Implication of Major Adverse Postoperative Events and Myocardial Injury on Disability and Survival: A Planned Subanalysis of the ENIGMA-II Trial.

BACKGROUND: Globally, >300 million patients have surgery annually, and ≤20% experience adverse postoperative events. We studied the impact of both cardiac and noncardiac adverse events on 1-year disability-free survival after noncardiac surgery. METHODS: We used the study cohort from the Evaluation of Nitrous oxide in Gas Mixture of Anesthesia (ENIGMA-II) trial, an international randomized trial of 6992 noncardiac surgical patients. All were ≥45 years of age and had moderate to high cardiac risk. The primary outcome was mortality within 1 postoperative year. We defined 4 separate types of postoperative adverse events. Major adverse cardiac events (MACEs) included myocardial infarction (MI), cardiac arrest, and myocardial revascularization with or without troponin elevation. MI was defined using the third Universal Definition and was blindly adjudicated. A second cohort consisted of patients with isolated troponin increases who did not meet the definition for MI. We also considered a cohort of patients who experienced major adverse postoperative events (MAPEs), including unplanned admission to intensive care, prolonged mechanical ventilation, wound infection, pulmonary embolism, and stroke. From this cohort, we identified a group without troponin elevation and another with troponin elevation that was not judged to be an MI. Multivariable Cox proportional hazard models for death at 1 year and assessments of proportionality of hazard functions were performed and expressed as an adjusted hazard ratio (aHR) and 95% confidence intervals (CIs). RESULTS: MACEs were observed in 469 patients, and another 754 patients had isolated troponin increases. MAPEs were observed in 631 patients. Compared with control patients, patients with a MACE were at increased risk of mortality (aHR, 3.36 [95% CI, 2.55-4.46]), similar to patients who suffered a MAPE without troponin elevation (n = 501) (aHR, 2.98 [95% CI, 2.26-3.92]). Patients who suffered a MAPE with troponin elevation but without MI had the highest risk of death (n = 116) (aHR, 4.29 [95% CI, 2.89-6.36]). These 4 types of adverse events similarly affected 1-year disability-free survival. CONCLUSIONS: MACEs and MAPEs occur at similar frequencies and affect survival to a similar degree. All 3 types of postoperative troponin elevation in this analysis were associated, to varying degrees, with increased risk of death and disability.

Identification of patients with cervical SCI suitable for early nerve transfer to achieve hand opening.

STUDY DESIGN: Retrospective audit. OBJECTIVES: The objective of this study was to identify the proportion of patients with cervical spinal cord injury who would potentially benefit from nerve transfer surgery to gain active hand opening, and to determine when a safe nerve transfer decision can be made. SETTING: Christchurch, New Zealand. METHODS: Case note review of the first 12 months following acute cervical spinal cord injury (2007-2012). Neurological assessment at 6 weeks, 12 weeks and 1 year following injury. RESULTS: Fifty-three patients had complete assessments and showed changes in the level of injury and severity of neurological injury between assessments. Forty-two percent of patients had motor complete C5-7 level injuries 12 weeks following injury and would benefit from consideration for nerve transfer to improve hand opening. Fewer (26%) would benefit 1 year following injury owing to a change in the neurological level of injury. CONCLUSIONS: Twelve-week neurological assessment identifies patients who may benefit from nerve transfer surgery. This enables referral for comprehensive upper limb assessment and reassessment of motor function to determine suitability for surgical intervention. Nerve transfer within the window of opportunity provides active hand opening for patients following cervical spinal cord injury.

Chronic postsurgical pain in the Evaluation of Nitrous Oxide in the Gas Mixture for Anaesthesia (ENIGMA)-II trial.

