Cholinergic Receptor Agonist Carbachol Research Articles

Effects of Carbachol on the Excitatory Muscarinic Cholinergic Receptors in Urinary Bladder and Plasma Inflammatory Mediators in Anesthetized Rats

To observe the effects of muscarinic cholinergic receptor agonist carbachol on urinary bladder function and plasma inflammatory mediators in anesthetized rats. A total of 20 rats were randomly divided into carbachol group and solvent control group, 10 rats in each group. The detection of bladder wall thickness and histological observation were conducted by light microscopy, the changes of urinary bladder function and plasma inflammatory mediators in rats were compared between the two groups. The maximum bladder capacity, static bladder pressure, compliance, bladder wet weight and bladder wall thickness in the carbachol group were significantly higher than those in the solvent control group. Light microscopic evaluation revealed that the detrusor cells in the solvent control group appeared to be in order in terms of size, arrangement and shape, while in the carbachol group the detrusor cells exhibited hypertrophy together with disordered arrangement, diverse morphology and vacuolar degeneration of muscle fiber. The levels of TNF-α and IL-10 in plasma of the carbachol group were significantly lower than those of the solvent control group. In anesthetized rats, the stimulation of muscarinic cholinergic receptors can significantly reduce urinary frequency, increase the volume of bladder storage and decrease systemic inflammatory response.

Cholinergic Overstimulation Attenuates Rule Selectivity in Macaque Prefrontal Cortex.

Acetylcholine is released in the prefrontal cortex (PFC) and is a key modulator of cognitive performance in primates. Cholinergic stimulation has been shown to have beneficial effects on performance of cognitive tasks, and cholinergic receptors are being actively explored as promising targets for ameliorating cognitive deficits in Alzheimer's disease. We hypothesized that cholinergic stimulation of PFC during performance of a cognitive task would augment neuronal activity and neuronal coding of task attributes. We iontophoretically applied the general cholinergic receptor agonist carbachol onto neurons in dorsolateral PFC (DLPFC) of male rhesus macaques performing rule-guided prosaccades and antisaccades, a well established oculomotor task for testing cognitive control. Carbachol application had heterogeneous effects on neuronal excitability, with both excitation and suppression observed in significant proportions. Contrary to our prediction, neurons with rule-selective activity exhibited a reduction in selectivity during carbachol application. Cholinergic stimulation disrupted rule selectivity regardless of whether it had suppressive or excitatory effects on these neurons. In addition, cholinergic stimulation excited putative pyramidal neurons, whereas the activity of putative interneurons remained unchanged. Moreover, cholinergic stimulation attenuated saccade direction selectivity in putative pyramidal neurons due to nonspecific increases in activity. Our results suggest excessive cholinergic stimulation has detrimental effects on DLPFC representations of task attributes. These findings delineate the complexity and heterogeneity of neuromodulation of cerebral cortex by cholinergic stimulation, an area of active exploration with respect to the development of cognitive enhancers.SIGNIFICANCE STATEMENT The neurotransmitter acetylcholine is known to be important for cognitive processes in the prefrontal cortex. Removal of acetylcholine from prefrontal cortex can disrupt short-term memory performance and is reminiscent of Alzheimer's disease, which is characterized by degeneration of acetylcholine-producing neurons. Stimulation of cholinergic receptors is being explored to create cognitive enhancers for the treatment of Alzheimer's disease and other psychiatric diseases. Here, we stimulated cholinergic receptors in prefrontal cortex and examined its effects on neurons that are engaged in cognitive behavior. Surprisingly, cholinergic stimulation decreased neurons' ability to discriminate between rules. This work suggests that overstimulation of acetylcholine receptors could disrupt neuronal processing during cognition and is relevant to the design of cognitive enhancers based on stimulating the cholinergic system.

Reversible differentiation of immortalized human bladder smooth muscle cells accompanied by actin bundle reorganization.

