The investigation of antibiotic residues, antibiotic resistance genes and antibiotic-resistant organisms in a drinking water reservoir system in Germany.

Between August 2018 and June 2019, a river system in Germany that supplies a drinking water reservoir and is subject to the discharge from two sewage treatment plants was monitored for antibiotic residues via liquid chromatography-tandem mass spectrometry, antibiotic resistance genes (including blaNDM, blaVIM, blaOXA-48, blaKPC, blaGIM, blaSME, blaIMI, blaIMP, blaSPM, blaSIM, blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58, mcr) via qualitative real-time PCR and antibiotic-resistant bacteria [belonging to the ESKAPE-group (Enterococcus faecium, Staphyhlococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter ssp.; with resistance against Carbapenemases, Cephalosporines and Colistin) and Escherichia coli] based on cultivation methods followed by a characterization via MALDI-TOF MS and susceptibility testing applying microdilution. Residues of macrolide antibiotics such as clarithromycin (up to 0.60 µg/L) and residues of sulfamethoxazole (up to 0.40 µg/L) and trimethoprim (up to 0.39 µg/L) were detected downstream of the sewage treatment plants. In addition, no antibiotic residues were detected upstream the respective sewage treatment plants, except for anhydroerythromycin (n = 1,

The occurrence of antimicrobial substances in toilet, sink and shower drainpipes of clinical units: A neglected source of antibiotic residues.

Antibiotics represent one of the most important drug groups used in the management of bacterial infections in humans and animals. Due to the increasing problem of antibiotic resistance, assurance of the antibacterial effectiveness of these substances has moved into the focus of public health. The reduction in antibiotic residues in wastewater and the environment may play a decisive role in the development of increasing rates of antibiotic resistance. The present study examines the wastewater of 31 patient rooms of various German clinics for possible residues of antibiotics, as well as the wastewater of five private households as a reference. To the best of our knowledge, this study shows for the first time that in hospitals with high antibiotic consumption rates, residues of these drugs can be regularly detected in toilets, sink siphons and shower drains at concentrations ranging from 0.02 µg·L-1 to a maximum of 79 mg·L-1. After complete flushing of the wastewater siphons, antibiotics are no longer detectable, but after temporal stagnation, the concentration of the active substances in the water phases of respective siphons increases again, suggesting that antibiotics persist through the washing process in biofilms. This study demonstrates that clinical wastewater systems offer further possibilities for the optimization of antibiotic resistance surveillance.

Isoform specific differences in phospholipase C beta 1 expression in the prefrontal cortex in schizophrenia and suicide.

Our previous study demonstrated that phospholipase C beta 1 mRNA was down-regulated in Brodmann's area 46 from subjects with schizophrenia. However, phospholipase C beta 1 protein has also been shown to be lower in Brodmann's area 8 and 9 from teenage suicide subjects, creating a potential confound in interpreting the findings in schizophrenia due to the high suicide rate associated with this disorder. To begin to reconcile and consolidate these findings, in this study, we measured mRNA and protein levels of phospholipase C beta 1 variants a and b in Brodmann's area 46 and Brodmann's area 9 from subjects with schizophrenia, many of whom were suicide completers, and determined the diagnostic specificity of observed findings. Consistent with our previous study, levels of phospholipase C beta 1 a and b mRNA, but not protein, were lower in Brodmann's area 46 from subjects with schizophrenia. In Brodmann's area 9, phospholipase C beta 1a protein levels were lower in subjects with schizophrenia, while phospholipase C beta 1b mRNA was higher and protein was lower in those that had died of suicide. Altered protein levels in Brodmann's area 9 appeared to be diagnostically specific, as we did not detect these changes in subjects with bipolar disorder, major depressive disorder or suicide completers with no diagnosis of mental illness. We further assessed the relationship between phospholipase C beta 1 and levels of muscarinic receptors (CHRMs) that signal through this protein, in both human and Chrm knockout mouse central nervous system tissue, and found no strong relationship between the two. Understanding central nervous system differences in downstream effector pathways in schizophrenia may lead to improved treatment strategies and help to identify those at risk of suicide.

M1 and m2 muscarinic receptor subtypes regulate antidepressant-like effects of the rapidly acting antidepressant scopolamine.

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2 (-/-) mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia.

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

SPINE bioinformatics and data-management aspects of high-throughput structural biology.