BACKGROUND: Previous animal and clinical studies showed that nitrous oxide may produce long-term analgesia. The aim of this study was to evaluate the effect of nitrous oxide in preventing chronic postsurgical pain. We also explored whether methylenetetrahydrofolate reductase gene polymorphisms (1298A>C, 667C>T) would enhance nitrous oxide analgesia. METHODS: We conducted a telephone interview at 12 months after surgery on 2924 (41.1%) patients enrolled in the Evaluation of Nitrous Oxide in the Gas Mixture for Anaesthesia-II trial. Pain at the wound site was recorded using the modified brief pain inventory and the neuropathic pain questionnaire. General health status was measured using the EQ-5D questionnaire. Genotyping was performed in a subset of 674 Asian patients in Hong Kong. RESULTS: At 12 months after surgery, 356 (12.2%) patients reported chronic postsurgical pain at the wound site and 112 (3.8%) patients had severe pain and required regular analgesic interventions. Nitrous oxide did not affect the rate of chronic postsurgical pain (11.8% nitrous oxide group; 12.5% no nitrous oxide group), relative risk (95% confidence intervals): 0.94 (0.75-1.17), P=0.57. However, in a planned subgroup analysis, nitrous oxide reduced the risk of chronic postsurgical pain in Asian patients, relative risk (95% confidence intervals): 0.70 (0.50-0.98), P=0.031. Patients who were homozygous for either gene polymorphism and who received nitrous oxide during surgery were less likely to report chronic postsurgical pain. CONCLUSIONS: Nitrous oxide administration had no impact on chronic postsurgical pain, but benefits may still be possible in Asian patients and patients with variants in methylenetetrahydrofolate reductase gene. CLINICAL TRIAL REGISTRATION: NCT00430989.

A study of the norcaradiene-cycloheptatriene equilibrium in a series of azulenones by NMR spectroscopy; the impact of substitution on the position of equilibrium.

A systematic investigation of the influence of substitution at positions C-2 and C-3 on the azulenone skeleton, based on NMR characterisation, is discussed with particular focus on the impact of the steric and electronic characteristics of substituents on the position of the norcaradiene-cycloheptatriene (NCD-CHT) equilibrium. Variable temperature (VT) NMR studies, undertaken to enable the resolution of signals for the equilibrating valence tautomers revealed, in addition, interesting shifts in the equilibrium.

In vivo delivery of DN:REST improves transcriptional changes of REST-regulated genes in HD mice.

Current therapeutic strategies for Huntington's disease (HD) are focused on symptom management of disease progression. Transcriptional dysregulation is one of the major characteristics in HD. REST is a transcriptional repressor that silences gene expression through binding to RE1/NRSE sites found in the regulatory regions of numerous neuronal genes. Dysregulation of REST and its targeted genes has been reported in different cell and mouse HD models, as well as in biopsies from human patients. In this work, we characterized transcriptional dysregulation associated with REST in two different HD mouse models and assessed the therapeutic effect of interfering with REST function by overexpressing a dominant-negative form (DN:REST). We show that delivery of DN:REST in the motor cortex restores brain-derived neurotrophic factor (BDNF) mRNA and protein levels by reducing endogenous REST occupancy at the Bdnf locus. Similarly, expression of other REST-regulated genes such as Synapsin I (Syn1), Proenkephalin (Penk1) and Cholinergic receptor muscarinic 4 (Chrm4) were restored to normal levels while non-REST-regulated genes were unaffected. This is the first study conducted to investigate REST's role in vivo in a neurodegenerative disease. Our data show that DN:REST in motor cortex reversed RESTs repressive effects on target genes. However, the lack of therapeutic effect on motor function suggests that a more widespread rescue of REST-regulated sites in the affected brain regions may be necessary.

The role of comorbidities on the uptake of systemic treatment and 3-year survival in older cancer patients.

BACKGROUND: Older patients are notably absent from clinical trials. Thus, observational studies are the primary avenue for understanding the role of comorbidity in cancer care and survival. We examined the impact of comorbidity on systemic treatment initiation and 3-year survival in a cohort of older cancer patients. PATIENTS AND METHODS: Our cohort comprised 2753 Australian veterans aged ≥65 years with full health coverage and a cancer registry notification for colorectal (CRC), breast, prostate or non-small-cell lung cancer (NSCLC). We established comorbidities based on drugs prescribed in the 6 months prior to cancer diagnosis. RESULTS: Patients with higher comorbidity burden were more likely to receive systemic treatment for prostate cancer [adjusted odds ratio 1.21, 95% confidence interval (CI) 1.05-1.39] but less likely for NSCLC (0.63, 95% CI 0.45-0.86). After adjusting for receipt of treatment, increased comorbidity resulted in shorter survival for CRC [adjusted hazard ratio (aHR) 1.16, 95% CI 1.07-1.26] and breast cancer (aHR 1.23, 95% CI 1.02-1.48). However, we did not demonstrate significant improvements in 3-year survival for patients receiving systemic treatment. CONCLUSION: Comorbidity influences systemic treatment uptake and adversely affects survival, with impact dependent upon comorbidity and cancer type. Clinical trials should be undertaken in older patients to better understand the risks and benefits of cancer treatments.