Previous studies have shown that phenotypic modulation of smooth muscle cells (SMCs) plays a pivotal role in human diseases. However, the molecular mechanisms underlying the reversible differentiation of SMCs remain elusive particularly because cultured SMCs that reproducibly exhibit bidirectional phenotypic modulation have not been established. Here we established an immortalized human bladder SMC line designated as hBS11. Under differentiation-inducing conditions, hBS11 cells underwent smooth muscle differentiation accompanied by the robust expression of smooth muscle differentiation markers and isoform-dependent reorganization of actin bundles. The cholinergic receptor agonist carbachol increased intracellular calcium in differentiated hBS11 cells in an acetylcholine muscarinic receptor-dependent manner. Differentiated hBS11 cells displayed contractile properties depending on the elevation in the levels of intracellular calcium. Depolarization of membrane potential triggered inward sodium current in differentiated hBS11 cells. However, differentiated hBS11 cells lost the differentiated phenotype and resumed mitosis when re-fed with growth medium. Our study provides direct evidence pertaining to the human bladder SMCs being able to retain the capacity of reversible differentiation and that the reorganization of actin bundles is involved in the reinstatement of contractility. Moreover, we have established a human SMC line retaining high proliferating potential without compromising differentiation potential.

Mechanisms underlying the reversal of acantholysis in pemphigus by a cholinergic receptor agonist

Objective To evaluate the reversal effect of a cholinergic receptor agonist on acantholysis in pemphigus, and to investigate its mechanism. Methods Human HaCaT keratinocytes were co-cultured with pemphigus vulgaris immunoglobulin G(PV-IgG) to establish a cell model of pemphigus, then classified into two groups to be incubated with the cholinergic receptor agonist carbachol for 12 hours (test group) or remain untreated (control group). Cell dissociation assay was performed to quantitatively estimate the reversal effect of carbachol on acantholysis, and immunofluorescence assay to qualitatively assess the changes of desmosomal proteins. Radio-immunoprecipitation assay (RIPA) lysis buffer and Triton X-100 were used to lyse HaCaT cells to obtain total proteins and cytoplasmic proteins, and Western blot was conducted to determine the expression levels of adhesion-related proteins desmoglein 3(Dsg3) and plakoglobin (PG) on the surface of HaCaT cells, as well as the phosphorylation levels of p38 mitogen activated protein kinase (p38 MAPK) and epidermal growth factor receptor (EGFR) at different time points. Quantitative polymerase chain reaction (qPCR) was performed to detect the mRNA expressions of the above surface proteins, and co-immunoprecipitation assay to qualitatively evaluate the interaction between Dsg3 and PG. Results The number of cell debris was significantly lower in the test group than in the control group (18.67±2.52 vs. 46.67±2.03,t=11.22, P<0.01). Immunofluorescence assay showed that carbachol could reverse the internalization of desmosomal molecules induced by PV-IgG. In the pemphigus cell model, the levels of total Dsg3 and PG as well as non-desmosomal Dsg3 were decreased, while the level of non-desmosomal PG increased, and the interaction between Dsg3 and PG was attenuated. When the pemphigus cell model was co-cultured with carbachol, these above changes were reversed. Carbachol also increased the mRNA levels (expressed as 2-ΔΔCt) of Dsg3 and PG from 1.428±0.215 and 1.563±0.247 in the control group to 4.974±0.948 (t=3.65,P=0.01) and 13.420±1.715 (t=6.85, P<0.01) in the test group respectively. In phosphorylation assay, carbachol inhibited the phosphorylation of EGFR, but had no significant effect on that of p38 MAPK. Conclusions The cholinergic receptor agonist carbachol can reverse acantholysis in pemphigus, likely by inhibiting the internalization of Dsg3 and PG, enhancing their expressions and interaction, and suppressing the phosphorylation of the key signaling molecule for acantholysis, EGFR. Key words: Pemphigus; Cholinergic agonists; Acantholysis; Desmoglein 3; gamma Catenin; HaCaT cells

Hippocampal long term memory: Effect of the cholinergic system on local protein synthesis

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1h, 4h or 24h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1h and 4h and of p70S6K activation at 4h, and long term memory impairment at 24h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1h and 4h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1h and 4h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.