SPINE (Structural Proteomics In Europe) was established in 2002 as an integrated research project to develop new methods and technologies for high-throughput structural biology. Development areas were broken down into workpackages and this article gives an overview of ongoing activity in the bioinformatics workpackage. Developments cover target selection, target registration, wet and dry laboratory data management and structure annotation as they pertain to high-throughput studies. Some individual projects and developments are discussed in detail, while those that are covered elsewhere in this issue are treated more briefly. In particular, this overview focuses on the infrastructure of the software that allows the experimentalist to move projects through different areas that are crucial to high-throughput studies, leading to the collation of large data sets which are managed and eventually archived and/or deposited.

Identification of a high-affinity binding site involved in the transport of endocannabinoids.

Phytocannabinoids, such as the principal bioactive component of marijuana, delta9-tetrahydrocannabinol, have been used for thousands of years for medical and recreational purposes. delta9-Tetrahydrocannabinol and endogenous cannabinoids (e.g., anandamide) initiate their agonist properties by stimulating the cannabinoid family of G protein-coupled receptors (CB1 and CB2). The biosynthesis and physiology of anandamide is well understood, but its mechanism of uptake (resulting in signal termination by fatty acid amide hydrolase) has been elusive. Mounting evidence points to the existence of a specific anandamide transport protein; however, no direct evidence for this protein has been provided. Here, we use a potent, competitive small molecule inhibitor of anandamide uptake (LY2318912, IC50 7.27 +/- 0.510 nM) to identify a high-affinity, saturable anandamide transporter binding site (LY2318912; K(d) = 7.62 +/- 1.18 nM, B(max) = 31.6 +/- 1.80 fmol/mg protein) that is distinct from fatty acid amide hydrolase. Systemic administration of the inhibitor into rodents elevates anandamide levels 5-fold in the brain and demonstrates efficacy in the formalin paw-licking model of persistent pain with no obvious adverse effects on motor function. Identification of the anandamide transporter binding site resolves a missing mechanistic link in endocannabinoid signaling, and in vivo results suggest that endocannabinoid transporter antagonists may provide a strategy for positive modulation of cannabinoid receptors.

Increase of oral methadone dose in methadone injecting patients: a pilot study.

A pilot study was initiated in seven methadone injecting patients to examine whether intravenous methadone use in patients in oral methadone maintenance treatment could be decreased by increased oral methadone dose. During the study, patients had a standardized methadone dose increase for three weeks, followed by a 12-week follow-up period. Mean methadone doses prior to, and at the end of the study, were 99 mg/day and 163 mg/day, respectively. On week 15, the mean frequency of injection and the mean proportion of methadone dose injected were reduced to 46% of the values measured at week 0 (p = 0.043 and p = 0.028, respectively). Two patients did not modify their fre- quency, nor their dose of injected methadone, four patients decreased their use of injectable methadone, while one completely stopped injecting methadone. Since the completion of the pilot study, an augmentation of oral methadone dose has been proposed as a therapeutic option to 18 other methadone injecting patients. Five patients did not change their frequency of injection. They did, however, either completely stop or decrease their illicit opiate consumption. Nine patients decreased their frequency of methadone injection from a mean 95% down to 35%. Finally, four patients completely stopped injecting methadone. Although the present results have to be confirmed by controlled studies including a larger number of patients, when considering the high frequency of methadone injection in some places and the associated problems, the therapeutic option of increasing methadone dose should be considered further.

M1-M5 muscarinic receptor knockout mice as novel tools to study the physiological roles of the muscarinic cholinergic system.

A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.

Dysregulated hippocampal acetylcholine neurotransmission and impaired cognition in M2, M4 and M2/M4 muscarinic receptor knockout mice.

