Muscarinic Agonist Pilocarpine Research Articles

Effects of the muscarinic agonist pilocarpine on vigilance states and locomotor activity in ring doves

Cholinergic systems play a significant role in regulating a variety of behavioral functions in mammals and birds. The aim of this work is to study the effects of the muscarinic agonist pilocarpine on behavioral states by visual inspection and electroencephalographic recording; also, locomotor activity was continuously recorded by infrared interruption system in ring doves. The current results in birds demonstrated that the muscarinic agonist pilocarpine (1 and 3mg/kg, i.p.) primarily induced theta activity in addition to promote passive waking, while diminished active waking, the EEG slow wave rhythm and REM sleep in ring doves. The locomotor activity recorded continuously in ring doves diminished after pilocarpine treatment, which was in good agreement with the observed reduction of active waking derived of the EEG study. Altogether, the current results are similar to the effects of pilocarpine previously reported in mammals. In conclusion, hippocampal theta rhythm in birds suggests that this rhythm is an ancestral property of hippocampal function and similar cholinergic mechanisms regulate vigilance states and theta generation in mammals and birds.

Pre-junctional muscarinic autoreceptors in bovine airways

We searched for pre-junctional inhibitory muscarinic receptors in isolated bovine trachealis strips and bronchial rings. Electric stimulation (ES)-induced tritiated acetylcholine ([(3)H]-ACh)-release and isometric contractions were determined in muscles incubated with the non-selective muscarinic agonist pilocarpine, the non-selective muscarinic antagonist atropine, the selective M(2)-receptor antagonists methoctramine and gallamine, or the selective M(4)-receptor antagonist PD102807. Electric field stimulation (EFS)-induced isometric contractile responses were assessed in trachealis strips and bronchial rings treated with 10(-9)-10(-5)M methoctramine, gallamine or PD102807. Pilocarpine (10(-6) and 10(-5)M) and atropine (10(-7)M) significantly decreased and increased ES-evoked [(3)H]-ACh-release, respectively. The enhancing effect of atropine on [(3)H]-ACh-release prevailed over the inhibitory effect of pilocarpine. M(2)- and M(4)-receptor antagonists did not increase EFS-induced contraction or ES-induced [(3)H]-ACh-release. However, 10(-7)M methoctramine, gallamine or PD102807 significantly attenuated the inhibitory effects of pilocarpine 10(-5)M on ES-induced [(3)H]-ACh-release. Muscarinic autoregulation is present in bovine airways but is not fully accounted for by M(2)- and M(4)-receptor subtypes.

Involvement of the dopaminergic receptors of the rat basolateral amygdala in anxiolytic-like effects of the cholinergic system

Cholinergic system stimulation in some parts of the brain may affect anxiety-related behaviors. This system has many interactions with dopaminergic neurotransmission in the brain. We have studied the effect of cholinergic system activation in the basolateral amygdala on anxiety-related behaviors in adult male wistar rats using the acetylcholinesterase inhibitor physostigmine. Furthermore, the possible involvement of dopamine D 1 and D 2 receptors of basolateral amygdala in physostigmine induced effects has been evaluated. The elevated plus-maze task was used to assess anxiety parameters and all drugs were delivered into basolateral amygdala via bilaterally implanted chronic cannulas. Physostigmine (20 μg/rat) increased the percentage of open arm time (%OAT) and open arm entries (%OAE), revealing an anxiolytic-like effect. However, muscarinic receptor antagonist scopolamine (8 μg/rat) decreased %OAT indicating anxiogenic-like effect. A sub-effective dose of scopolamine (2 μg/rat) plus physostigmine decreased %OAT and %OAE in comparison to saline plus physostigmine (20 μg/rat). Muscarinic receptor agonist pilocarpine (5 μg/rat), dopamine D 1 receptor antagonist SCH23390 (1 μg/rat) and dopamine D 2 receptor antagonist sulpiride (5 μg/rat) significantly increased %OAT which may show anxiolytic-like effects of drugs. Sulpiride (5 μg/rat) also increased %OAE parameter. Pre-treatment with SCH23390 (0.5 and 1 μg/rat) or sulpiride (5 μg/rat) blocked anxiolytic-like effect of physostigmine (20 μg/rat). All drugs were devoid of any significant effect on locomotor activity. It is concluded that intra-basolateral amygdala administration of physostigmine has anxiolytic-like effects which may be via muscarinic mechanisms. Furthermore, dopaminergic system activation probably via dopamine D 1 and D 2 receptors is necessary for mediating anxiolytic-like effects of physostigmine.

