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Alzheimer's disease (AD) is the most common form of dementia, an eversible, progressive disease that causes problems with memory, thinking, language, planning, and behavior. There are a number of risk factors associated with developing AD but the exact cause remains unknown. The predominant theory is that excessive build-up of amyloid protein leads to cell death, brain atrophy, and cognitive and functional decline. However, the amyloid hypothesis has not led to a single successful treatment. The recent failure of Solanezumab, a monoclonal antibody to amyloid, in a large phase III trial was emblematic of the repeated failure of anti-amyloid therapeutics. New disease targets are urgently needed. The innate immune system is increasingly being implicated in the pathology of number of chronic diseases. This focused review will summarize the role of transcription factor nuclear factor-kappa B (NF-κB), a key regulator of innate immunity, in the major genetic and environmental risk factors in cellular, invertebrate and vertebrate models of AD. The paper will also explore the relationship between NF-κB and emerging environmental risk factors in an attempt to assess the potential for this transcription factor to be targeted for disease prevention.

Key Points: A reduction in Kindlin-2 levels in endothelial cells compromises vascular barrier function. Kindlin-2 is a previously unrecognized component of endothelial adherens junctions. By interacting directly and simultaneously with β- or γ-catenin and cortical actin filaments, Kindlin-2 stabilizes adherens junctions. The Kindlin-2 binding sites for β- and γ-catenin reside within its F1 and F3 subdomains. Although Kindlin-2 does not associate directly with tight junctions, its downregulation also destabilizes these junctions. Thus, impairment of both adherens and tight junctions may contribute to enhanced leakiness of vasculature in Kindlin-2 +/- mice., Abstract: Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischaemia or sepsis and cause severe organ dysfunction. Kindlin-2, which is primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2 +/- mice aortic endothelial cells (MAECs) from these mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and platelet-activating factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted the Kindlin-2 +/- MAECs barrier to BSA and dextran and reduced transendothelial resistance compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with vascular endothelial cadherin-based complexes, including β- and γ-catenin and actin, components of adherens junctions (AJ). Direct interaction of Kindlin-2 with β- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The β- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2 but not the integrin binding site in F3. These results establish a previously unrecognized and vital role of Kindlin-2 with respect to maintaining the vascular barrier by linking Vascuar endothelial cadherin-based complexes to cortical actin and thereby stabilizing AJ., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

The postnatal period appears to be associated with higher rates of adjustment disorder, generalised anxiety disorder, and depression. Women who have a history of serious mental illness are at higher risk of developing a postpartum relapse, even if they have been well during pregnancy. Psychiatric causes of maternal death are more common than some direct causes of death. UK rates increased from 13/100,000 in 2006-2008 to 16/100,000 in 2010-2012, higher than, for example, mortality caused by haemorrhage or anaesthetic complications of childbirth. Postnatal depression is more severe than baby blues, follows a chronic course and may relapse outside the perinatal period. Although 13% of patients already have depression in pregnancy, the majority tend to be diagnosed after delivery; up to 19% from childbirth to three months postpartum. NICE recommends using the Two Question Depression Screen and the Generalized Anxiety Disorder scale from the booking visit through to one year postpartum. A positive response to depression or anxiety questions warrants a full assessment using either PHQ-9 or the Edinburgh Postnatal Depression Scale. Bipolar disorder may present as a first depressive episode in pregnancy or the postnatal period. In the postpartum period women have a high risk of severe relapse. Postpartum psychosis has a sudden and dramatic presentation with delusions, mania, severe depression, or mixed episodes with wide fluctuations of symptoms and severe mood swings.

This article describes and contrasts the public health response to two human rabies cases: one organ recipient diagnosed within days of symptom onset and the transplant donor who was diagnosed 18 months post-symptom onset. In response to an organ-transplant-related rabies case diagnosed in 2013, organ donor and recipient investigations were conducted by multiple public health agencies. Persons with potential exposure to infectious patient materials were assessed for rabies virus exposure. An exposure investigation was conducted to determine the source of the organ donor's infection. Over 100 persons from more than 20 agencies spent over 2700 h conducting contact investigations in healthcare, military and community settings. The 564 persons assessed include 417 healthcare workers [5.8% recommended for post-exposure prophylaxis (PEP)], 96 community contacts (15.6% recommended for PEP), 30 autopsy personnel (50% recommended for PEP), and 21 other persons (4.8% recommended for PEP). Donor contacts represented 188 assessed with 20.2% recommended for PEP, compared with 5.6% of 306 recipient contacts recommended for PEP. Human rabies cases result in substantial use of public health and medical resources, especially when diagnosis is delayed. Although rare, clinicians should consider rabies in cases of encephalitis of unexplained aetiology, particularly for cases that may result in organ donation., (Published 2014. This article is a U.S. Government work and is in the public domain in the USA.)