Comparison of Properties of Medial Entorhinal Cortex Layer II Neurons in Two Anatomical Dimensions with and without Cholinergic Activation

Mechanisms underlying grid cell firing in the medial entorhinal cortex (MEC) still remain unknown. Computational modeling studies have suggested that cellular properties such as spike frequency adaptation and persistent firing might underlie the grid cell firing. Recent in vivo studies also suggest that cholinergic activation influences grid cell firing. Here we investigated the anatomical distribution of firing frequency adaptation, the medium spike after hyperpolarization potential (mAHP), subthreshold membrane potential oscillations, sag potential, input resistance and persistent firing, in MEC layer II principal cells using in vitro whole-cell patch clamp recordings in rats. Anatomical distributions of these properties were compared along both the dorso-ventral and medio-lateral axes, both with and without the cholinergic receptor agonist carbachol. We found that spike frequency adaptation is significantly stronger in ventral than in dorsal neurons both with and without carbachol. Spike frequency adaptation was significantly correlated with the duration of the mAHP, which also showed a gradient along the dorso-ventral axis. In carbachol, we found that about 50% of MEC layer II neurons show persistent firing which lasted more than 30 seconds. Persistent firing of MEC layer II neurons might contribute to grid cell firing by providing the excitatory drive. Dorso-ventral differences in spike frequency adaptation we report here are opposite from previous predictions by a computational model. We discuss an alternative mechanism as to how dorso-ventral differences in spike frequency adaptation could contribute to different scales of grid spacing.

Muscarinic receptors stimulate cell proliferation in the human urothelium-derived cell line UROtsa

The widespread non-neuronal synthesis of acetylcholine (ACh) has changed the paradigm of ACh acting solely as a neurotransmitter. Indeed, the presence of ACh in many types of proliferating cells suggests a role for this neurotransmitter in the control of cell division. The parasympathetic system is a major pathway regulating micturition, but ACh-mediated control plays a more complex role than previously described, acting not only in the detrusor muscle, but also influencing detrusor function through the activity of urothelial muscarinic receptors. Here we investigated the role of muscarinic receptors in mediating cell proliferation in the human UROtsa cell line, which is a widely used experimental model to study urothelium physiology and pathophysiology. Our results demonstrate that UROtsa cells express the machinery for ACh synthesis and that muscarinic receptors, with the rank order of M 3 > M 2 > M 5 > M 1 = M 4, are present and functionally linked to their known second messengers. Indeed, the cholinergic receptor agonist carbachol (CCh) (1–100 μM) concentration-dependently raised IP 3 levels, reaching 66 ± 5% over basal. The forskolin-mediated adenylyl cyclase activation was reduced by CCh exposure (forskolin: 1.4 ± 0.14 pmol/ml; forskolin + 100 μM CCh: 0.84 ± 0.12 pmol/ml). CCh (1–100 μM) concentration-dependently increased UROtsa cell proliferation and this effect was inhibited by the non-selective antagonist atropine and the M 3-selective antagonists darifenacin and J104129. Finally, CCh-induced cell proliferation was blocked by selective PI-3 kinase and ERK activation inhibitors, strongly suggesting that these intracellular pathways mediate, at least in part, the muscarinic receptor-mediated cell proliferation.

Cholinergic and glutamatergic agonists induce gamma frequency activity in dorsal subcoeruleus nucleus neurons

The dorsal subcoeruleus nucleus (SubCD) is involved in generating two signs of rapid eye movement (REM) sleep: muscle atonia and ponto-geniculo-occipital (PGO) waves. We tested the hypothesis that single cell and/or population responses of SubCD neurons are capable of generating gamma frequency activity in response to intracellular stimulation or receptor agonist activation. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. All SubCD neurons (n = 103) fired at gamma frequency when subjected to depolarizing steps. Two statistically distinct populations of neurons were observed, which were distinguished by their high (>80 Hz, n = 24) versus low (35-80 Hz, n = 16) initial firing frequencies. Both cell types exhibited subthreshold oscillations in the gamma range (n = 43), which may underlie the gamma band firing properties of these neurons. The subthreshold oscillations were blocked by the sodium channel blockers tetrodotoxin (TTX, n = 21) extracellularly and N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide (QX-314) intracellularly (n = 5), indicating they were sodium channel dependent. Gamma frequency subthreshold oscillations were observed in response to the nonspecific cholinergic receptor agonist carbachol (CAR, n = 11, d = 1.08) and the glutamate receptor agonists N-methyl-d-aspartic acid (NMDA, n = 12, d = 1.09) and kainic acid (KA, n = 13, d = 0.96), indicating that cholinergic and glutamatergic inputs may be involved in the activation of these subthreshold currents. Gamma band activity also was observed in population responses following application of CAR (n = 4, P < 0.05), NMDA (n = 4, P < 0.05) and KA (n = 4, P < 0.05). Voltage-sensitive, sodium channel-dependent gamma band activity appears to be a part of the intrinsic membrane properties of SubCD neurons.