CD38/ADP-ribosyl cyclase in the rat sublingual gland: Subcellular localization under resting and saliva-secreting conditions

CD38 is a 42–45kDa transmembrane glycoprotein that exhibits ADP-ribosyl cyclase enzyme activity. In the rat, we have previously reported strong ADP-ribosyl cyclase activity in the sublingual salivary gland (Masuda W. and Noguchi T. Biochem. Biophys. Res. Commun. (2000) 270, 469–472). Here, we have examined the specific localization of CD38/ADP-ribosyl cyclase activity in this gland and whether that localization changes upon saliva-secretary stimulation. Under resting conditions, CD38/ADP-ribosyl cyclase activity in the post-nuclear fraction of SLG homogenates was separated into two major peaks by sucrose density gradient centrifugation. The first peak included the plasma membrane proteins Na+/K+ ATPase and aquaporin 5, while the second peak included mucous secretory protein mucin and vesicle-associated membrane protein 2. When rats were subjected to the muscarinic agonist pilocarpine, the CD38/ADP-ribosyl cyclase activity disappeared from the second peak, as did mucin and vesicle-associated membrane protein 2. Pre-treatment of rats with the muscarinic antagonist atropine before pilocarpine administration, or adrenergic stimulation with isoproterenol, the sucrose density gradient separation profiles were same as that seen under resting condition. Using an immunofluorescent strategy, we observed the preferential localization of CD38 in the basolateral plasma membrane and intracellular granule-like membrane in sublingual acinar cells under resting conditions.

Noninvasive assessment of vascular structure and function in conscious rats based on in vivo imaging of the albino iris

Experimental techniques allowing longitudinal studies of vascular disease progression or treatment effects are not readily available for most animal models. Thus, most existing studies are destined to either study individual time points or use large cohorts of animals. Here we describe a noninvasive technique for studying vascular disease that is based on in vivo imaging of the long posterior ciliary artery (LPCA) in the iris of albino rats. Using a slit-lamp biomicroscope, images of the LPCA were taken weekly in conscious normotensive Wistar Kyoto rats (WKY, n = 10) and spontaneously hypertensive rats (SHR, n = 10) for 10 wk. Using imaging software, we found that lumen diameter was significantly smaller and the wall-to-lumen (W/L) ratio larger in SHR than in WKY. Wall thickness was not different. Blood pressure correlated with the W/L ratio. Histology of the abdominal aorta also revealed a smaller lumen diameter and greater W/L ratio in SHR compared with WKY. Corneal application of the muscarinic receptor agonist pilocarpine elicited a dose-dependent vasodilation of the LPCA that could be antagonized by inhibition of nitric oxide synthase, suggesting that the pilocarpine response is mainly mediated by endothelium-derived nitric oxide. Consistent with endothelial dysfunction in SHR, pilocarpine-induced vasodilation was greater in WKY rats than in SHR. These findings indicate that in vivo imaging of the LPCA allows assessment of several structural and functional vascular parameters in conscious rats and that the LPCA responds to disease insults and pharmacologic treatments in a fashion that will make it a useful model for further studies.