Mutations in Kir2.1 inwardly rectifying potassium channels are associated with Andersen syndrome, a disease characterized by potentially fatal cardiac arrhythmias. While several Andersen-associated mutations affect membrane expression, the cytoplasmic signals that regulate Kir2.1 trafficking are poorly understood. Here, we investigated whether the Rho-family of small GTPases regulates trafficking of Kir2.1 channels expressed in HEK-293 cells. Treatment with Clostridium difficile toxin B, an inhibitor of Rho-family GTPases, or co-expression of the dominant-negative mutant of Rac1 (Rac1(DN)) increased Kir2.1 channels approximately 2-fold. However, the dominant-negative forms of other Rho-family GTPases, RhoA or Cdc42, did not alter Kir2.1 currents, suggesting a selective effect of Rac1 on Kir2.1 channels. Single-channel properties (gamma, tau(o), tau(c)) and total protein levels of Kir2.1 were unchanged with co-expression of Rac1(DN); however, studies using TIRF microscopy and CFP-tagged Kir2.1 revealed increased channel surface expression. Immunohistochemical detection of extracellularly tagged HA-Kir2.1 channels showed that Rac1(DN) reduced channel internalization when co-expressed. Finally, the dominant-negative mutant of dynamin, which interferes with endocytosis, occluded the Rac1(DN)-induced potentiation of Kir2.1 currents. These data suggest that inhibition of Rac1 increases Kir2.1 surface expression by interfering with endocytosis, likely via a dynamin-dependent pathway. Surprisingly, Rac1(DN) did not alter Kir2.2 current density or internalization, suggesting subunit specific modulation of Kir2.1 channels. Consistent with this, construction of Kir2.1/2.2 chimeras implicated the C-terminal domain of Kir2.1 in mediating the potentiating effect of Rac1(DN). This novel pathway for regulating surface expression of cardiac Kir2.1 channels could have implications for normal and diseased cardiac states., ((c) 2008 Wiley-Liss, Inc.)

Olfactory learning in humans leads to enhanced perceptual discrimination of odor cues. Examining mouse models of both aversive and appetitive conditioning, we demonstrate a mechanism which may underlie this adult learning phenomenon. Topographically unique spatial wiring of the olfactory system allowed us to demonstrate that emotional learning of odor cues alters the primary sensory representation within the nose and brain of adult mice. Transgenic mice labeled at the M71 odorant receptor (specifically activated by the odorant acetophenone) were behaviorally trained with olfactory-dependent fear conditioning or conditioned place preference using acetophenone. Odor-trained mice had larger M71-specific glomeruli and an increase in M71-specific sensory neurons within the nose compared with mice that were untrained, trained to a non-M71 activating odorant, or had nonassociative pairings of acetophenone. These data indicate that the primary sensory neuron population and its projections may remain plastic in adults, providing a structural mechanism for learning-enhanced olfactory sensitivity and discrimination.

We examined brain-derived neurotrophic factor (BDNF) mRNA expression across the olfactory system following fear conditioning. Mice received 10 pairings of odor with footshock or equivalent unpaired odors and shocks. We found increased BDNF mRNA in animals receiving paired footshocks in the multiple regions examined including the posterior piriform cortex (PPC) and basolateral amygdala (BLA). This was in contrast to the unpaired and odor-alone treatments, where BDNF mRNA was increased in the olfactory bulb (OB) and anterior piriform cortex (APC) only, but not the higher olfactory areas. We propose that odor exposure increases expression of BDNF in the OB and APC while the PPC and BLA increase BDNF mRNA only when associative learning occurs.

Studies on olfactory receptor (OR) pharmacology have been hindered by the poor plasma membrane localization of most ORs in heterologous cells. We previously reported that association with the beta(2)-adrenergic receptor (beta(2)-AR) facilitates functional expression of the OR M71 at the plasma membrane of HEK-293 cells. In the present study, we examined the specificity of M71 interactions with other G protein-coupled receptors (GPCRs). M71 was co-expressed in HEK-293 cells with 42 distinct GPCRs, and the vast majority of these receptors had no significant effect on M71 surface expression. However, co-expression with three subtypes of purinergic receptor (P2Y(1)R, P2Y(2)R, and A(2A)R) resulted in markedly enhanced plasma membrane localization of M71. Agonist stimulation of M71 co-expressed with P2Y(1)R and P2Y(2)R activated the mitogen-activated protein kinase pathway via coupling of M71 to Galpha(o). We also examined the ability of beta(2)-AR, P2Y(1)R, P2Y(2)R, and A(2A)Rto interact with and regulate ORs beyond M71. We found that co-expression of beta(2)-AR or the purinergic receptors enhanced the surface expression for an M71 subfamily member but not for several other ORs from different subfamilies. In addition, through chimeric receptor studies, we determined that the second transmembrane domain of beta(2)-AR is necessary for beta(2)-AR facilitation of M71 plasma membrane localization. These studies shed light on the specificity of OR interactions with other GPCRs and the mechanisms governing olfactory receptor trafficking.