Cholinergic control of GABA release by carbachol at perisomatic inhibitory synapses in the CA3 region of mouse hippocampus

Event Abstract Back to Event Cholinergic control of GABA release by carbachol at perisomatic inhibitory synapses in the CA3 region of mouse hippocampus G. G. Szabó1, N. Holderith1, A. I. Gulyás1, T. F. Freund1 and N. Hájos1* 1 Hungarian Academy of Sciences, Institute of Experimental Medicine, Hungary Action potential generation of hippocampal pyramidal neurons is controlled by perisomatic inhibition, originating from fast-spiking basket (FSBC)- and axo-axonic cells (AACs) as well as from regular spiking basket cells (RSBCs). These neurons play a role in feedforward and feedback inhibition and are tuned by cholinergic receptor activation. We aimed to clarify the impact of cholinergic receptor activation on synaptic communication between these perisomatic inhibitory neurons (PINs) and pyramidal cells (PCs). By performing paired recordings between the three types of PINs and PCs in mouse hippocampal slices, we investigated the release properties and their sensitivity to the cholinergic receptor agonist carbachol (CCh). Synaptic currents originating from the distinct types of PINs had different properties recorded in CA3 PCs. Analyzes of the decay kinetics suggested that at AAC-PC pairs synaptic cross-talk may occur between adjacent release sites. RSBCs showed robust asynchronous release, whereas the other two types of PINs did not. CCh decreased the GABA release significantly from all the three types of PINs to a different extent and via different receptor activation. In addition, CCh significantly reduced the short-term depression of synaptic transmission at FSBC- and AAC-PC pairs.These findings suggest that the contribution of different types of PINs to feedforward and feedback inhibition might be distinct that could be altered by cholinergic receptor activation. Keywords: Neurophysiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Neurophysiology Citation: Szabó GG, Holderith N, Gulyás AI, Freund TF and Hájos N (2011). Cholinergic control of GABA release by carbachol at perisomatic inhibitory synapses in the CA3 region of mouse hippocampus. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00038 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. N. Hájos, Hungarian Academy of Sciences, Institute of Experimental Medicine, Budapest, Hungary, hajos@koki.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos Google G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos Google Scholar G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos PubMed G. G Szabó N. Holderith A. I Gulyás T. F Freund N. Hájos Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Gamma Band Unit Activity and Population Responses in the Pedunculopontine Nucleus

The pedunculopontine nucleus (PPN) is involved in the activated states of waking and paradoxical sleep, forming part of the reticular activating system (RAS). The studies described tested the hypothesis that single unit and/or population responses of PPN neurons are capable of generating gamma band frequency activity. Whole cell patch clamp recordings (immersion chamber) and population responses (interface chamber) were conducted on 9- to 20-day-old rat brain stem slices. Regardless of cell type (I, II, or III) or type of response to the nonselective cholinergic receptor agonist carbachol (excitation, inhibition, biphasic), almost all PPN neurons fired at gamma band frequency, but no higher, when subjected to depolarizing steps (50 +/- 2 Hz, mean +/- SE). Nonaccommodating neurons fired at 18-100 Hz throughout depolarizing steps, while most accommodating neurons exhibited gamma band frequency of action potentials followed by gamma band membrane oscillations. These oscillations were blocked by the sodium channel blocker tetrodotoxin (TTX), suggesting that at least some are mediated by sodium currents. Population responses in the PPN showed that carbachol induced peaks of activation in the theta and gamma range, while glutamatergic receptor agonists induced overall increases in activity at theta and gamma frequencies, although in differing patterns. Gamma band activity appears to be a part of the intrinsic membrane properties of PPN neurons, and the population as a whole generates different patterns of gamma band activity under the influence of specific transmitters. Given sufficient excitation, the PPN may impart gamma band activation on its targets.