Parotid gland function after radiotherapy

Radiotherapy is a common treatment for head and neck cancer patients. Unfortunately, it produces serious acute and long-term side effects to the oral cavity. One severe complication is the loss of salivary gland function, which can persists for many years. Saliva has multiple functions relating to speech, taste perception, mastication, and swallowing and bolus formation. Cleansing and dental and mucosal protection are also important functions. In this thesis, a detailed report is given on the effects of radiotherapy on changes in the parotid gland function. Different methods of measuring gland function are described. Moreover, a preventive measure to limit the gland toxicity was investigated in rodents. The radiation field and, in particular, the volume of the parotid gland tissue exposed to radiation is of notable importance with regard to the development of gland damage and hypofunction. Therefore, it is important to achieve detailed information about the size and position of the parotid gland. Great differences in size and position of the parotid glands between the patients were seen. In order to exactly define the localisation of the glands, it is essential to use CT-based simulation of radiation fields. The radiation tolerance of the parotid glands as a function of dose and volume irradiated was accurately described. The best parameter for evaluation of the parotid gland function appeared to be the flow measurement using the Lashley cups. However, if direct flow measurements are not feasible, 99mTc-pertechnetate scintigraphy might be a good alternative. Flow reduction depended on the mean parotid gland dose. For a post treatment parotid flow ratio a 25%, the TD50 (the dose to the whole organ leading to a complication probability of 50%) was found to be 31, 35 and 39 Gy at 6 weeks, 6 months and 1 year after radiotherapy respectively. The consequences of parotid gland injury are still difficult to manage. Prophylactic treatment with sialogogues like the muscarinic receptor agonist pilocarpine has been shown to have radioprotective potential. The effects of preirradiation treatment of pilocarpine on rat parotid gland function were investigated in relation to radiation dose. Pilocarpine induced compensation, which at least underlies the radioprotective effect of the drug. This effect seemed to be dependent on the amount of damage induced. Therefore, the type of fractionation scheme and the volume of the gland that lies within the radiation portal will be crucial for the effectiveness of a prophylactic pilocarpine treatment. With the more detailed knowledge on the dose/volume effects of radiation on parotid function, we can try to focus on sparing the parotid gland function. The prevention of radiation-induced loss of parotid gland function will depend on both optimal sparing radiation therapy techniques and on pharmacological agents, which can selectively interfere with the radiation-induced effects. These preventing strategies will need a joint effort of radiation-oncologists, clinical physicists and radiobiologists.

Oral tremor induced by the muscarinic agonist pilocarpine is suppressed by the adenosine A 2A antagonists MSX-3 and SCH58261, but not the adenosine A 1 antagonist DPCPX

Tremulous jaw movements in rats, which can be induced by dopamine (DA) antagonists, DA depletion, and cholinomimetics, have served as a useful model for studies of tremor. Although adenosine A 2A antagonists can reduce the tremulous jaw movements induced by DA antagonists and DA depletion, there are conflicting reports about the interaction between adenosine antagonists and cholinomimetic drugs. The present studies investigated the ability of adenosine antagonists to reverse the tremorogenic effect of the muscarinic agonist pilocarpine. While the adenosine A 2A antagonist MSX-3 was incapable of reversing the tremulous jaw movements induced by the 4.0 mg/kg dose of pilocarpine, both MSX-3 and the adenosine A 2A antagonist SCH58261 reversed the tremulous jaw movements elicited by 0.5 mg/kg pilocarpine. Systemic administration of the adenosine A 1 antagonist DPCPX failed to reverse the tremulous jaw movements induced by either an acute 0.5 mg/kg dose of the cholinomimetic pilocarpine or the DA D2 antagonist pimozide, indicating that the tremorolytic effects of adenosine antagonists may be receptor subtype specific. Behaviorally active doses of MSX-3 and SCH 58261 showed substantial in vivo occupancy of A 2A receptors, but DPCPX did not. The results of these studies support the use of adenosine A 2A antagonists for the treatment of tremor.

Electroencephalogram functional connectivity between rat hippocampus and cortex after pilocarpine treatment

The muscarinic agonist pilocarpine has been shown to increase the duration and total number of episodes presenting theta rhythm—simultaneously in hippocampus and cortex—in rats during the waking states. Theta waves are suggested to be involved in the flow of information between hippocampus and cortex during memory processes. This work investigates this functional interdependence using the spectral and phase synchronization analysis of the electroencephalogram (EEG) theta band recorded in these brain structures of rats after pilocarpine treatment. Pilocarpine was used at doses devoid of epilepticus-like seizures effects in conscious freely moving rats. The results showed that pilocarpine administration significantly increased the relative theta power during the waking states in the cortex, but not in the hippocampus of rats. Additionally, the EEG coherence between the hippocampal EEG theta band and that arising at the frontal cortex increased after pilocarpine treatment but only during the waking states. This result reveals an increase of the linear correlation between the theta waves of these two brain structures after pilocarpine treatment during the waking states. Moreover, phase synchronization results showed an effective phase locking with non-zero phase difference between hippocampus and frontal cortex theta waves that remained after pilocarpine treatment. Therefore, pilocarpine seems to reinforce the neural transmission waves from the hippocampus toward the cortex during waking. In conclusion, the present EEG study could suggest an effect of the muscarinic cholinergic agonist pilocarpine on the hippocampal-cortical functional connectivity.

