Activation Of M1 Muscarinic Receptors Research Articles

An in vivo biosensor for neurotransmitter release and in situ receptor activity

Tools from molecular biology, in combination with in vivo optical imaging techniques, provide new mechanisms to noninvasively observe brain processing. Current approaches primarily probe cell-based variables, such as cytosolic calcium or membrane potential, but not cell-to-cell signaling. Here we introduce CNiFERs, cell-based neurotransmitter fluorescent engineered reporters, to address this challenge and monitor in situ neurotransmitter receptor activation. CNiFERs are cultured cells that are engineered to express a chosen metabotropic receptor, make use of the Gq protein-coupled receptor cascade to transform receptor activity into a rise in cytosolic [Ca2+], and report [Ca2+] with a genetically encoded fluorescent Ca2+ sensor. The initial realization of CNiFERs detects acetylcholine release via activation of M1 muscarinic receptors. Chronic implantation of M1-CNiFERs in frontal cortex of the adult rat is used to elucidate the muscarinic action of the atypical neuroleptics clozapine and olanzapine. We show that these drugs potently inhibit in situ muscarinic receptor activity.

G Protein Subunit Dissociation and Translocation Regulate Cellular Response to Receptor Stimulation

We examined the role of G proteins in modulating the response of living cells to receptor activation. The response of an effector, phospholipase C-β to M3 muscarinic receptor activation was measured using sensors that detect the generation of inositol triphosphate or diacylglycerol. The recently discovered translocation of Gβγ from plasma membrane to endomembranes on receptor activation attenuated this response. A FRET based G protein sensor suggested that in contrast to translocating Gβγ, non-translocating Gβγ subunits do not dissociate from the αq subunit on receptor activation leading to prolonged retention of the heterotrimer state and an accentuated response. M3 receptors with tethered αq induced differential responses to receptor activation in cells with or without an endogenous translocation capable γ subunit. G protein heterotrimer dissociation and βγ translocation are thus unanticipated modulators of the intensity of a cell's response to an extracellular signal.

Activation of M2 muscarinic receptors leads to sustained suppression of hippocampal transmission in the medial prefrontal cortex

Cholinergic innervation of the prefrontal cortex is critically involved in arousal, learning and memory. Dysfunction of muscarinic acetylcholine receptors and their downstream signalling pathways has been identified in mental retardation. To assess the role played by the muscarinic receptors at the hippocampal-frontal cortex synapses, an important relay in information storage, we used a newly developed frontal slice preparation in which hippocampal afferent fibres are preserved. Transient activation of muscarinic receptors by carbachol results in a long-lasting depression of synaptic efficacy at the hippocampal but not cortical pathways or local circuitry. On the basis of a combination of electrophysiological, pharmacological and anatomical results, this input-specific muscarinic modulation can be partially attributed to the M2 subtype of muscarinic receptors, possibly through a combination of pre- and postsynaptic mechanisms.

Immunoglobulins from scleroderma patients inhibit the muscarinic receptor activation in internal anal sphincter smooth muscle cells

Systemic sclerosis (SSc) IgGs affecting the M(3)-muscarinic receptor (M(3)-R) have been proposed to be responsible for the gastrointestinal (GI) dysmotility in this disease. However, the effect of SSc IgGs on smooth muscle cell (SMC) function has not been studied. We determined the effect of SSc IgGs on the muscarinic receptor activation by bethanechol (BeCh; methyl derivate of carbachol) in SMC and smooth muscle strips from rat internal anal sphincter. IgGs were purified from GI-symptomatic SSc patients and normal volunteers, with protein G-Sepharose columns. SMC lengths were determined via computerized digital micrometry. The presence of M(3)-R and IgG-M(3)-R complex was determined by Western blot. IgGs from SSc patients but not from normal volunteers caused significant and concentration-dependent inhibition of BeCh response (P < 0.05). The maximal shortening of 22.2 +/- 1.2% caused by 10(-4) M BeCh was significantly attenuated to 8.3 +/- 1.2% by 1 mg/ml of SSc IgGs (P < 0.05). Experiments performed in smooth muscle strips revealed a similar effect of SSc IgG that was fully reversible. In contrast to the effect on BeCh, the SSc IgGs caused no significant effect (P > 0.05) on K(+) depolarization and alpha(1)-adrenoceptor activation by phenylephrine. Western blot studies revealed the specific presence of SSc IgG-M(3)-R complex. SSc IgGs attenuated M(3)-R activation, which was reversible with antibody removal. These data suggest that SSc GI dysmotility may be caused by autoantibodies that inhibit the muscarinic neurotransmission. Future treatment of SSc patients may be directed at the removal or neutralization of these antibodies.