Validity of referral hospitals for the toxicovigilance of acute poisoning in Sri Lanka.

OBJECTIVE: To identify the hospital admission data set that best captures the incidence of acute poisoning in rural Sri Lanka. METHODS: Data were collected on all acute poisoning cases admitted to 34 primary and 1 referral hospital in Anuradhapura district from September 2008 to January 2010. Three admission data sets were compared with the "true" incidence of acute poisoning to determine the systematic bias inherent to each data set. "True" incidence was calculated by adding all direct admissions (not transfers) to primary hospitals and to the referral hospital. The three data sets were: (i) all admissions to primary hospitals only; (ii) all admissions to the referral hospital only (direct and referrals), and (iii) all admissions to both primary hospitals and the referral hospital ("all admissions"). The third is the government's routine statistical method but counts transfers twice, so for the study transferred patients were counted only once through data linkage. FINDINGS: Of 3813 patients admitted for poisoning, 3111 first presented to a primary hospital and 2287 (73.5%) were later transferred to the referral hospital, where most deaths (161/177) occurred. All data sets were representative demographically and in poisoning type, but referral hospital data yielded a more accurate case-fatality rate than primary hospital data or "all admissions" data. Admissions to primary hospitals only or to the referral hospital only underestimated the incidence of acute poisoning by about 20%, and data on "all admissions" overestimated it by 60%. CONCLUSION: Admission data from referral hospitals are easily obtainable and accurately reflect the true poisoning incidence.

Modulation of thiopental-induced vascular relaxation and contraction by perivascular adipose tissue and endothelium.

BACKGROUND: Thiopental induces relaxation of vascular smooth muscle cells through its direct and/or indirect vasodilator effects. The perivascular adipose tissue (PVAT) and the endothelium are known to attenuate vascular contraction, and we have recently reported that PVAT potentiates the relaxation effect of propofol through endothelium-dependent and -independent mechanisms. Here, we studied the mechanisms of thiopental-induced vascular responses in relation to the involvement of PVAT and endothelium. METHODS: Thoracic aortic rings from male Wistar rats were prepared with or without PVAT (PVAT+ and PVAT-) and with an intact endothelium (E+) or with the endothelium removed (E-) for functional studies. The contraction and relaxation responses of these vessels to thiopental in the presence of agonists and various receptor antagonists and channel blockers were studied. RESULTS: In vessels pre-contracted with phenylephrine or KCl, thiopental-induced relaxation was highest in vessels denuded of both PVAT and the endothelium. PVAT attenuated the relaxation response to thiopental, and this attenuation effect was reduced by both angiotensin II (Ang II) type 1 receptor antagonists CV-11974 (2-n-butyl-4-choloro-5-hydroxymethyl-1-[2'-(1H-tetrazol-5-yl)biphenyl-methyl]-imidazole) or losartan and the angiotensin-converting enzyme inhibitor enalaprilat. Thiopental at high concentration (3 × 10(-3) M) caused a contraction through an endothelin-dependent mechanism. CONCLUSIONS: Thiopental induced relaxation in rat aorta through an endothelium-independent pathway and the presence of PVAT, endothelium, or both attenuated this relaxation response through Ang II-dependent and endothelin-dependent mechanisms, respectively.

Deliberate self-poisoning in Australian adolescents is increased on school days.