Pilocarpine protects cobalt chloride-induced apoptosis of RGC-5 cells: involvement of muscarinic receptors and HIF-1 alpha pathway.

The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl(2))-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1 alpha (HIF-1 alpha), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 microM CoCl(2) for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 microM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 microM pilocarpine could significantly prevent CoCl(2)-induced HIF-1 alpha translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1 alpha, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1 alpha pathway. The findings suggest that HIF-1 alpha pathway as a "master switch" may be used as a therapeutic target in the cholinergic treatment of glaucoma.

The pilocarpine model of epilepsy: what have we learned?

The systemic administration of a potent muscarinic agonist pilocarpine in rats promotes sequential behavioral and electrographic changes that can be divided into 3 distinct periods: (a) an acute period that built up progressively into a limbic status epilepticus and that lasts 24 h, (b) a silent period with a progressive normalization of EEG and behavior which varies from 4 to 44 days, and (c) a chronic period with spontaneous recurrent seizures (SRSs). The main features of the SRSs observed during the long-term period resemble those of human complex partial seizures and recurs 2-3 times per week per animal. Therefore, the pilocarpine model of epilepsy is a valuable tool not only to study the pathogenesis of temporal lobe epilepsy in human condition, but also to evaluate potential antiepileptogenic drugs. This review concentrates on data from pilocarpine model of epilepsy.

Retinoic acid prevents virus-induced airway hyperreactivity and M2 receptor dysfunction via anti-inflammatory and antiviral effects

Inhibitory M(2) muscarinic receptors on airway parasympathetic nerves normally limit acetylcholine release. Viral infections decrease M(2) receptor function, increasing vagally mediated bronchoconstriction. Since retinoic acid deficiency causes M(2) receptor dysfunction, we tested whether retinoic acid would prevent virus-induced airway hyperreactivity and prevent M(2) receptor dysfunction. Guinea pigs infected with parainfluenza virus were hyperreactive to electrical stimulation of the vagus nerves, but not to intravenous acetylcholine, indicating that hyperreactivity was due to increased release of acetylcholine from parasympathetic nerves. The muscarinic agonist pilocarpine, which inhibits vagally mediated bronchoconstriction in control animals, no longer inhibited vagally induced bronchoconstriction, demonstrating M(2) receptor dysfunction. Treatment with all-trans retinoic acid (1 mg/kg) prevented virus-induced hyperreactivity and M(2) receptor dysfunction. However, retinoic acid also significantly reduced viral titers in the lungs and attenuated virus-induced lung inflammation. In vitro, retinoic acid decreased M(2) receptor mRNA expression in both human neuroblastoma cells and primary cultures of airway parasympathetic neurons. Thus, the protective effects of retinoic acid on airway function during viral infection appear to be due to anti-inflammatory and antiviral mechanisms, rather than to direct effects on M(2) receptor gene expression.

Muscarinic activation attenuates abnormal processing of β-amyloid precursor protein induced by cobalt chloride-mimetic hypoxia in retinal ganglion cells

β-Amyloid peptide (Aβ), the major pathological factor in Alzheimer’s disease, has recently been reported to be implicated in the development of glaucoma. In this study, we explored the effect of muscarinic activation on abnormal processing of β-amyloid precursor protein (APP) induced by a risk factor hypoxia in retinal ganglion cells. Hypoxia mimetic compound cobalt chloride could increase the generation of Aβ via up-regulating the expression of APP as well as the expression of β-secretase and γ-secretase, whereas muscarinic receptor agonist pilocarpine could significantly attenuate this abnormal pathway, thereby resulting in a decreased amyloidogenic cleavage of APP. This finding may provide an insight into better understanding of pathophysiology for the retinal neurodegenerative disease and searching for its new modifying approach.