Cholinergic modulation of GABAergic and glutamatergic transmission in the dorsal subcoeruleus: mechanisms for REM sleep control.

Dorsal subcoeruleus (SubCD) neurons are thought to promote PGO waves and to be modulated by cholinergic afferents during REM sleep. We examined the differential effect of the cholinergic agonist carbachol (CAR) on excitatory and inhibitory postsynaptic currents (PSCs), and investigated the effects of CAR on SubCD neurons during the developmental decrease in REM sleep. Whole-cell patch clamp recordings were conducted on brainstem slices of 7- to 20-day-old rats. CAR acted directly on 50% of SubCD neurons by inducing an inward current, via both nicotinic and muscarinic M1 receptors. CAR induced a potassium mediated outward current via activation of M2 muscarinic receptors in 43% of SubCD cells. Evoked stimulation established the presence of NMDA, AMPA, GABA, and glycinergic PSCs in the SubCD. CAR was found to decrease the amplitude of evoked EPSCs in 31 of 34 SubCD cells, but decreased the amplitude of evoked IPSCs in only 1 of 13 SubCD cells tested. Spontaneous EPSCs were decreased by CAR in 55% of cells recorded, while spontaneous IPSCs were increased in 27% of SubCD cells. These findings indicate that CAR exerts a predominantly inhibitory role on fast synaptic glutamatergic activity and a predominantly excitatory role on fast synaptic GABAergic/glycinergic activity in the SubCD. We hypothesize that during REM sleep, cholinergic "REM-on" neurons that project to the SubCD induce an excitation of inhibitory interneurons and inhibition of excitatory events leading to the production of coordinated activity in SubCD projection neurons. The coordination of these projection neurons may be essential for the production of REM sleep signs such as PGO waves.

Modulation of GABAergic Transmission by Muscarinic Receptors in the Entorhinal Cortex of Juvenile Rats

Whereas the entorhinal cortex (EC) receives profuse cholinergic innervations from the basal forebrain and activation of cholinergic receptors has been shown to modulate the activities of the principal neurons and promote the intrinsic oscillations in the EC, the effects of cholinergic receptor activation on GABAergic transmission in this brain region have not been determined. We examined the effects of muscarinic receptor activation on GABA(A) receptor-mediated synaptic transmission in the superficial layers of the EC. Application of muscarine dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III via activation of M(3) muscarinic receptors. Muscarine slightly reduced the frequency but had no effects on the amplitude of miniature IPSCs recorded in the presence of tetrodotoxin. Muscarine reduced the amplitude of IPSCs evoked by extracellular field stimulation and by depolarization of GABAergic interneurons in synaptically connected interneuron and pyramidal neuron pairs. Application of muscarine generated membrane depolarization and increased action potential firing frequency but reduced the amplitude of action potentials in GABAergic interneurons. Muscarine-induced depolarization of GABAergic interneurons was mediated by inhibition of background K(+) channels and independent of phospholipase C, intracellular Ca(2+) release, and protein kinase C. Our results demonstrate that activation of muscarinic receptors exerts diverse effects on GABAergic transmission in the EC.

Cannabinoid CB1 receptor elevation of intracellular calcium in neuroblastoma SH-SY5Y cells: Interactions with muscarinic and δ-opioid receptors