Deliberate self-poisoning (DSP) in adolescents is increasing dramatically. Life at school is one of the most important life influences for this age group. This study aimed to investigate whether the frequency of DSP is higher during school term compared to holidays and whether this difference has become greater over time. This is an ecological study using Poisons Information Centre (PIC) data for all DSPs in 10-19 year olds from New South Wales, Tasmania and Australian Capital Territory that occurred between 2005 and 2018. For each call, the date of the poisoning was assigned as 'term' or 'holiday'. To control for population growth, calls were expressed as per 100,000 of the population per day. Multivariable Poisson regression was performed to investigate the combined impact of various predictors (state, sex, year, holiday/term, day of week, age) on call number. 26,432 calls were included in the analysis (73.6% female, 24.1% male and 2.3% unknown). Poisson regression showed significant effects for all predictors, with an increased likelihood of DSP during the school term compared with holidays and on Monday-Thursday compared with Saturday but only during the school term. DSP doubled between 2012 and 2017 and the disparity between DSP that occurs during term vs. holiday increased over that time frame. We conclude that some of the increase in DSP is likely due to school-specific stressors, hence the school environment is the ideal setting for self-harm prevention initiatives.

Acute alcohol use in Australian coronial suicide cases, 2010-2015.

BACKGROUND: Acute use of alcohol is a robust risk factor for suicide, reported in approximately one- to two-fifths of suicide cases. Comparisons of risk factors between suicides with and without prior acute alcohol consumption have not been investigated in Australia. This study addresses the gap by examining individual factors (age, sex, employment status, method of suicide) and environmental factors (month of death, jurisdiction) between alcohol and non-alcohol suicide. METHODS: Data for all suicide deaths (aged 15 and over) in Australia were obtained from the National Coronial Information System (NCIS). Blood alcohol concentrations (BAC) were extracted from coronial reports, along with demographic information. Alcohol consumption prior to suicide was assumed if BAC ≥ 0.05 g/100 mL. We compared case characteristics between alcohol related and non-alcohol related suicides using logistic regression. RESULTS: 26.7% of suicide deaths in Australia had a BAC ≥ 0.05 g/100 mL. Alcohol use prior to suicide was associated with male gender (adjusted odds ratio [AOR]: 1.14, 95% confidence interval [95%CI]: 1.03, 1.26), being aged between 35-44 years (AOR: 1.26, 95%CI: 1.08, 1.46) and hangings (AOR: 1.53, 95%CI: 1.08, 1.46). Mean suicides per month over the timeframe demonstrated significant seasonality. Mean counts per month for alcohol related suicides peaked in December, compared to a peak in September for non-alcohol related suicides. CONCLUSIONS: This study highlights differences between alcohol related and non-alcohol related suicides including sex, age, method of death, time of year and location within Australia. Targeting alcohol related suicide should be a key priority in comprehensive suicide prevention strategies.

Effect of age on the severity of chronic lithium poisoning.

Objectives: Severe lithium toxicity is commonly observed in older people. We aimed to determine the extent to which age is associated with increased severity of chronic lithium poisoning and of which a range of possible factors might explain the associations.Method: We did a retrospective review of patients aged ≥15 years old with serum lithium concentrations ≥1.3 mmol/L from three hospitals. Clinical details, treatment and outcomes were recorded. eGFR, creatinine and lithium clearance were calculated. The severity of lithium toxicity was graded into five categories (Amdisen score). ANOVA was used to quantify the association between age and severity. Spearman correlation coefficient was used to explore relationships between age and different factors expected to alter severity. Ordinal regression analysis was used to determine the interdependence of age and these factors and age on severity of lithium toxicity.Results: From 2008-2018, there were 242 patients with a median age of 56.5 years (IQR: 41-69). There were 156 females (64%). There was a statistically significant association between Amdisen severity scores and age (p = .0004). The median calculated eGFR was 65 mL/min/1.73 m2 (IQR: 41-91) with a corresponding estimated lithium clearance of 18 mL/min (IQR: 13.8-22.8). There was no correlation of age with initial serum lithium concentration (p = .76). There was a strong correlation between age and estimated lithium clearance (r = -0.72, 95% CI: -0.78 to -0.66, p < .001), lithium daily dose (r = -0.65, 95% CI: -0.72 to -0.57, p < .0001) and lithium concentration/dose (r = 0.62, 95% CI: 0.53-0.69, p < .0001). There was a weak correlation between age and infection (r = 0.18, 95% CI: 0.04-0.31, p = .009) and drug interactions (r = 0.25, 95% CI: 0.11-0.37, p = .0003). Ordinal regression indicated the independent predictors for severity of lithium toxicity were lithium concentration (p < .0001) and lithium clearance (p = .03) adjusted for age and dose.Conclusions: Despite lower lithium doses, older patients had more severe toxicity. Increased severity of lithium toxicity in the elderly is largely explainable by decreased lithium clearance from multiple factors such as age-related decline in renal function, drug interactions and infection.