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.

Relationship between carbachol hyperstimulation-induced pancreatic intracelluar trypsinogen and NF-κB activation in rats in vitro

The relationship between intracellular trypsinogen activation and NF-kappa B activation in rat pancreatic acinar cells induced by M3 cholinergic receptor agonist (carbachol) hyperstimulation was studied. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, the active protease inhibitor (pefabloc) and NF-kappa B inhibitor (PDTC) in vitro. Intracellular trypsin activity was measured by using a fluorogenic substrate. The activity of NF-kappa B was monitored by using electrophoretic mobility shift assay. The results showed that after pretreatment with 2 mmol/L pefabloc, the activities of trypsin and NF-kappa B in pancreatic acinar cells treated with high concentrations of carbachol (10(-3) mol/L) in vitro was significantly decreased as compared with control group (P<0.01). The addition of 10(-2) mol/L PDTC resulted in a significant decrease of NF-kappa B activities in pancreatic acinar cells after treated with high concentrations of carbachol (10(-3) mol/L) in vitro, but the intracellular trypsinogen activity was not obviously inhibited (P>0.05). It was concluded that intracellular trypsinogen activation is likely involved in the regulation of high concentrations of carbachol-induced NF-kappa B activation in pancreatic acinar cells in vitro. NF-kappa B activation is likely not necessary for high concentrations of carbachol-induced trypsinogen activation in pancreatic acinar cells in vitro.

Electrotonic Coupling in the Nucleus SubCoeruleus. Focus on “Evidence for Electrical Coupling in the SubCoeruleus (SubC) Nucleus”

New findings by Heister et al. presented in this issue of the Journal of Neurophysiology (p. 3142–3147) suggest that neurons in the rat pontine subcoeruleus (SubC) nucleus are electrotonically coupled. Electrical neurotransmission in the CNS is frequently generated by members of the connexin (Cx)

Ontogeny of excitatory and inhibitory control of gastrointestinal motility in the African clawed frog,Xenopus laevis

The transparent body wall of Xenopus laevis larvae during the first developmental stages allows in vivo studies of gastrointestinal tract activity. The purpose of this study was to chart the ontogeny of gut motility in Xenopus larvae and to identify the most important control systems during the first developmental stages. Coordinated descending contraction waves first occurred in the gut at Nieuwkoop and Faber stage 43 [0.8 +/- 0.1 contractions/min (cpm)] and increased to 4.9 +/- 0.1 cpm at stage 47. The cholinergic receptor agonist carbachol (5-10 microM) increased contraction frequency already at stage 43, as did neurokinin A (NKA, 0.3-1 microM). The muscarinic antagonist atropine (100 microM) first affected contraction frequency at stage 45, which coincides with the onset of feeding. The tachykinin antagonist MEN-10,376 (6 microM) blocked NKA-induced contractions but not spontaneous motility. Both sodium nitroprusside [nitric oxide (NO) donor, 1-10 microM] and vasoactive intestinal peptide (VIP, 0.1-1 microM) inhibited contractions from the earliest stage onward. Blocking NO synthesis using NG-nitro-L-arginine methyl ester (100 microM) had no effect per se, but antagonized VIP evoked inhibition at stage 47. We conclude that gastrointestinal motility is well developed in the Xenopus laevis larvae before the onset of feeding. Functional muscarinic and tachykinin receptors are present already at the onset of motility, whereas a cholinergic tone develops around the onset of feeding. No endogenous tachykinin tone was found. Functional VIP receptors mediate inhibition at the onset of motility. NO seems to mediate the VIP effect at later stages.