Although coupled to Gi/o proteins, cannabinoid CB1 receptors can also activate intracellular Ca2+ ([Ca2+]i) accumulation through not fully understood mechanisms. We report that in, human neuroblastoma SH-SY5Y cells, CB1 activation with the specific agonist arachidonoylchloroethanolamide (ACEA), weakly elevates [Ca2+]i and that this effect, when using low (1–100 nM) concentrations of ACEA, is enhanced by the previous activation of Gq/11-coupled M3 muscarinic receptors with carbachol, dose-dependently and up to ∼8-fold. A similar behaviour was also observed with carbachol and the Gi/o-coupled δ-opioid receptor. Furthermore, stimulation of CB1 receptors produced a concentration-dependent leftward shift of the elevation of [Ca2+]i by δ-opioid receptors. These stimulatory effects were variedly attenuated by selective antagonists of each receptor, pertussis toxin, inhibitors of phospholipase C (U73122 and D609), and, when assessed in the presence of extracellular Ca2+, by the block of voltage-activated calcium channels. Cholera toxin only slightly inhibited the Gq/11-Gi/o-mediated cross-talk, but induced a stronger inhibition of the Gi/o-Gi/o-mediated interaction. These findings suggest that activation of M3 muscarinic receptors might produce a qualitative alteration of the signaling associated with Gi/o-coupled receptors, and that sequential activation of CB1 and δ-opioid receptors, both coupled to Gi/o, produces instead synergistic effects on [Ca2+]i elevation.

Dynamics of PIP2 regulation and metabolism seen with PIP2‐sensitive ion channels and optical probes.

KCNQ2/3 K+ channels require PIP2 to function. Activation of muscarinic M1 receptors (M1R) turns these channels off by Gq, PLC activation, and PIP2 depletion (Suh &amp; Hille, 2002). To monitor steps of this signaling cascade and plasma membrane PIP2 dynamics, we use FRET pairs, translocation of probes, and the channel current in intact living tsA cells. M1Rs acting through PLC deplete PIP2, make DAG, and shut channels in ~10 s. Following FRET pairs in the receptor signaling pathway shows that receptor activation takes &lt;0.1 s, receptor‐G‐protein interactions ~0.3 s, G protein activation ~2 s, and PLC is activated shortly after. Thus most of the delay in signaling to channels is the time it takes PLC to deplete PIP2 pools. Recovery of PIP2 after M1R agonist takes 100‐200 s. This recovery is wortmannin sensitive so PI(4)P needs to be regenerated as well as PIP2. Recovery time shortens with overexpression of PI 4‐kinase but not PIP 5‐kinase. PIP2 can also be depleted in a few seconds by activating a PIP2 5‐phosphatase with voltage steps (VSP) or with chemical dimerization, generating PI(4)P rather than IP3/DAG. Recovery from the perturbation by VSP takes only 15 s and becomes even faster if PIP 5‐kinase is overexpressed. It is not wortmannin sensitive. Thus the PIP 5‐kinase step in the synthesis of PIP2 is normally significantly faster than the PI 4‐kinase step. Supported by NIH grants RO1 NS08174, RO1 GM83913, T32 GM07108, and the HFSP.

Fluorescence changes reveal kinetic steps of muscarinic receptor–mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

G protein–coupled receptors initiate signaling cascades. M1 muscarinic receptor (M1R) activation couples through Gαq to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2). Depletion of PIP2 closes PIP2-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M1R activation, M1R/Gβ interaction, Gαq/Gβ separation, Gαq/PLC interaction, and PIP2 hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M1R activation (<100 ms) and M1R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gαq/Gβ separation and Gαq/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP2 hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Gαq/PLC interaction. Evidently, channel release of PIP2 and closure are rapid, and the availability of active PLC limits the rate of M current suppression.

Gating Current Measurements Reveal Ligand-Selective Conformational Changes in the M2 Muscarinic Receptor

In addition to external stimuli, membrane voltage modulates the activity of several G-protein-coupled receptors (GPCRs). For example, membrane depolarization reduces the affinity of the M2 muscarinic receptor (M2R) for acetylcholine (ACh). The intrinsic capacity of muscarinic receptors to “sense” transmembrane potential was confirmed by the recording of “gating” currents that reflect reorientation of charges within the receptor in response to changes in voltage. We studied the effects of membrane voltage on agonist activation of M2R in atrial myocytes and on gating currents in oocytes heterologously expressing M2R. Depolarization decreased the apparent affinity of M2R for ACh, but increased the apparent affinity and efficacy of pilocarpine (PILO), as measured by acetylcholine-activated K+ current, IKACh. Voltage-induced conformational changes in M2R (as measured by gating currents) were modified in a ligand-selective manner. For example, ACh reduced gating charge displacement while PILO increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent IKACh modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor and confirmed a link between voltage sensing and changes in agonist affinity. Our data suggest that voltage modulates the conformation of the ligand binding site, while ligand binding induces ligand-specific conformational changes in the receptor. Voltage-dependent GPCR modulation has important implications for cellular signaling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany ligand binding and changes in membrane voltage.

