Inhibition of m3 muscarinic acetylcholine receptors by local anaesthetics

1. Muscarinic m1 receptors are inhibited by local anaesthetics (LA) at nM concentrations. To elucidate in more detail the site(s) of LA interaction, we compared these findings with LA effects on m3 muscarinic receptors. 2. We expressed receptors in Xenopus oocytes. Using two-electrode voltage clamp, we measured the effects of lidocaine, QX314 (permanently charged) and benzocaine (permanently uncharged) on Ca(2+)-activated Cl(-)-currents (I(Cl(Ca))), elicited by acetyl-beta-methylcholine bromide (MCh). We also characterized the interaction of lidocaine with [(3)H]-quinuclydinyl benzylate ([(3)H]-QNB) binding to m3 receptors. Antisense-injection was used to determine the role of specific G-protein alpha subunits in mediating the inhibitory effects of LA. Using chimeric receptor constructs we investigated which domains of the muscarinic receptors contribute to the binding site for LA. 3. Lidocaine inhibited m3-signalling in a concentration-dependent, reversible, non-competitive manner with an IC(50) of 370 nM, approximately 21 fold higher than the IC(50) (18 nM) reported for m1 receptors. Intracellular inhibition of both signalling pathways by LA was similar, and dependent on the G(q)- protein alpha subunit. In contrast to results reported for the m1 receptor, the m3 receptor lacks the major extracellular binding site for charged LA. The N-terminus and third extracellular loop of the m1 muscarinic receptor molecule were identified as requirements to obtain extracellular inhibition by charged LA.     

Genetic perspectives on the serotonin transporter

The serotonin transporter (5-HTT) is most well known as the site of action of the serotonin reuptake inhibitors, which were initially developed as antidepressants, but now are the most widely used agents in the treatment of many additional neuropsychiatric and related disorders. The discovery that the gene that expresses the 5-HTT possesses a functional promoter-region polymorphism, which is associated with temperament and personality traits such as anxiety and negative emotionality as well as some behaviors, led to many studies examining this polymorphism in individuals with different neuropsychiatric disorders. The subsequent development of mice with a targeted disruption of the 5-HTT in our laboratory has provided an experimental model to examine the many consequences of diminished (in +/-, heterozygote mice) or absent (in -/-, homozygote knockout mice) function of the 5-HTT. The 5-HTT-deficient mice were also crossed with other knockout mice, allowing the study of multiple neurobiologic dysfunctions. As multiple genes are probably involved in the expression of complex behaviors such as anxiety, as well as neuropsychiatric disorders, these more genetically complex mice may more closely model disorders with complex etiologies. Thus, the combination of these comparative human and mouse studies may extend the opportunities to examine genetic alterations from a novel "bottom-up" approach [gene knockout or partial gene knockout in a combinational gene x gene x (yet unknown) gene approach], which is complementary to the traditional "top-down" genetic approach based upon studies of individuals with diagnosed neuropsychiatric disorders and their family members.     

Unnecessary emergency caesarean section due to silent CTG during anaesthesia?

We present a case of a probably unnecessary Caesarean sectiondue to misinterpretation of the cardiotocography (CTG) traceduring general anaesthesia. A 27-yr-old patient in her 30thweek of an uneventful, normal first pregnancy presented witha deep venous thrombosis in the pelvic region. She was to undergoan emergency thrombectomy under general anaesthesia. Duringthe operation, the CTG showed a lack of beat-to-beat heart ratevariation (silent pattern CTG) with normal fetal heart rate.This silent CTG pattern was probably a result of the effectof general anaesthesia on the fetus. The CTG pattern was interpretedas indicating fetal distress, and an emergency Caesarean sectionwas performed after the thrombectomy. The infant was apnoeicand had to be resuscitated and admitted to the neonatal intensivecare unit. The pH at delivery was 7.23 and the baby was extubated2 days later. Mother and child recovered without short-termsequelae. In the absence of alternative explanations, reducedfetal beat-to-beat variability with a normal baseline heartrate during general anaesthesia is probably normal. Br J Anaesth 2001; 87: 791–3     

Merkel cell carcinoma of the eyelid: histological and immunohistochemical features with special respect to differential diagnosis

· Background: Merkel cell carcinomas (MCC) not infrequently involve the periorbital region and the eyelids. Clinically, they are relatively characteristic but often unsuspected. Histologically, MCC are often misdiagnosed as lymphoma, melanoma, or metastatic small cell carcinoma of the lung (SCCL). · Methods: We present clinical, histological, and immunohistochemical data on six eyelid cases (all females; age 63–102 years; one with concomitant CLL) from our files of 77 MCC with special respect to differential diagnosis. For comparison, 22 SCCL were analyzed. Immunohistochemistry was done with antibodies against pan-cytokeratin (pan-CK), cytokeratin-20 (CK-20), neurofilament protein (NF), neuron-specific enolase (NSE), chromogranin (CHR), and S100 protein (S100). · Results: Morphologically, five of six MCC were prototypic, one was of the small cell variant. Immunohistochemically, dot-like positivities for pan-CK and CK-20 were seen in all six MCC, and for NF in five tumors. None of the 22 SCCL stained positively for CK-20 or NF but 21/22 cases were positive for pan-CK. Only 1/21 SCCL showed dot-like patterns for pan-CK; 20/21 reacted diffusely. All MCC and 13/22 SCCL displayed CHR-positive cells. All MCC and all SCCL were positive for NSE and negative for S100. · Conclusions: Dot-like positivities for CK-20 or NF are important to prove MCC and to exclude SCCL in clinically and morphologically doubtful cases. Dot-like positivities for pan-CK favor MCC, but do not always exclude SCCL. NSE and CHR are of no value for the differential diagnosis of MCC and SCCL. Melanoma and lymphoma are ruled out by negativity for S100 and pan-CK, respectively. Received: 8 July 1997 Revised version received: 10 December 1997 Accepted: 17 December 1997     