Among the five different muscarinic receptors that have been cloned and characterized, M2 and M4 receptors are localized both post- and presynaptically and are believed to have a pronounced autoreceptor role. The functional importance of these receptors in the regulation of acetylcholine release in the hippocampus and in cognitive processes was investigated by using M2 and M4 receptor single knockout (KO) as well as M2/M4 receptor double KO mice. We found profound alterations in acetylcholine homeostasis in the hippocampus of both M2- and M4-KO mice as well as of the combined M2/M4-KOs, as assessed by in vivo microdialysis. Basal acetylcholine efflux in the hippocampus was significantly increased in M4-KO and was elevated further in M2/M4-KOs. The increase in hippocampal acetylcholine induced by local administration of scopolamine was markedly reduced in M2-KO and completely abolished in M2/M4-KOs. In M2-KO and much more in M2/M4-KOs, the increase in hippocampal acetylcholine triggered by exposure to a novel environment was more pronounced both in amplitude and duration, with a similar trend observed for M4-KOs. Dysregulation of cholinergic function in the hippocampus, as it could result from perturbed autoreceptor function, may be associated with cognitive deficits. Importantly, M2- and M2/M4-KO, but not M4-KO, animals showed an impaired performance in the passive avoidance test. Together these results suggest a crucial role for muscarinic M2 and M4 receptors in the tonic and phasic regulation of acetylcholine efflux in the hippocampus as well as in cognitive processes.

Muscarinic agonist-mediated increases in serum corticosterone levels are abolished in m(2) muscarinic acetylcholine receptor knockout mice.

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions in the central and peripheral nervous system. Due to the lack of receptor subtype-selective ligands, however, the physiological roles of individual muscarinic receptor subtypes remain to be determined. In this study, we examined the effects of the muscarinic M(2)/M(4) receptor-preferring agonist [5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.2.1]-octane (BuTAC) on serum corticosterone levels in M(2) and M(4) receptor single knockout (KO) and M(2,4) receptor double KO mice. Responses were compared with those obtained with the corresponding wild-type (WT) mice. BuTAC (0.03-0.3 mg/kg s.c.) dose dependently and significantly increased serum corticosterone concentrations in WT mice to 5-fold or greater levels compared with vehicle controls. In muscarinic M(2) and M(2,4) KO mice, however, BuTAC had no significant effect on corticosterone concentrations at doses of 0.1, 0.3, and 1 mg/kg s.c. In both WT and muscarinic M(4) KO mice increases in serum corticosterone concentrations induced by BuTAC (0.1 and 0.3 mg/kg) were not significantly different and were blocked by scopolamine. In summary, the muscarinic M(2,4)-preferring agonist BuTAC had no effect on corticosterone levels in mice lacking functional muscarinic M(2) receptors. These data suggest that the muscarinic M(2) receptor subtype mediates muscarinic agonist-induced activation of the hypothalamic-pituitary-adrenocortical axis in mice.

International Union of Pharmacology. XXVII. Classification of cannabinoid receptors.

Two types of cannabinoid receptor have been discovered so far, CB(1) (2.1: CBD:1:CB1:), cloned in 1990, and CB(2) (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB(1) and CB(2) exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB(1) receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB(1) agonists probably serve as retrograde synaptic messengers. CB(2) receptors are present mainly on immune cells. Such cells also express CB(1) receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid molecules are the only, or primary, endogenous agonists. Hence, we consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacology Committee on Receptor Nomenclature and Drug Classification. Although pharmacological evidence for the existence of additional types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.

Role of the cholinergic muscarinic system in bipolar disorder and related mechanism of action of antipsychotic agents.

The evidence for the involvement of cholinergic muscarinic receptors in mania and depression is reviewed. Small pilot trials with cholinesterase inhibitors and muscarinic agonists suggest that stimulation of muscarinic receptors may produce an antimanic effect, possibly by activation of muscarinic M(4) receptors. It is concluded that it is not likely that currently used mood stabilizers, such as lithium, valproic acid and carbamazepine, work directly through muscarinic receptor mechanisms. Furthermore, the evidence indicates that antipsychotic agents used for mania are working through the common mechanism of antagonism of dopamine D(2) receptors, and interactions with muscarinic receptors do not play a key role. Finally, it is hypothesized that olanzapine has robust antimanic activity, due to blockade of dopamine D(2) receptors and antagonism of other monoaminergic receptors. Olanzapine may normalize mood due to antidepressant-like activities, such as 5-HT(2A) receptor antagonism and increasing cortical norepinephrine and dopamine.

Cannabinoid CB(1) receptor expression, activation and detection of endogenous ligand in trabecular meshwork and ciliary process tissues.

