Approaches are being explored to restore accommodation to the presbyopic eye. Some of these approaches can be tested in monkeys by stimulating accommodation in various ways including using carbachol iontophoresis. Knowledge of the repeatability of carbachol iontophoresis stimulated accommodation in the monkey phakic eye is necessary to understand the variability of this method of evaluating accommodation. Data from 9 to 10 separate carbachol iontophoresis experiments performed on phakic eyes from 8 monkeys were retrospectively analyzed. For each experiment, carbachol was applied iontophoretically to the eyes of anesthetized monkeys and refraction generally measured every two minutes until accommodation reached a plateau. Repeated experiments were performed in each monkey over periods ranging from 10 to 18 months. Maximum accommodation measured for each monkey ranged from 11.1 D to 18.3 D with standard deviations from 0.8 D to 2.1 D and differences in accommodative amplitude varying from 2.2 D to 7.5 D. Time to reach maximum accommodation ranged from 18 to 64 min in individual experiments. Averaged time-courses indicate that maximum accommodation is generally achieved between 10 and 20 min after carbachol administration. Although carbachol iontophoresis is considered a reliable method to stimulate maximum accommodation in anesthetized monkeys, the amplitude achieved typically varies by more than 2 D. Presbyopia treatments evaluated in this way in phakic monkeys would need to show an increase in accommodation of over 2 D to clearly demonstrate that the treatments work when being tested with carbachol iontophoresis stimulation.

We have expanded neuroepithelial cells dissociated from the embryonic rat telencephalon in serum-free defined medium containing basic fibroblast growth factor (bFGF) in order to generate a model neuroepithelium to study the interaction of ethanol with both growth factor- and transmitter-stimulated proliferation. Ethanol blocked proliferation stimulated by bFGF and by carbachol, an agonist at muscarinic acetylcholine receptors, in a dose-dependent manner. In addition, ethanol attenuated autonomous expansion of neuroepithelial cells occurring following withdrawal of bFGF. The latter effect was associated with an increase in the number of apoptotic cells identified by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling labeling. We studied the effects of ethanol on carbachol-stimulated signaling pathways critical to its proliferative effects. Ethanol significantly reduced carbachol-stimulated Ca(2+) signaling, as well as Erk1/Erk2, Akt and cyclic AMP-response element-binding phosphorylations in a dose-dependent manner. Comparison of the potency of ethanol in attenuating carbachol-stimulated proliferation and signal transduction showed that mitogen-activated protein kinase phosphorylation was less sensitive to ethanol than the other parameters. The results indicate that ethanol's suppression of proliferation induced by carbachol in this model neuroepithelium likely involves multiple signaling pathways. These effects in vitro may help to explain the devastating effects of prenatal ethanol exposure in vivo, which contribute to the fetal alcohol syndrome.

We employed simultaneous field potential recordings from CA3, subiculum and entorhinal cortex in an in vitro brain slice preparation to understand the involvement of these limbic areas in the generation of the field potential oscillations that are induced by bath application of the muscarinic receptor agonist carbachol. Regularly spaced oscillations that mainly presented at theta frequency range (5-12Hz) occurred synchronously in all three structures in the presence of carbachol. These oscillations, which disappeared when slices were perfused with pirenzepine or with glutamatergic receptor antagonists, were categorized as short (<4s) and long (>4s) with short events oscillating at higher frequencies than long events. Field oscillations were highly synchronized between regions and latency analysis revealed that they often initiated in the entorhinal cortex later than in the other two structures. Blocking GABAA receptors modified the activity patterns of both short and long oscillations and decreased their coherence in the theta frequency range. Finally, blocking KCC2 activity disclosed a pattern of recurrent short oscillations. Our results suggest that in the presence of carbachol both subiculum and CA3 most often drive theta generators in the entorhinal cortex and that these oscillations are influenced but not abolished by altering GABAA receptor signaling.

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1-5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior. Classification, Molecular and cellular pharmacology.

Asthma exacerbations evoke emergency room visits, progressive loss of lung function and increased mortality. Environmental and industrial toxicants exacerbate asthma, although the underlying mechanisms are unknown. We assessed whether 3 distinct toxicants, salicylic acid (SA), toluene diisocyanate (TDI), and 1-chloro-2,4-dinitrobenzene (DNCB) induced airway hyperresponsiveness (AHR) through modulating excitation-contraction coupling in human airway smooth muscle (HASM) cells. The toxicants include a non-sensitizing irritant (SA), respiratory sensitizer (TDI) and dermal sensitizer (DNCB), respectively. We hypothesized that these toxicants induce AHR by modulating excitation-contraction (EC) coupling in airway smooth muscle (ASM) cells.