Characterization of EBV-carrying B-cell populations in healthy seropositive individuals with regard to density, release of transforming virus and spontaneous outgrowth

Peripheral or tonsil lymphocyte populations of EBV-seropositive donors give rise to EBV-carrying LCLs upon in vitro explantation. Such lines can arise either by a 2-step mechanism, namely release of virus from some of the explanted cells followed by infection of previously uninfected B cells, or by direct outgrowth of virus-harboring B cells (Rickinson et al., 1974; Dalens et al., 1975; Hinuma and Katsuki 1978; Katsuki et al., 1979). We observed that cells responsible for both the 2-step mechanism and for direct outgrowth are found in the purified B-cell compartment. Virus release was more frequent than direct outgrowth. The majority of virus-releasing cells were found in the low-density fraction that contains large, activated B blasts. Cells that were capable of spontaneous outgrowth in the presence of the viral inhibitor PFA and of virus-neutralizing antibody gave rise to cell lines that carried the sex chromosome marker of the original donor, rather than that of admixed cord blood lymphocyte of the opposite sex. Such cells were found in both the low- and the high-density fractions. The majority of the EBV-carrying B cells in vivo are thus low-density blasts. Rare small B cells of high density harboring EBV were capable of spontaneous outgrowth. This may be indicative of a host control mechanism that is removed upon cultivation in vitro.     

The influence of the lower patellar pole in the pathogenesis of chronic patellar tendinopathy

Resection of the lower patellar pole provides good results in the treatment of jumper’s knee. Therefore we hypothesized that the length of the lower patellar pole is increased in patients with chronic patellar tendinopathy. Cohort study, level of evidence 2. Between 2000 and 2005, 25 patients with chronic patellar tendinopathy underwent conservative and surgical treatment in our clinic. All of them had preoperative MRI were three independent examiners measured the Caton Index, the length and the ratio of the articular and non-articular patellar surface, tendon length and thickness and the thickness and length of the hypodens lesions in the patellar tendon. The measurements were compared with 50 MRI of a control group with no clinical patellofemoral disorders or patellar tendinopathy. Significant changes in tendon thickness (9.42 ± 2.87 vs. 4.88 ± 1.13; P < 0.0001), a longer non-articular surface of the patella (10.62 ± 2.86 vs. 7.098 ± 2.53; P < 0.0001) and significant higher ratio between the articular and the non-articular patellar surface (0.32 vs. 0.24; P < 0.0001) were found in the jumper’s knee group. No significant changes were seen in the length of the articular surface or the Caton Index. The development of chronic patellar tendinopathy in athletes might be associated with a longer lower patellar pole as patients with jumper’s knee showed a longer non-articular patellar surface compared with the control group.     

Esophageal stenosis in childhood: dystrophic epidermolysis bullosa without skin blistering due to collagen VII mutations.

We report a 9-year-old girl who experienced recurrent dysphagia since infancy. Crohn's disease was suspected because she had aphthous ulcers of the mouth and anal dermatitis with hematochezia. After bougienages of esophageal stenoses and medication for inflammatory bowel disease proved unsuccessful, interdisciplinary re-examination revealed the cause of the symptoms to be an extracutaneous form of dystrophic epidermolysis bullosa, a genetic skin fragility disorder. Dystrophic epidermolysis bullosa is caused by mutations in the COL7A1 gene encoding collagen VII, a protein of the epidermal attachment complex, and typically manifests with trauma-induced skin blistering, scarring, nail dystrophy, and, in some cases, mucosal involvement. The present proband never developed skin blisters but had nail dystrophy and erosions of the oral, esophageal, and genitoanal mucosa, which healed with slight scarring. Mutation analysis disclosed compound heterozygosity for recessive mutations in the COL7A1 gene. The paternal mutation 425 A-->G caused abnormal splicing resulting in a premature stop codon. The maternal mutation G2775S led to the substitution of a glycine by a serine in the triple helical domain of collagen VII. This case shows that mucosal disease and esophageal strictures in childhood are not always acquired, but can also represent a genetic defect of dermal-epidermal adhesion, even in the absence of skin blistering.     

Magnetic field inhibition of morphine-induced analgesia and behavioral activity in mice: Evidence for involvement of calcium ions

An exposure for 60 min to a 0.5 Hz rotating magnetic field (1.5-90 G) significantly reduced the day-time analgesic (in CF-1 mice) and locomotory (in C-57BL mice) effects of morphine (10 mg/kg). Intracerebroventricular (i.c.v.) injections of a calcium chelator, EGTA, blocked these effects, while administration of the calcium ionophore, A23187, potentiated the inhibitory actions. In a parallel fashion, i.c.v. administration of Ca2+ reduced, in a dose-related manner, the analgesic and locomotory effects of morphine in control CF-1 and C57 mice. These latter inhibitory effects could also be blocked by EGTA and augmented by A23187, indicating that opiate effects on activity and nociception are both sensitive to antagonism by calcium. Taken together these results suggest that exposure to magnetic stimuli may alter morphine-induced responses in mice, in a manner compatible and consistent with effects on Ca2+ and possibly other divalent ions.