Elevated intraocular pressure is the primary risk factor for glaucoma. Cannabinoids interact with molecular targets in the eye and lower intraocular pressure by an unknown mechanism. The purpose of the present study was to examine eye tissues for functional cannabinoid receptors of the neuronal, CB(1) class, and an endogenous ligand, anandamide. The trabecular meshwork and ciliary processes are the primary structures of the eye that contribute to intraocular pressure and thus were our focus. Total RNA, frozen sections, cellular membranes and primary cultures of cells were prepared from both bovine and cadaveric human tissues. Using cannabinoid CB(1) receptor-specific oligodeoxynucleotide primers, cannabinoid CB(1) receptor antiserum, and cannabinoid-specific compounds (CP-55,940, WIN55,212-2 and SR-141716A), the presence of cannabinoid CB(1) receptors in ciliary processes and trabecular meshwork was determined. Using reverse transcription-polymerase chain reaction, we identified mRNA encoding cannabinoid CB(1) receptor protein in ciliary process and trabecular meshwork cells. Specific binding of anti-CB(1) immunoglobulin-G in tissue sections localized cannabinoid CB(1) receptor protein to the non-pigmented epithelial cells of the ciliary process and cells of the trabecular meshwork. While CP-55,940 and WIN55,212-2 failed to stimulate [(35)S]GTP gamma S binding in membrane preparations from trabecular meshwork and ciliary process, CP-55,940 significantly stimulated whole cell [(35)S]GTP gamma S binding by 51% over basal in ciliary process epithelial cells and 69% over basal in trabecular meshwork cells permeabilized with 5 microM digitonin (p<0.001). Specificity of agonist stimulation was verified by complete blockade with the specific cannabinoid CB(1) receptor antagonist, SR-141716A. Moreover, activation of cannabinoid CB(1) receptors by CP-55,940 resulted in a 2.3+/-0.3 and 1.7+/-0.3-fold stimulation of cAMP accumulation in trabecular meshwork and ciliary process cells, respectively (p<0.01). Lastly, anandamide was detected in human trabecular meshwork (3.08 pmol/g), ciliary process (49.42 pmol/g) and neurosensory retinal (4.48 pmol/g) tissues. These data, for the first time, demonstrate in a single study the presence of both CB(1) mRNA and protein in trabecular meshwork and ciliary processes from two different species. Activation of heterotrimeric G-proteins and stimulation of cAMP accumulation by cannabinoids in vitro suggest that their intraocular pressure-lowering effects in vivo result from activation of cannabinoid CB(1) receptors in the trabecular meshwork and increase aqueous outflow.

Non-MAO A binding of clorgyline in white matter in human brain.

Clorgyline is an irreversible inhibitor of monoamine oxidase (MAO A) which has been labeled with carbon-11 (C-11) and used to measure human brain MAO A with positron emission tomography (PET). In this study we compared [11C]clorgyline and deuterium-substituted [11C]clorgyline ([11C]clorgyline-D2) to better understand the molecular link between [11C]clorgyline binding and MAO A. In PET studies of five normal healthy volunteers scanned with [11C]clorgyline and [11C]clorgyline-D2 2 h apart, deuterium substitution generally produced the expected reductions in the brain uptake of [11C]clorgyline. However, the reduction was not uniform with the C-11 binding in white matter being significantly less sensitive to deuterium substitution than other brain regions. The percentages of the total binding attributable to MAO A is largest for the thalamus and smallest for the white matter and this is clearly seen in PET images with [11C]clorgyline-D2. Thus deuterium-substituted [11C]clorgyline selectively reduces the MAO A binding component of clorgyline in the human brain revealing non-MAO A binding which is most apparent in the white matter. The characterization of the non-MAO A binding component of this widely used MAO A inhibitor merits further investigation.

A current review of olanzapine's safety in the geriatric patient: from pre-clinical pharmacology to clinical data.

OBJECTIVE: Olanzapine (OLZ) is unique among currently available antipsychotic medications in its antagonism of a range of receptor systems including dopamine, norepinephrine, serotonin, acetylcholine, and histamine. Olanzapine's mechanistic complexity provides a broad efficacy profile in patients with schizophrenia and acute, pure or mixed mania. Patients experience symptomatic relief of mania, anxiety, hallucinations, delusions, and agitation/aggression and reduced depressive, negative, and some cognitive symptoms. This paper will review the safety profile of OLZ, focusing on the elderly, where data are available. METHOD: Preclinical and clinical studies of OLZ are reviewed, with emphasis on its possible effects on the cholinergic system and the histamine H(1) receptor. Weight change and related metabolic considerations, cardiac and cardiovascular safety, and motor function during treatment with OLZ are also reviewed. RESULTS AND CONCLUSION: In vitro receptor characterization methods, when done using physiologically relevant conditions allow accurate prediction of the relatively low rate of anticholinergic-like adverse events, extrapyramidal symptoms, and cardiovascular adverse events during treatment with OLZ. Currently available clinical data suggest olanzapine is predictably safe in treating adult patients of any age with schizophrenia and acute bipolar mania, as well as in treatment of patients with some types of neurodegenerative disorders.