Modulation of Neuritogenesis by Astrocyte Muscarinic Receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.

SPATIAL AND TEMPORAL ASPECTS OF cAMP SIGNALLING IN CARDIAC MYOCYTES

1. beta(1)-Adrenoceptor and M(2) muscarinic receptor regulation of cAMP production plays a pivotal role in autonomic regulation of cardiac myocyte function. However, not all responses are easily explained by a uniform increase or decrease in cAMP activity throughout the entire cell. 2. Adenovirus expression of fluorescence resonance energy transfer (FRET)-based biosensors can be used to monitor cAMP activity in protein kinase A (PKA) signalling domains, as well as the bulk cytoplasmic domain of intact adult cardiac myocytes. 3. Data obtained using FRET-based biosensors expressed in different cellular microdomains have been used to develop a computational model of compartmentalized cAMP signalling. 4. A systems biology approach that uses quantitative computational modelling together with experimental data obtained using FRET-based biosensors has been used to provide evidence for the idea that compartmentation of cAMP signalling is necessary to explain the stimulatory responses to beta(1)-adrenoceptor activation as well as the complex temporal responses to M(2) muscarinic receptor activation.

Interaction of M3 receptors and calcium channels in feline bladder smooth muscle.

It is known that muscarinic receptors and calcium channels play roles in smooth muscle contraction, but it is unclear which receptor and which calcium channel types are most important. Recent studies have shown that M3 receptors may have a direct interaction with L‐type calcium channels. It is hypothesized that if stimulus evoked activation of muscarinic M3 receptors causes an influx of extracellular calcium by opening of L‐type calcium channels, then administration of L‐type calcium channel inhibitors will attenuate evoked contractions of bladder smooth muscle. This hypothesis was tested using an M3 specific antagonist, DAU 5884 (DAU), and an L‐type calcium channel inhibitor, verapamil (Ver). Using an anesthetized cat model, bladder contractions, evoked by pelvic nerve stimulation (PNS) and exogenous administration of acetylcholine, ATP or beta, gamma methylene ATP (APPCP), before and after administration of DAU, or Ver, were measured using DATAQ data acquisition hardware and software. The results show that DAU decreased cholinergic, but not purinergic contractions. Ver after DAU had no effect on any evoked contractions. Ver alone decreased cholinergic contractions, but not purinergic contractions. DAU after Ver decreased cholinergic, but not purinergic contractions. This data suggests that there is an interaction between M3 receptors and calcium channels, as indicated by the effect of these agents on cholinergic evoked bladder contractions.

The role of Wnt signaling in neuronal dysfunction in Alzheimer's Disease.

Recent evidence supports a neuroprotective role for Wnt signaling in neurodegenerative disorders such as Alzheimer's Disease (AD). In fact, a relationship between amyloid-β-peptide (Aβ)-induced neurotoxicity and a decrease in the cytoplasmic levels of β-catenin has been observed. Apparently Aβ binds to the extracellular cysteine-rich domain of the Frizzled receptor (Fz) inhibiting Wnt/β-catenin signaling. Cross-talk with other signaling cascades that regulate Wnt/β-catenin signaling, including the activation of M1 muscarinic receptor and PKC, the use of Ibuprofen-ChE bi-functional compounds, PPAR α, γ agonists, nicotine and some antioxidants, results in neuroprotection against Aβ. These studies indicate that a sustained loss of Wnt signaling function may be involved in the Aβ-dependent neurodegeneration observed in Alzheimer's brain. In conclusion the activation of the Wnt signaling pathway could be proposed as a therapeutic target for the treatment of AD.

Mechanism of Salivary Secretion Enhancement by Byakkokaninjinto

Byakkokaninjinto (BN) is a Kampo preparation used for the treatment of xerostomia induced by drug, ageing, Sjogren syndrome, etc. The mechanism for BN to induce salivary secretion has not been made clear. In this study, various rat thirst models were prepared using muscarinic receptor blockers, such as 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) and atropine, or adrenoceptor blockers, such as phentolamine and propranolol, in order to investigate the efficiency of BN. When BN was orally administered to the rats in the dose range of 100 to 300 mg/kg, the salivary secretion increased in a dose-dependent manner. The suppression of salivary secretion induced by phentolamine, atropine, and 4-DAMP was recovered by the additional treatment of BN. Interestingly, BN treatment increased the expression of aquaporin 5 in rats, which is known to regulate salivary secretion from the submandibular gland. These results suggested that BN increased the expression of aquaporin 5 through activation of muscarinic M3 receptor and enhanced salivary secretion.