Antennal motor activity induced by pilocarpine in the American cockroach

The antennal motor system is activated by the muscarinic agonist pilocarpine in the American cockroach Periplaneta americana, and its output patterns were examined both in restrained intact animals and in isolated CNS preparations. The three-dimensional antennal movements induced by the hemocoelic drug injection were analyzed in in vivo preparations. Pilocarpine effectively induced prolonged rhythmic movements of both antennae. The antennae tended to describe a spatially patterned trajectory, forming loops or the symbol of infinity (infinity). Such spatial regularity is comparable to that during spontaneous tethered-walking. Rhythmic bursting activities of the antennal motor nerves in in vitro preparations were also elicited by bath application of pilocarpine. Cross-correlation analyses of the bursting spike activities revealed significant couplings among certain motor units, implying the spatial regularity of the antennal trajectory. The pilocarpine-induced rhythmic activity of antennal motor nerves was effectively suppressed by the muscarinic antagonist atropine. These results indicate that the activation of the antennal motor system is mediated by muscarinic receptors.

Long‐term changes in dopamine‐stimulated gene expression after single‐day methamphetamine exposure

Methamphetamine (mAMPH) is a highly addictive psychostimulant drug that injures monoaminergic neurons and results in behavioral impairments in humans and animals. Although evidence exists for changes in cortical volume, metabolism, and blood oxygenation levels in human mAMPH abusers, animal models have instead emphasized this drug's long-lasting influence on ascending monoaminergic (dopamine, serotonin) projections. The aim of this study was to investigate cortical and subcortical function in rats long after administration of a single-day mAMPH regimen known to damage monoaminergic systems, at a time point when behavioral impairments are still evident. Rats were given either saline or a neurotoxic (4 x 4 mg/kg, sc) mAMPH regimen. Five weeks later, they were given pharmacological treatments that stimulate cortical gene expression: either the dopaminergic agonist apomorphine (3 mg/kg, sc) or the muscarinic acetylcholine agonist pilocarpine (25 mg/kg, ip). Cortical and subcortical immediate early gene (IEG) responses were measured by immunocytochemical analysis of Fos or JunB, protein products of the IEGs, c-fos and junB. Compared with saline-pretreated controls, mAMPH-pretreated animals had about 50-70% fewer Fos- and JunB-immunoreactive cells in anterior cingulate, infralimbic, orbital, somatosensory, and rhinal cortices as well as caudate-putamen and nucleus accumbens, 90 min after apomorphine challenge. By contrast, mAMPH-pretreated rats had no reductions in the numbers of Fos or JunB-positive cells following pilocarpine challenge. This study demonstrates the profound and enduring effects of mAMPH administration on dopamine-stimulated cortical function in animals.

Loss of AKAP150 perturbs distinct neuronal processes in mice

A-Kinase Anchoring Proteins (AKAPs) ensure the fidelity of second messenger signaling events by directing protein kinases and phosphatases toward their preferred substrates. AKAP150 brings protein kinase A (PKA), the calcium/calmodulin dependent phosphatase PP2B and protein kinase C (PKC) to postsynaptic membranes where they facilitate the phosphorylation dependent modulation of certain ion channels. Immunofluorescence and electrophysiological recordings were combined with behavioral analyses to assess whether removal of AKAP150 by gene targeting in mice changes the signaling environment to affect excitatory and inhibitory neuronal processes. Mislocalization of PKA in AKAP150 null hippocampal neurons alters the bidirectional modulation of postsynaptic AMPA receptors with concomitant changes in synaptic transmission and memory retention. AKAP150 null mice also exhibit deficits in motor coordination and strength that are consistent with a role for the anchoring protein in the cerebellum. Loss of AKAP150 in sympathetic cervical ganglion (SCG) neurons reduces muscarinic suppression of inhibitory M currents and provides these animals with a measure of resistance to seizures induced by the non-selective muscarinic agonist pilocarpine. These studies argue that distinct AKAP150-enzyme complexes regulate context-dependent neuronal signaling events in vivo.