We reported previously that many neurodegenerative changes characteristic of apoptosis could be induced by a short fragment of beta-amyloid protein, A(beta31-35), in cultured newborn mice cortical neurons, and that these changes were accompanied with alterations in expression of some genes. This study was designed to examine whether the apoptotic processes and related gene modulations in this model could be affected by coadministration of carbachol by electrophoretic analysis for DNA ladder formation and by RT-PCR assays for genomic modulation. The results showed that (1) simultaneous incubation with carbachol dose- and time-dependently blocked the specific DNA ladder formation induced by exposure to A(beta31-35) and (2) the A(beta31-35)-induced downregulation of bcl-2 and upregulations of bax, p53, and c-fos genes were reversed or ameliorated by the coadministration of carbachol. It is proposed that A(beta31-35)-induced apoptosis can be prevented by carbachol through mechanisms that modulate the expression of related genes.

BACKGROUND Safety concerns about drugs used intracamerally during cataract surgery have been the subject of many studies. In this study, the effect of using intracameral carbachol and epinephrine on choroidal thickness was evaluated. MATERIAL AND METHODS This prospective interventional study included 81 eyes of 81 patients undergoing cataract surgery. During cataract surgery, intracameral carbachol was administered to 27 eyes, intracameral epinephrine was administered to 20 eyes, and 34 eyes were the control group. Macular choroidal thickness measurement was performed before, 1 day, and 1 week after phacoemulsification surgery in all patients using optical coherence tomography. RESULTS Subfoveal choroidal thickness was significantly reduced at day 1 and week 1 in the group receiving intraoperative carbachol compared with preoperative measurement (P=0.016). In addition, choroidal thickness in the 500 µm nasal fovea was significantly reduced in the carbachol group at 1st week compared to the preoperative measurement (P=0.008). There was no significant difference in postoperative subfoveal thickness in the intraoperative epinephrine group and control group (P=0.179 and P=0.953, respectively). Choroidal thickness at 1000 µm nasal fovea was significantly higher in the epinephrine group at postoperative 1st day than preoperative and postoperative 1st week values (P=0.009). CONCLUSIONS The use of intracameral epinephrine caused an increase in choroidal thickness 1000 µm nasal of the fovea, while intracameral carbachol caused thinning in the subfoveal and 500 µm nasal quadrant. Intracameral drug administration during cataract surgery may be associated with posterior segment complications.

To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process.

Airway fibrosis is one of the pathological features of chronic obstructive pulmonary disease (COPD), and recent studies revealed that acetylcholine plays an important role in the development of airway remodeling by stimulating proliferation and collagen synthesis of lung fibroblasts. This study was designed to examine the effects of a long-acting muscarinic receptor antagonist (LAMA) glycopyrronium and a long-acting β2 adrenergic receptor agonist (LABA) indacaterol on acetylcholine-mediated fibrotic responses in lung fibroblasts.

Calcium-activated chloride channels (CaCCs) mediate numerous physiological functions and are best known for the transport of electrolytes and water in epithelia. In the intestine, CaCC currents are considered necessary for the secretion of fluid to protect the intestinal epithelium. Although genetic ablation of ANO1/TMEM16A, a gene encoding a CaCC, reduces the carbachol-induced secretion of intestinal fluid, its mechanism of action is still unknown. Here, we confirm that ANO1 is essential for the secretion of intestinal fluid. Carbachol-induced transepithelial currents were reduced in the proximal colon of Ano1-deficient mice. Surprisingly, cholera toxin-induced and cAMP-induced fluid secretion, believed to be mediated by CFTR, were also significantly reduced in the intestine of Ano1-deficient mice. ANO1 is largely expressed in the apical membranes of intestines, as predicted for CaCCs. The Ano1-deficient colons became edematous under basal conditions and had a greater susceptibility to dextran sodium sulfate-induced colitis. However, Ano1 depletion failed to affect tumor development in a model of colorectal cancer. We thus conclude that ANO1 is necessary for cAMP- and carbachol-induced Cl- secretion in the intestine, which is essential for the protection of the intestinal epithelium from colitis.