A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM.

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.

Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts.

Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-1-phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling pathways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-1-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-1-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-1-phosphate.

Effect of cell-cell interactions on drug sensitivity and growth of drug-sensitive and -resistant tumor cells in spheroids.

Multicellular spheroids were grown from mixtures of rat brain tumor cells sensitive (9L) and resistant (R3) to 1,3-bis(2-chloroethyl)-1-nitrosourea. Percentages of each cell subpopulation in these spheroids were estimated with the sister chromatid exchange assay and were found to be approximately the same as those used to initiate spheroids. Spheroids grown from 9L cells alone had a higher growth rate than spheroids grown from R3 cells alone. However, the growth rate of mixed-cell spheroids was essentially the same as that of pure 9L spheroids and was independent of the percentages of R3 cells in mixed-cell spheroids. The sensitivity of 9L cells in mixed-cell spheroids treated with 1,3-bis(2-chloroethyl)-1-nitrosourea, estimated by changes in the number of sister chromatid exchanges per metaphase induced by treatment, decreased as the percentage of R3 cells increased. These effects are probably the result of an interaction between the two cell subpopulations held in three-dimensional contact, a situation similar to that in tumors in situ. The results suggest why one cell subpopulation of tumors does not become dominant during growth and indicate that interactions between cell subpopulations can influence the sensitivity of one subpopulation to 1,3-bis(2-chloroethyl)-1-nitrosourea.

Identification of a family of muscarinic acetylcholine receptor genes.

Complementary DNAs for three different muscarinic acetylcholine receptors were isolated from a rat cerebral cortex library, and the cloned receptors were expressed in mammalian cells. Analysis of human and rat genomic clones indicates that there are at least four functional muscarinic receptor genes and that these genes lack introns in the coding sequence. This gene family provides a new basis for evaluating the diversity of muscarinic mechanisms in the nervous system.

Potential for methotrexate exposure through contamination during parenteral use as an immunosuppressant.

BACKGROUND: To evaluate whether the risk of methotrexate (MTX) exposure through skin contamination using parenteral doses of 25 mg warrants special oncology handling precautions during administration. METHODS: We conducted a study with six human volunteers deliberately exposed to an entire dose of 25 mg MTX solution on their skin for 30 min. Serum levels of MTX were measured at baseline, 2, 4, 8, 12 and 24 h as well as serum homocysteine at baseline and 24 h after clinical exposure. Twenty-four-hour urinary excretion of MTX and possible local or systemic signs of toxicity were also recorded. RESULTS: All MTX serum concentrations were less than 0.02 micromol/L within the 24-h period. This is 500 times below the recommended serum concentration for which folinic acid supplementation is recommended. There was also no significant increase in homocysteine level to suggest MTX toxicity. The only adverse effects were mild local dermal reactions in three female volunteers. CONCLUSION: Deliberate skin contamination and possible inhalation of a 25 mg MTX solution failed to show significant or quantifiable serum and urine concentrations to suggest MTX toxicity. Precautions to prevent contact with MTX designed for oncology protocols are unnecessary for our rheumatology patients or their carers using these much lower immunosuppressant doses for autoimmune diseases.

TBX2 interacts with heterochromatin protein 1 to recruit a novel repression complex to EGR1-targeted promoters to drive the proliferation of breast cancer cells.