A muscarinic cholinergic mechanism underlies activation of the central pattern generator for locust flight

A central question in behavioural control is how central pattern generators (CPGs) for locomotion are activated. This paper disputes the key role generally accredited to octopamine in activating the CPG for insect flight. In deafferented locusts, fictive flight was initiated by bath application of the muscarinic agonist pilocarpine, the acetylcholine analogue carbachol, and the acetylcholinesterase blocker eserine, but not by nicotine. Furthermore, in addition to octopamine, various other amines including dopamine, tyramine and histamine all induced fictive flight, but not serotonin or the amine-precursor amino acid tyrosine. However, flight initiation was not reversibly blocked by aminergic antagonists, and was still readily elicited by both natural stimulation (wind) and pilocarpine in reserpinized, amine-depleted locusts. By contrast, the muscarinic antagonists atropine and scopolamine reversibly blocked flight initiated by wind, cholinergic agonists, octopamine, and by selective stimulation of a flight-initiating interneurone (TCG). The short delay from TCG stimulation to flight onset suggests that TCG acts directly on the flight CPG, and accordingly that TCG, or its follower cell within the flight generating circuit, is cholinergic. We conclude that acetylcholine acting via muscarinic receptors is the key neurotransmitter in the mechanism underlying the natural activation of the locust flight CPG. Amines are not essential for this, but must be considered as potential neuromodulators for facilitating flight release and tuning the motor pattern. We speculate that muscarinic activation coupled to aminergic facilitation may be a general feature of behavioural control in insects for ensuring conditional recruitment of individual motor programs in accordance with momentary adaptive requirements.

Sleep-deprivation enhances in adult rats the antagonistic effects of pilocarpine on cortical spreading depression: A dose–response study

Conditions that facilitate epilepsy, such as sleep deprivation or the cholinergic muscarinic agonist pilocarpine (PILO), have been experimentally studied, as they are relevant for brain excitability control and could help in understanding the development of epilepsy in humans. In this work, following to 72h deprivation (D) of sleep by the water tank technique, adult Wistar rats were anesthetized with a 1g/kg urethane+40mg/kg chloralose mixture and cortical spreading depression (CSD) was recorded for a 1-2h period in the parietal surface. CSD propagation velocity was calculated based on the time spent for a CSD-wave to pass the interelectrode distance. After this "baseline" recording, D-animals were divided in three groups and respectively injected with 45, 95 and 190mg/kg of PILO, i.p., and the CSD recording continued for two more hours. Three groups of non-deprived (ND) rats were also injected with the three doses of PILO and studied regarding CSD features. D-animals presented higher baseline CSD velocities than the corresponding ND-controls, confirming previous observations that D-condition facilitates CSD propagation. After PILO, the ECoG amplitudes were markedly and lastingly reduced in all animals, at the three doses used. In the groups treated with 95 and 190mg/kg PILO, the CSD velocity of propagation significantly (P<0.05) decreased, and the incidence of PILO-associated CSD-propagation failure increased in the D rats, but not in the ND rats, as compared with the pre-drug values. The data suggest the existence of one or more cholinergic, muscarinic-mediated antagonistic processes influencing CSD, which are dose-dependent and are modulated by sleep-deprivation.

Distinct pathways of ERK activation by the muscarinic agonists pilocarpine and carbachol in a human salivary cell line

Cholinergic-muscarinic receptor agonists are used to alleviate mouth dryness, although the cellular signals mediating the actions of these agents on salivary glands have not been identified. We examined the activation of ERK1/2 by two muscarinic agonists, pilocarpine and carbachol, in a human salivary cell line (HSY). Immunoblot analysis revealed that both agonists induced transient activation of ERK1/2. Whereas pilocarpine induced phosphorylation of the epidermal growth factor (EGF) receptor, carbachol did not. Moreover, ERK activation by pilocarpine, but not carbachol, was abolished by the EGF receptor inhibitor AG-1478. Downregulation of PKC by prolonged treatment of cells with the phorbol ester PMA diminished carbachol-induced ERK phosphorylation but had no effect on pilocarpine responsiveness. Depletion of intracellular Ca2+ ([Ca2+]i by EGTA did not affect ERK activation by either agent. In contrast to carbachol, pilocarpine did not elicit [Ca2+]i mobilization in HSY cells. Treatment of cells with the muscarinic receptor subtype 3 (M3) antagonist N-(3-chloropropyl)-4-piperidnyl diphenylacetate decreased ERK responsiveness to both agents, whereas the subtype 1 (M1) antagonist pirenzepine reduced only the carbachol response. Stimulation of ERKs by pilocarpine was also decreased by M3, but not M1, receptor small interfering RNA. The Src inhibitor PP2 blocked pilocarpine-induced ERK activation and EGF receptor phosphorylation, without affecting ERK activation by carbachol. Our results demonstrate that the actions of pilocarpine and carbachol in salivary cells are mediated through two distinct signaling mechanisms-pilocarpine acting via M3 receptors and Src-dependent transactivation of EGF receptors, and carbachol via M1/M3 receptors and PKC-converging on the ERK pathway.

