IP3)-sensitive calcium stores. However, extracellular calcium also contributes to the carbachol-induced increase in the intracellular calcium concentration.

Muscarinic receptor subtype controlling the carbachol-induced muscle contraction in guinea pig gastric antrum

Stimulation of muscarinic receptors by carbachol (CCh) in the circular smooth muscle of the guinea pig gastric antrum causes muscle contraction. In the present study, muscarinic receptor subtype controlling the muscle contraction in response to CCh was studied using putative muscarinic receptor antagonists. Isometric force of the isolated circular muscle strips was measured in an organ bath. CCh contracted the muscle in a dose-dependent way, and each of the three muscarinic receptor antagonists, 4-diphenylacetoxy-N-methylpeperdine methiodide (4-DAMP), methoctramine and pirenzepine shifted the concentration-response curves to the right without significantly reducing the maximum force. The affinities of the muscarinic antagonists (pA2 values) obtained from Schild plot analysis were 10.15, 7.05 and 6.84 for 4-DAMP, methoctramine and pirenzepine, respectively. These results suggest that the M3-subtype mainly mediate the muscle contraction in response to CCh in guinea pig gastric antrum.

Effects of Ketamine on Carbachol and Epidermal Growth Factor Contraction in Rat Trachea / 대한마취과학회지

BACKGROUND: It is well known that ketamine, a dissociative anesthetic, inhibits smooth muscle contractions induced by high KCl and receptor agonists such as carbachol, histamine and endothelin. It has been reported that the inhibitory effect is mediated by a decrease in Ca2+ influx through the plasma membrane in smooth muscle. However, the mechanism of action for ketamine is not fully understood. In the present study, the effect of ketamine on Ca2+ -dependent and -independent carbachol and epidermal growth factor (EGF) contractions was investigated in rat tracheal smooth muscle. METHODS: Tracheal smooth muscle strips from Sprague-Dawley rats were mounted in an organ bath with physiological salt solution (PSS, pH 7.4, 37oC). When high KCl and carbachol (1, 10 micrometer) induced contraction and verapamil and ketamine attenuated contraction, and when EGF or carbachol alone and EGF plus carbachol in Ca2+ free media induced contration and verapamil and ketamine inhibited contration, the changes in tention of the muscle strips were measured. RESULTS: 1) Ketamine inhibited the contractions stimulated by high KCl (70 mM) and carbachol (1, 10 micrometer) in a concentration-dependent manner. The inhibitory effect was more marked in the 1 micrometer carbachol preparation than in the 10 micrometer carbachol preparation. 2) Although the contraction induced by carbachol (1 micrometer) was attenuated by verapamil (10 micrometer), a voltage-dependent Ca2+ channel blocker, the degree of inhibition was not complete, but only partial. The verapamil-insensitive portion of the contraction induced by carbachol was completely inhibited by the cumulative application of ketamine. 3) EGF (1 mg/ml), a tyrosine kinase-associated growth factor, or carbachol alone did not induce contraction in Ca2+ -free PSS (1 mM EGTA). However EGF plus carbachol contracted tracheal smooth muscle in the Ca2+ -free media and it was completely inhibited by ketamine but not by verapamil. CONCLUSIONS: These results suggest that ketamine inhibits the smooth muscle contraction including both verapamil-sensitive and -insensitive Ca2+ increments and by Ca2+ -independent contraction mechanisms.