Dopaminergic and GABAergic modulation of glutamate release from rat subthalamic nucleus efferents to the substantia nigra

The regulation of the glutamatergic projection from the subthalamic nucleus (STN) to the substantia nigra (SN) was investigated using dual-probe microdialysis in the awake behaving rat. Reverse dialysis of the cholinergic receptor agonist carbachol (1 mM) into the STN caused an increase in the extracellular concentrations of glutamate and dopamine in the SN. The increase in glutamate was transient and returned toward basal values despite the continued perfusions of the STN with carbachol. Carbachol-stimulated glutamate release was prolonged by perfusion of the selective D 2 dopamine receptor antagonist raclopride (100 μM) into the SN and was attenuated by the perfusion of the selective D 2-like receptor agonist quinpirole (10 μM). In contrast, perfusion of the D 1 dopamine receptor antagonist SCH-23390 (100 μM) did not alter the carbachol-stimulated glutamate release even though it increased basal glutamate concentrations. Perfusion of the GABA A receptor antagonist bicuculline (10 μM) into the SN prolonged the carbachol-stimulated glutamate release in similar fashion as raclopride. The present findings suggest that somatodendritically released dopamine in the SN regulates glutamate release from subthalamic axon terminals by differentially activating dopamine D 2 and D 1 receptors. Activation of D 2 heteroreceptors, located on STN axon terminals, provides a negative feedback control on stimulated subthalamic glutamate release, while D 1 receptor activation preferentially regulates basal glutamate concentrations. The findings of the present study also indicate that GABA exerts an inhibitory control on glutamate release in the SN through GABA A receptors.

Relationship between carbachol hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen or NF-κB activation in ratsin vitro

The relationship between M3 cholinergic receptor agonist (carbachol) hyperstimulation-induced pancreatic acinar cellular injury and trypsinogen activation or NF-kappaB 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-kappaB 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-kappaB activation may not be involved in pancreatic acinar cellular injury induced by carbachol hyperstimulation in vitro.

L- and T-Type Voltage-Gated Ca2+Channels in Human Granulosa Cells: Functional Characterization and Cholinergic Regulation

Using the whole-cell configuration of the patch-clamp technique, we have characterized two types of ionic currents through voltage-dependent Ca2+ channels in human granulosa cells. One is long-lasting, activates at approximately -20 mV, reaches the peak at approximately +20 mV, has an inactivation time constant of 132.5 +/- 5.6 msec at 20 mV, and is sensitive to dihydropyridines. The other is transient, activates at approximately -40 mV, peaks at approximately -10 mV, has an inactivation time constant of 38.8 +/- 1.8 msec at -10 mV, displays a voltage-dependent inactivation, and is sensitive to 100 microm Ni2+, but not to dihydropyridines. Biophysical and pharmacological properties of these currents indicate that they are gated through L- and T-type calcium channels, respectively. The cholinergic receptor agonist carbachol (50 microm) reduces the amplitude of the currents through both L-type (-34.7 +/- 6.4%; n = 10) and T-type (-52.6 +/- 7.4%; n = 8) channels, suggesting a possible role of these channels in the cholinergic regulation of human ovarian functions.

Retrograde signaling changes the venue of postsynaptic inhibition in rat substantia nigra

Both endocannabinoids through cannabinoid receptor type I (CB1) receptors and dopamine through dopamine receptor type D1 receptors modulate postsynaptic inhibition in substantia nigra by changing GABA release from striatonigral terminals. By recording from visually identified pars compacta and pars reticulata neurons we searched for a possible co-release and interaction of endocannabinoids and dopamine. Depolarization of a neuron in pars reticulata or in pars compacta transiently suppressed evoked synaptic currents which were blocked by GABA A receptor antagonists (inhibitory postsynaptic currents [IPSCs]). This depolarization-induced suppression of inhibition (DSI) was abrogated by the cannabinoid CB1 receptor antagonist AM251 (1 μM). A correlation existed between the degree of DSI and the degree of reduction of evoked IPSCs by the CB1 receptor agonist WIN55,212–2 (1 μM). The cholinergic receptor agonist carbachol (0.5–5 μM) enhanced DSI, but suppression of spontaneous IPSCs was barely detectable pointing to the existence of GABA release sites without CB1 receptors. In dopamine, but not in GABAergic neurons DSI was enhanced by the dopamine D1 receptor antagonist SCH23390 (3–10 μM). Both the antagonist for CB1 receptors and the antagonist for dopamine D1 receptors enhanced or reduced, respectively, the amplitudes of evoked IPSCs. This tonic influence persisted if the receptor for the other ligand was blocked. We conclude that endocannabinoids and dopamine can be co-released. Retrograde signaling through endocannabinoids and dopamine changes inhibition independently from each other. Activation of dopamine D1 receptors emphasizes extrinsic inhibition and activation of CB1 receptors promotes intrinsic inhibition.