PGE2 is a potent bronchodilator, but the mechanisms underlying this effect have not been fully elucidated. Acetylcholine-induced contractions of airway smooth muscle (ASM) are associated with the generation of repetitive Ca2+ oscillations in airway smooth muscle cells (ASMC) and the force of contraction is positively correlated with the frequency of the underlying Ca2+ oscillations. The purpose of the present study was to examine if carbachol-evoked Ca2+ oscillations in isolated ASMC were inhibited by PGE2. Isolated murine ASMC loaded with fluo4-AM were imaged with a Nipkow spinning disk confocal microscope. Cells responded to application of CCh (1 μM) by generating an initial Ca2+ transient followed by a series of Ca2+ oscillations. This activity was abolished by PGE2 (300 nM) and the EP2R agonist (R)-butaprost (3 μM) and the inhibitory effects of PGE2 were reversed by application of the EP2R antagonist PF-04418948 (100 nM). Activation of adenylate cyclase using forskolin (1 μM) mimicked the effects of PGE2. The PKA activator, 6-MB-cAMP (300 μM) reduced the frequency of CCh-induced Ca2+ oscillations by 33% and the PKA inhibitor Rp-8-CPT-cAMPs partially reversed the inhibitory effects of PGE2. The EPAC activator 007-AM (10 μM) reduced the frequency of the oscillations by 60% and joint application of 007-AM and 6-MB-cAMP reduced oscillation frequency by ∼85%. CCh-induced Ca2+ oscillations were inhibited by 2-APB and tetracaine, but caffeine-evoked Ca2+ transients were resistant to PGE2. These data suggest that PGE2 inhibits CCh-induced Ca2+ oscillations in murine ASMC via stimulation of EP2Rs and a mechanism involving activation of PKA and EPAC.

To investigate protein content of saliva produced in vitro by samples of human submandibular gland following stimulation with the muscarinic agent carbachol.

The present study aimed to investigate the effect of carbachol on the intestinal tight-junction barrier in a rat model of severe acute pancreatitis (SAP) without aggravating pancreatic injury, and to determine whether cell division cycle 42 (Cdc42)/F-actin could have a regulatory role. Rats were separated into a sham-operation (SO) group (n=10), SO + carbachol group (n=10), SAP group (n=60) and SAP + carbachol group (n=60). Sodium taurocholate (5%) was retrogradely injected into the biliopancreatic duct of rats to induce SAP. Subsequently, 16S rRNA sequencing was used to detect bacterial translocation (BT) in the gut of surviving animals. Hematoxylin and eosin staining was used to detect morphological changes in the pancreas and intestine. The expression of F-actin and tight junction proteins was analyzed by western blotting and immunofluorescence, and Cdc42 expression was analyzed by immunohistochemistry and western blotting. The results demonstrated that the intestinal injury in SO and SO + carbachol groups was lower than that in the SAP + carbachol group (P<0.05); however, the intestinal injury was similar in the SO and SO + carbachol groups (P>0.05), and was significantly more severe in the SAP group compared with the SAP + carbachol group (P<0.05). Similarly, pancreatic injury in the SAP and SAP + carbachol groups was significantly higher compared with the SO and SO + carbachol groups (P<0.05); however, pancreatic injury was similar in the SAP and SAP + carbachol groups (P>0.05), and in the SO and SO + carbachol groups (P>0.05). Furthermore, the mortality rate and BT in the SAP group were significantly higher compared with the SAP + carbachol group (mortality rate, 50% vs. 30%, P<0.05; BT, 60% vs. 33.3%, P<0.05). In addition, the expression of Cdc42, F-actin and claudin-2 was significantly higher in the SAP and SAP + carbachol groups compared with the SO and SO + carbachol groups (P<0.05), and the expression of occludin and zonula occludens-1 were significantly higher in the SO and SO + carbachol groups compared with the SAP and SAP + carbachol groups (P<0.05). In conclusion, these findings demonstrated that carbachol may protect the intestinal barrier in the SAP rat model without aggravating pancreatic injury via regulation of Cdc42/F-actin expression.

Cholinergic mechanism is involved in motor behavior. In songbirds, the robust nucleus of the arcopallium (RA) is a song premotor nucleus in the pallium and receives cholinergic inputs from the basal forebrain. The activity of projection neurons in RA determines song motor behavior. Although many evidences suggest that cholinergic system is implicated in song production, the cholinergic modulation of RA is not clear until now. In the present study, the electrophysiological effects of carbachol, a nonselective cholinergic receptor agonist, were investigated on the RA projection neurons of adult male zebra finches through whole-cell patch-clamp techniques in vitro. Our results show that carbachol produced a significant decrease in the spontaneous and evoked action potential (AP) firing frequency of RA projection neurons, accompanying a hyperpolarization of the membrane potential, an increase in the evoked AP latency, afterhyperpolarization (AHP) peak amplitude, and AHP time to peak, and a decrease in the membrane input resistance, membrane time constant, and membrane capacitance. These results indicate that carbachol reduces the activity of RA projection neurons by hyperpolarizing the resting membrane potential and increasing the AHP and the membrane conductance, suggesting that the cholinergic modulation of RA may play an important role in song production.