Heterologous up-regulation of the histamine H1 receptor by M3 muscarinic receptor-mediated activation of H1-receptor gene transcription

Histamine H(1) receptor (H1R) level varies under various pathological conditions, and these changes may be responsible for some pathogenesis, such as allergic rhinitis. Previously, we showed that H1R was heterologously down-regulated (through degradation of H1R) by prolonged stimulation with muscarinic M(3) receptor (M3R) in Chinese hamster ovary (CHO) cells stably expressing H1R and M3R. However, this cell was inadequate for studying the effects on H1R gene regulation, because the cell expresses H1R, which is under the control of the SV40 promoter. Therefore, in this study, we have investigated the possible role of M3R stimulation in the H1R gene transcription and H1R mRNA stability by using U373 astrocytoma cells that express endogenous H1R and transfected M3R. Stimulation of M3R significantly increased H1R promoter activity and H1R mRNA level without alteration in H1R mRNA stability. The H1R level was also up-regulated by M3R activation (150% of control by treatment with carbachol for 24 h). These M3R-mediated events were almost completely blocked by the protein kinase C (PKC) inhibitor, Ro 31-8220, suggesting the involvement of PKC. These results indicated that M3R was involved in the up-regulation of H1R by activating H1R gene transcription through a PKC-dependent process.

Subcellular Targeting of RGS9-2 Is Controlled by Multiple Molecular Determinants on Its Membrane Anchor, R7BP

RGS9-2, a member of the R7 regulators of G protein signaling (RGS) protein family of neuronal RGS, is a critical regulator of G protein signaling. In striatal neurons, RGS9-2 is tightly associated with a novel palmitoylated protein, R7BP (R7 family binding protein). Here we report that R7BP acts to target the localization of RGS9-2 to the plasma membrane. Examination of the subcellular distribution in native striatal neurons revealed that both R7BP and RGS9-2 are almost entirely associated with the neuronal membranes. In addition to the plasma membrane, a large portion of RGS9-2 was found in the neuronal specializations, the postsynaptic densities, where it forms complexes with R7BP and its constitutive partner Gbeta5. Using site-directed mutagenesis we found that the molecular determinants that specify the subcellular targeting of RGS9-2.Gbeta5.R7BP complex are contained within the 21 C-terminal amino acids of R7BP. This function of the C terminus was found to require the synergistic contributions of its two distinct elements, a polybasic motif and palmitoylated cysteines, which when combined are sufficient for directing the intracellular localization of the constituent protein. In differentiated neurons, the C-terminal targeting motif of R7BP was found to be essential for mediating its postsynaptic localization. In addition to the plasma membrane targeting elements, we identified two functional nuclear localization sequences that can mediate the import of R7BP into the nucleus upon depalmitoylation. These findings provide a mechanism for the subcellular targeting of RGS9-2 in neurons.

Sympathetic Sprouting Drives Hippocampal Cholinergic Reinnervation That Prevents Loss of a Muscarinic Receptor-Dependent Long-Term Depression at CA3–CA1 Synapses