Interactions of suboesophageal ganglion and frontal ganglion motor patterns in the locust

Although locust feeding has been well studied, our understanding of the neural basis of feeding-related motor patterns is still far from complete. This paper focuses on interactions between the pattern of rhythmic movements of the mouth appendages, governed by the suboesophageal ganglion (SOG), and the foregut movements, controlled by the frontal ganglion (FG), in the desert locust. In vitro simultaneous extracellular nerve recordings were made from totally isolated ganglia as well as from fully interconnected SOG–FG and brain–SOG–FG preparations. SOG-confined bath application of the nitric oxide donor, SNP, or the phosphodiesterase antagonist, IBMX, each followed by the muscarinic agonist pilocarpine, consistently induced robust fictive motor patterns in the SOG. This was observed in both isolated and interconnected preparations. In the brain-SOG–FG configuration the SOG-confined modulator application had an indirect excitatory effect on spontaneous FG rhythmic activity. Correlation between fictive motor patterns of the two ganglia was demonstrated by simultaneous changes in burst frequency. These interactions were found to be brain-mediated. Our results indicate the presence of intricate neuromodulation-mediated circuit interactions, even in the absence of sensory inputs. These interactions may be instrumental in generating the complex rhythmic motor patterns of the mandibles and gut muscles during locust feeding or ecdysis-related air swallowing.

Differential Coupling of M1 Muscarinic and α7 Nicotinic Receptors to Inhibition of Pemphigus Acantholysis

The mechanisms mediating and regulating assembly and disassembly of intercellular junctions is a subject of intensive research. The IgG autoantibodies produced in patients with the immunoblistering skin disease pemphigus vulgaris (PV) can induce keratinocyte (KC) dyshesion (acantholysis) via mechanisms that involve signaling kinases targeting intercellular adhesion molecules, thus providing a useful model to study the physiologic regulation of KC cohesion. Previous studies showed that activation of Src and protein kinase C are the earliest events in the PV IgG-induced intracellular phosphorylation cascades and that cholinergic agonists are effective for treating patients with pemphigus. In this study, we sought to elucidate the molecular mechanisms allowing cholinergic agonists to inhibit PV IgG-induced acantholysis and phosphorylation of KC adhesion molecules. The extent of acantholysis in KC monolayers correlated closely with the degree of PV IgG-induced phosphorylation of p120- and beta-catenins, with classic isoforms of protein kinase C mediating serine phosphorylation of beta-catenin and Src-tyrosine phosphorylation of p120-catenin. The M(1) muscarinic agonist pilocarpine blocked phosphorylation of both catenins, which could be abolised by the M(1) antagonist MT7. The alpha7 nicotinic agonist AR-R17779 inhibited phosphorylation of P120-cateinin. The alpha7 antagonist methyllycaconitine abolished the effect of AR-R17779. Okadaic acid abrogated protective effects of agonists on phosphorylation of beta-catenin, and pervanadate, on that of p120-catenin. Stimulation of KCs with pilocarpine significantly (p < 0.05) elevated both serine/threonine and tyrosine phosphatase activities in KCs. AR-R17779 both stimulated tyrosine phosphatase and decreased PV IgG-induced Src activity. Methyllycaconitine released Src activity in intact KCs and caused acantholysis. Thus, downstream signaling from M(1) abolished PV IgG-dependent catenin phosphorylation due to activation of both serine/threonine and tyrosine phosphatases, whereas alpha7 action involved both activation of tyrosine phosphatase and inhibition of Src. These findings identified novel paradigm of regulation of signaling kinases associated with cholinergic receptors and provided mechanistic explanation of therapeutic activity of cholinomimetics in PV patients.