Although the nucleus accumbens (NAc) typically is not considered a primary component of the circuitry underlying either the acquisition or retrieval of conditioned fear, evidence suggests that this region may play some role in modulating fear-related behaviors. The goal of the present study was to explore a potential role for NAc cholinergic receptors in the expression of fear-potentiated startle (FPS) and baseline startle reactivity. Intra-NAc infusion of the broad-acting cholinergic receptor agonist, carbachol, suppressed FPS elicited by re-exposure to both a discrete odor previously paired with footshock and the conditioning context. Although carbachol elevated spontaneous motor activity, activity bouts did not account for startle suppression in carbachol-treated Ss. In addition, intra-NAc carbachol suppressed baseline startle over a range of acoustic pulse intensities in the absence of explicit fear conditioning. Collectively, these findings suggest that NAc cholinergic receptors play a role in the modulation of baseline startle reactivity, rather than in the retrieval of learned fear, and that this role is independent of overt motor activity.

In this paper we have determined the different signal pathways involved in M(1) and M(3) muscarinic acetylcholine receptor (mAChR) dependent stimulation of cyclo-oxygenase 1 (cox-1) mRNA gene expression and PGE(2) production on rat cerebral frontal cortex. Carbachol stimulation of M(1) and M(3) mAChR exerts an increase in cox-1 mRNA gene expression without affecting cox-2 mRNA expression and increased PGE(2) generation. Besides, increased phosphoinositide (PI) turnover and stimulation of nitric oxide synthase (NOS) and cyclic GMP (cGMP) production. Inhibitors of phospholipase A(2) (PLA(2)), COX and phospholipase C (PLC), calcium/calmodulin (CaM), NOS and soluble guanylate cyclase prevent the carbachol effect. These results suggest that carbachol-activation of M(1) and M(3) mAChR increased PGE(2) release associated with an increased expression of cox-1 and NO-cGMP production. The mechanism appears to occur directly to PLC stimulation and indirectly to PLA(2) activation. These results may contribute to understand the effects and side effect of non-steroidal anti-inflammatory drugs in patients with cerebral degenerative diseases.

The role of the brainstem cholinergic system in the regulation of sleep-wake states has been studied extensively but relatively little is known about the role of cholinergic mechanisms in prefrontal cortex in the regulation of sleep-wake states. In a recent study, we showed that prefrontal cholinergic stimulation in anesthetized rat can reverse the traits associated with anesthesia and restore a wake-like state, thereby providing evidence for a causal role for prefrontal cholinergic mechanisms in modulating level of arousal. However, the effect of increase in prefrontal cholinergic tone on spontaneous sleep-wake states has yet to be demonstrated. Therefore, in this study, we tested the hypothesis that delivery of cholinergic agonists - carbachol or nicotine - into prefrontal cortex of rat during slow wave sleep (SWS) would produce behavioral arousal and increase the time spent in wake state. We show that unilateral microinjection (200 nL) of carbachol (1 mM) or nicotine (100 mM) into prefrontal cortex during SWS decreased the latency to the onset of wake state (p = 0.03 for carbachol, p = 0.03 for nicotine) and increased the latency to the onset of rapid eye movement sleep (p = 0.008 for carbachol, p = 0.006 for nicotine). Although the infusion of 1 mM carbachol increased the time spent in wake state (p = 0.01) and decreased the time spent in SWS (p = 0.01), infusion of 10 or 100 mM nicotine did not produce any statistically significant change in sleep-wake architecture. These data demonstrate a differential role of prefrontal cholinergic receptors in modulating spontaneous sleep-wake states.

1. The acid secretagogue effect of gastrin is mainly mediated by the release of enterochromaffin-like (ECL) cell histamine, but the mechanism of muscarinic stimulation of acid secretion remains unclear. The results of studying aminopyrine uptake in isolated parietal cells, and histamine release in isolated ECL cells suggest that muscarinic agents may act both directly on the parietal cell and indirectly via histamine release from ECL cells. 2. We examined parietal and ECL cell responses to the muscarinic agent carbamylcholine (carbachol) in conscious rats and in rat isolated vascularly perfused stomachs. 3. Intravenous carbachol stimulated acid secretion in conscious gastric fistula rats and increased H+K+ ATPase mRNA abundance, indicating activation of parietal cells. In these experiments there was no increase in portal venous histamine, or in oxyntic mucosal histidine decarboxylase (HDC) enzyme activity and HDC mRNA abundance. 4. In rat isolated stomachs stimulated with carbachol in the dose range 10 nM(-1) mM only the 1 microM concentration increased venous histamine significantly. 5. We concluded that the muscarinic agent carbachol stimulates acid secretion and H+K+ ATPase mRNA in vivo by a direct effect on the parietal cell, that does not depend on the release of ECL cell histamine.

In intestinal epithelial cells, acute regulation of the brush border Na(+)/H(+) exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca(2+) ([Ca(2+)]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin.

To test and compare in a masked fashion the efficacy of using a parasympathomimetic drug (3% carbachol) and an alpha-2 agonist (0.2% brimonidine) in both combined and separate forms to create optically beneficial miosis to pharmacologically improve vision in presbyopia.