Degeneration of septohippocampal cholinergic neurons results in memory deficits attributable to loss of cholinergic modulation of hippocampal synaptic circuits. A remarkable consequence of cholinergic degeneration is the sprouting of noradrenergic sympathetic fibers from the superior cervical ganglia into hippocampus. The functional impact of sympathetic ingrowth on synaptic physiology has never been investigated. Here, we report that, at CA3-CA1 synapses, a Hebbian form of long-term depression (LTD) induced by muscarinic M1 receptor activation (mLTD) is lost after medial septal lesion. Unexpectedly, expression of mLTD is rescued by sympathetic sprouting. These effects are specific because LTP and other forms of LTD are unaffected. The rescue of mLTD expression is coupled temporally with the reappearance of cholinergic fibers in hippocampus, as assessed by the immunostaining of fibers for VAChT (vesicular acetylcholine transporter). Both the cholinergic reinnervation and mLTD rescue are prevented by bilateral superior cervical ganglionectomy, which also prevents the noradrenergic sympathetic sprouting. The new cholinergic fibers likely originate from the superior cervical ganglia because unilateral ganglionectomy, performed when cholinergic reinnervation is well established, removes the reinnervation on the ipsilateral side. Thus, the temporal coupling of the cholinergic reinnervation with mLTD rescue, together with the absence of reinnervation and mLTD expression after ganglionectomy, demonstrate that the autonomic-driven cholinergic reinnervation is essential for maintaining mLTD after central cholinergic cell death. We have discovered a novel phenomenon whereby the autonomic and central nervous systems experience structural rearrangement to replace lost cholinergic innervation in hippocampus, with the consequence of preserving a form of LTD that would otherwise be lost as a result of cholinergic degeneration.

Acetyl- l-carnitine protects striatal neurons against in vitro ischemia: The role of endogenous acetylcholine

The neuronal death after ischemia is closely linked to the essential role of mitochondrial metabolism. Inhibition of mitochondrial respiratory chain reduces ATP generation leading to a dysregulation of ion metabolism. Acetyl- l-carnitine (ALC) influences the maintenance of key mitochondrial proteins for maximum energy production and it may play a neuroprotective role in some pathological conditions. In this study we have analyzed ALC-mediated neuroprotection on an in vitro model of brain ischemia. Field potential recordings were obtained from a rat corticostriatal slice preparation. In vitro ischemia (oxygen and glucose deprivation) was delivered by switching to a solution in which glucose was omitted and oxygen was replaced with N 2. Ten minutes of in vitro ischemia caused an irreversible loss of the field potential amplitude. Pretreatment with ALC produced a progressive and dose-dependent recovery of the field potential amplitude following in vitro ischemia. The neuroprotective effect of ALC was stereospecific since the pretreatment with two different carnitine-related compounds did not cause neuroprotection. The choline transporter inhibitor hemicholinium-3 blocked the neuroprotective effect of ALC. ALC-mediated neuroprotection was also prevented either by the non-selective muscarinic antagonist scopolamine, or by the putative M2-like receptor antagonist methoctramine. Conversely, the effect of ALC was not altered by the M1-like receptor antagonist pirenzepine. These findings show that ALC exert a neuroprotective action against in vitro ischemia. This neuroprotective effect requires the activity of choline uptake system and the activation of M2 muscarinic receptors.

Effects of Pirenzepine on Dai-kenchu-to-Induced Elevation of the Plasma Neuropeptide Levels in Humans

Dai-kenchu-to has been used for the treatment of abdominal obstructions, including bowel obstructions and a feeling of coldness in the abdomen. We reported that Dai-kenchu-to increases plasma neuropeptide [motilin, vasoactive intestinal polypeptide (VIP), serotonin, calcitonin gene-related peptide (CGRP), and substance P]-like immunoreactive substances (IS) levels and that its pharmacologic effects on the gastrointestine are due to changes in gastrointestinal mucosa-regulatory peptide levels. We examined the effects of the selective M(1) muscarinic receptor antagonist pirenzepine on the elevation of Dai-kenchu-to-induced plasma neuropeptide (gastrin, motilin, somatostatin, VIP, CGRP, substance P)-IS levels in human volunteers and the area under the plasma neuropeptide concentration-time curve from 0 to 240 min (AUC(0-->240 min)), which were calculated from the plasma neuropeptide concentration-time curves from each volunteers. Oral pretreatment with pirenzepine reduced the Dai-kenchu-to-induced elevation of plasma motilin and VIP-IS levels and AUC(0-->240 min). Combined treatment with Dai-kenchu-to and pirenzepine increased plasma somatostatin-IS levels and decreased plasma gastrin-IS levels and had no effects on plasma CGRP- and substance P-IS levels and AUC(0-->240 min) compared with administration of Dai-kenchu-to alone. Dai-kenchu-to appeared to induce the release of motilin and VIP into plasma mainly through the activation of M(1) muscarinic receptors, and pirenzepine may affect the pharmacologic action of Dai-kenchu-to by elevation of plasma motilin and VIP levels.