Muscarinic receptors control K+ secretion in inner ear strial marginal cells.

K+ secretion in strial marginal cells (SMC) of stria vascularis (SV) is stimulated by beta1-adrenergic receptors. The aim of the present study was to determine, whether SMC from the gerbil inner ear contain muscarinic receptors that inhibit K+ secretion. Receptors were identified with pharmacological tools in functional studies where K+ secretion was monitored as transepithelial current (Isc). The cytosolic Ca2+ concentration ([Ca2+]i) was measured as fluo-4 fluorescence and cAMP production with a colorimetric immunoassay. Further, receptors were identified in SV as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. The cholinergic receptor agonist carbachol (CCh) caused a transient increase in [Ca2+]i with a half-maximal concentration value (EC50) of (5 +/- 6) x 10(-6) m (n = 29) and a decrease in basal and stimulated cAMP production. Apical CCh had no effect on Isc but basolateral CCh caused a transient increase in Isc with an EC50 of (3 +/- 1) x 10(-6) m and a sustained decrease of Isc with an EC50 of (1.2 +/- 0.2) x 10(-5) m (n = 129). The effects of CCh on Isc and [Ca2+]i were inhibited in the presence of muscarinic antagonist 10(-6) m atropine. Further, the muscarinic antagonists pirenzipine, methoctramine and para-fluoro-hexahydo-sila-defenidol (pFHHSiD) inhibited the CCh-induced transient increase of Isc with affinity constants (KDB) of 3 x 10(-8) m (pKDB = 7.54 +/- 0.19, n = 17), 2 x 10(-6) m (pKDB = 5.71 +/- 0.26, n = 19) and 2 x 10(-8) m (pKDB = 7.65 +/- 0.28, n = 19) and the sustained decrease of Isc with KDB of 7 x 10(-8) m (pKDB = 7.05 +/- 0.09, n = 33), 6 x 10(-6) m (pKDB = 5.21 +/- 0.13, n = 23), 5 x 10(-8) m (pKDB = 7.34 +/- 0.13, n = 31), respectively. RT-PCR of total RNA isolated from SV using primers specific for the M1-M5 muscarinic receptors revealed products of the predicted sizes for the M3- and M4- but not the M1-, M2- and M5-muscarinic receptor subtypes. Sequence analysis confirmed that amplified cDNA fragments encoded gene-specific nucleotide sequences. These results suggest that K+ secretion in SMC is under the control of M3- and M4-muscarinic receptors that may be located in the basolateral membrane of strial marginal cells.

Cholinergic, noradrenergic, and serotonergic inhibition of fast synaptic transmission in spinal lumbar dorsal horn of rat

It is known that spinal nociceptive sensory transmission receives descending inhibitory and facilitatory modulation from supraspinal structures. Glutamate is the major fast excitatory transmitter between primary afferent fibers and spinal dorsal horn neurons. In whole-cell patch clamp recordings from dorsal horn neurons in spinal slices, we investigated synaptic mechanisms for inhibitory modulation at the lumbar level of the spinal cord. Application of the cholinergic receptor agonist carbachol produced a dose-dependent inhibition of glutamate-mediated excitatory postsynaptic currents (EPSCs) (IC 50 13 μM). Postsynaptic injection of two different types of G-protein inhibitors, guanosine 5′-O-2-thiophosphate or guanosine 5′-O-3-thiotriphosphate, blocked the inhibition produced by carbachol. Clonidine, a selective α-adrenergic receptor agonist, also produced a dose-dependent inhibition of EPSCs (IC 50 7 μM) that was reduced by postsynaptic inhibition of G-proteins. The inhibitory effect of serotonin was likewise mediated by postsynaptic G-proteins. Our results suggest that activation of postsynaptic neurotransmitter receptors plays a critical role in inhibition of glutamate mediated sensory responses by acetylcholine, norepinephrine, and serotonin. Our results support the hypothesis that descending sensory modulation may be mediated by multiple neurotransmitter receptors in the spinal cord.