Early Growth Response 1 (EGR1) is a stress response transcription factor with multiple tumour suppressor roles in breast tissue, whose expression is often lost in breast cancers. We have previously shown that the breast cancer oncogene TBX2 (T-BOX2) interacts with EGR1 to co-repress EGR1-target genes including the breast tumour suppressor NDRG1. Here, we show the mechanistic basis of this TBX2 repression complex. We show that siRNA knockdown of TBX2, EGR1, Heterochromatin Protein 1 (HP1) isoforms and the generic HP1-associated corepressor protein KAP1 all resulted in growth inhibition of TBX2-expressing breast cancer cells. We show that TBX2 interacts with HP1 through a conserved HP1-binding motif in its N-terminus, which in turn leads to the recruitment of KAP1 and other associated proteins. Mutation of the TBX2 HP1 binding domain abrogates the TBX2-HP1 interaction and loss of repression of target genes such as NDRG1. Chromatin-immunoprecipitation (ChIP) assays showed that TBX2 establishes a repressive chromatin mark, specifically H3K9me3, around the NDRG1 proximal promoter coincident with the recruitment of the DNA methyltransferase DNMT3B and histone methyltransferase (HMT) complex components (G9A, Enhancer of Zeste 2 (EZH2) and Suppressor of Zeste 12 (SUZ12)). Knockdown of G9A, EZH2 or SUZ12 resulted in upregulation of TBX2/EGR1 co-regulated targets accompanied by a dramatic inhibition of cell proliferation. We show that a generic inhibitor of HMT activity, DzNep, phenocopies expression of an inducible dominant negative TBX2. Knockdown of TBX2, KAP1 or HP1 inhibited NDRG1 promoter decoration specifically with the H3K9me3 repression mark. Correspondingly, treatment with a G9A inhibitor effectively reversed TBX2 repression of NDRG1 and synergistically downregulated cell proliferation following TBX2 functional inhibition. These data demonstrate that TBX2 promotes suppression of normal growth control mechanisms through recruitment of a large repression complex to EGR1-responsive promoters leading to the uncontrolled proliferation of breast cancer cells.

Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes.

The human and rat genes for a fifth muscarinic receptor have been cloned and expressed in mammalian cells. The 532 amino acid human protein has 89% sequence identity to the 531 amino acid rat protein and is most closely related to the m3 receptor. Both proteins are encoded by single exons. The receptor has intermediate affinity for pirenzepine and low affinity for AF-DX 116, and it increases metabolism of phosphatidylinositol when stimulated with carbachol. Expression of mRNA has yet to be observed in brain or selected peripheral tissues, suggesting that either it is substantially less abundant than m1-m4 or its distribution is quite different.

Bradykinin excites rat sympathetic neurons by inhibition of M current through a mechanism involving B2 receptors and G alpha q/11.

Bradykinin (BK) is a peptide mediator released in inflammation that potently excites sympathetic neurons. We have studied the mechanism of this excitation in dissociated rat sympathetic neurons and found that at low nanomolar (EC50 = 0.9 nM) concentrations, BK inhibited the M-type K+ current IK(M). Studies with the selective antagonist Hoe140 revealed that this effect was mediated via the B2 receptor subtype, and mRNA encoding this receptor was identified in these neurons by RT-PCR. IK(M) inhibition was unaffected by Pertussis toxin or microinjection of antibodies to G alpha o but was selectively inhibited by microinjection of antibodies to G alpha q/11. Thus, BK is the most potent M current inhibitor yet described in mammalian neurons, and BK inhibition of M current is mediated by a G protein pathway similar to that activated by muscarinic acetylcholine receptors.

The peripheral cannabinoid receptor knockout mice: an update.

This review gives an overview of the CB2 receptor (CB2R) knockout (CB2R-/-) mice phenotype and the work that has been carried out using this mutant mouse. Using the CB2R-/- mice, investigators have discovered the involvement of CB2R on immune cell function and development, infection, embryonic development, bone loss, liver disorders, pain, autoimmune inflammation, allergic dermatitis, atherosclerosis, apoptosis and chemotaxis. Using the CB2R-/- mice, investigators have also found that this receptor is not involved in cannabinoid-induced hypotension. In addition, the CB2R-/- mice have been used to determine specific tissue CB2R expression. The specificity of synthetic cannabinoid agonists, antagonists and anti-CB2R antibodies has been screened using tissues from CB2R-/- mice. Thus, the use of this mouse model has greatly helped reveal the diverse events involving the CB2R, and has aided in drug and antibody screening.