Cholinergic dilation of cerebral blood vessels is abolished in M(5) muscarinic acetylcholine receptor knockout mice.

The M(5) muscarinic receptor is the most recent member of the muscarinic acetylcholine receptor family (M(1)-M(5)) to be cloned. At present, the physiological relevance of this receptor subtype remains unknown, primarily because of its low expression levels and the lack of M(5) receptor-selective ligands. To circumvent these difficulties, we used gene targeting technology to generate M(5) receptor-deficient mice (M5R(-/-) mice). M5R(-/-) mice did not differ from their wild-type littermates in various behavioral and pharmacologic tests. However, in vitro neurotransmitter release experiments showed that M(5) receptors play a role in facilitating muscarinic agonist-induced dopamine release in the striatum. Because M(5) receptor mRNA has been detected in several blood vessels, we also investigated whether the lack of M(5) receptors led to changes in vascular tone by using several in vivo and in vitro vascular preparations. Strikingly, acetylcholine, a powerful dilator of most vascular beds, virtually lost the ability to dilate cerebral arteries and arterioles in M5R(-/-) mice. This effect was specific for cerebral blood vessels, because acetylcholine-mediated dilation of extra-cerebral arteries remained fully intact in M5R(-/-) mice. Our findings provide direct evidence that M(5) muscarinic receptors are physiologically relevant. Because it has been suggested that impaired cholinergic dilation of cerebral blood vessels may play a role in the pathophysiology of Alzheimer's disease and focal cerebral ischemia, cerebrovascular M(5) receptors may represent an attractive therapeutic target.

Receptor reserve of phosphoinositide-coupled muscarinic receptors in mouse hippocampus in vivo.

The ability of the partial muscarinic agonist pilocarpine to increase in vivo phosphoinositide (PI) hydrolysis in mouse brain was compared to two full agonists. Pilocarpine increased in vivo phosphoinositide (PI) hydrolysis in cortex, striatum, and to the greatest extent in the hippocampus. Pilocarpine injected either subcutaneously or intracerebroventricularly robustly increased in vivo PI hydrolysis in hippocampus up to 500% of control levels and the increases were blocked by the muscarinic antagonist scopolamine. The increases in vivo PI hydrolysis induced by pilocarpine were 60-75% of the magnitude of the full muscarinic agonists oxotremorine-M and cis-dioxolane. The muscarinic M(1) preferring antagonist pirenzepine potently blocked pilocarpine-induced increases in in vivo PI hydrolysis, consistent with the increase being mediated by M(1) receptors. Since pilocarpine is a relatively weak partial agonist, these data suggest a substantial level of receptor reserve for the PI response in mouse hippocampus.

The endocannabinoid nervous system: unique opportunities for therapeutic intervention.

The active principle in marijuana, Delta(9)-tetrahydrocannabinol (THC), has been shown to have wide therapeutic application for a number of important medical conditions, including pain, anxiety, glaucoma, nausea, emesis, muscle spasms, and wasting diseases. Delta(9)-THC binds to and activates two known cannabinoid receptors found in mammalian tissue, CB1 and CB2. The development of cannabinoid-based therapeutics has focused predominantly on the CB1 receptor, based on its predominant and abundant localization in the CNS. Like most of the known cannabinoid agonists, Delta(9)-THC is lipophilic and relatively nonselective for both receptor subtypes. Clinical studies show that nonselective cannabinoid agonists are relatively safe and provide therapeutic efficacy, but that they also induce psychotropic side effects. Recent studies of the biosynthesis, release, transport, and disposition of anandamide are beginning to provide an understanding of the role of lipid transmitters in the CNS. This review attempts to link current understanding of the basic biology of the endocannabinoid nervous system to novel opportunities for therapeutic intervention. This new knowledge may facilitate the development of cannabinoid receptor-targeted therapeutics with improved safety and efficacy profiles.

Generation and pharmacological analysis of M2 and M4 muscarinic receptor knockout mice.

Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.