Inwardly rectifying potassium channels Kir2.1-Kir2.3 are important regulators of membrane potential and, thus, control cellular excitability. However, little is known about the regulation of these channels. Therefore, we studied the mechanisms mediating the regulation of Kir2.1-Kir2.3 by the G-protein-coupled m1 muscarinic receptor using the whole-cell patch-clamp technique and recombinant expression in the tsA201 mammalian cell line. Stimulation of the m1 muscarinic receptor inhibited all subtypes of inward rectifier tested, Kir2.1-Kir2.3. The inhibition of each channel subtype was reversible and was attenuated by the muscarinic receptor antagonist, atropine. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) mimicked the effects of m1 receptor activation by inhibiting Kir2.1 currents. However, PMA had no effect on Kir2.2 or Kir2.3. Inclusion of 200-microM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) in the patch pipette solution prevented the effects of m1 muscarinic receptor stimulation on all three of the channel subtypes tested, confirming the mediation of the responses by G-proteins. Cotransfection with the activated mutant of the small GTPase Rho reduced current density, while C3 exoenzyme, a selective inhibitor of Rho, attenuated the m1 muscarinic receptor-induced inhibition of Kir2.1-Kir2.3. Also, buffering the intracellular calcium concentration with a high concentration of EGTA abolished the m1 receptor-induced inhibition of Kir2.1-Kir2.3, implicating a role for calcium in these responses. These results indicate that all three of the Kir2 channels are similarly inhibited by m1 muscarinic receptor stimulation through calcium-dependent activation of the small GTPase Rho.

Hyperthyroidism has been associated with atrial fibrillation (AF); however, hyperthyroidism-induced ion channel changes that may predispose to AF have not been fully elucidated. To understand the electrophysiological changes that occur in left and right atria with hyperthyroidism, the patch-clamp technique was used to compare action potential duration (APD) and whole cell currents in myocytes from left and right atria from both control and hyperthyroid mice. Additionally, RNase protection assays and immunoblotting were performed to evaluate the mRNA and protein expression levels of K(+) channel alpha-subunits in left and right atria. The results showed that 1) in control mice, the APD was shorter and the ultra-rapid delayed rectifier K(+) conductance (I(Kur)) and the sustained delayed rectifier K(+) conductance (I(ss)) were larger in the left than in the right atrium; also, mRNA and protein expression levels of Kv1.5 and Kv2.1 were higher in the left atrium; 2) in hyperthyroid mice, the APD was shortened and I(Kur) and I(ss) were increased in both left and right atrial myocytes, and the protein expression levels of Kv1.5 and Kv2.1 were increased significantly in both atria; and 3) the influence of hyperthyroidism on APD and delayed rectifier K(+) currents was more prominent in right than in left atrium, which minimized the interatrial APD difference. In conclusion, hyperthyroidism resulted in more significant APD shortening and greater delayed rectifier K(+) current increases in the right vs. the left atrium, which can contribute to the propensity for atrial arrhythmia in hyperthyroid heart.

This study demonstrates that mice display olfactory-cued fear as measured with both freezing and fear-potentiated startle. Following a preconditioning test to measure any unconditioned responses to odor, mice received 5 pairings of a 10-s odor with a 0.25-s, 0.4-mA footshock. The next day, startle and freezing were measured in the presence and absence of the odor. Both fear measures increased after training with amyl acetate (Experiment 1) and acetophenone (Experiment 2). The enhancement of startle did not occur when the same number of odors and shocks were presented in an unpaired fashion (Experiment 3). Furthermore, mice were able to discriminate between an odor paired with shock and a nonreinforced odor (Experiment 4)., (Copyright 2005 APA.)

Olfactory receptors (ORs) comprise more than half of the large class I G protein-coupled receptor (GPCR) superfamily. Although cloned over a decade ago, little is known about their properties because wild-type ORs do not efficiently reach the cell surface following heterologous expression. Receptor-receptor interactions strongly influence surface trafficking of other GPCRs, and we examined whether a similar mechanism might be involved in OR surface expression. Olfactory neurons are known to express beta-adrenergic receptors (ARs), and we found that coexpression with beta(2)-ARs, but not any other AR subtypes, dramatically increased mouse 71 (M71) OR surface expression in human embryonic kidney 293 cells. A persistent physical interaction between M71 ORs and beta(2)-ARs was shown by coimmunoprecipitation and by cointernalization of the two receptors in response to their specific ligands. Also, coexpression of wild-type M71 ORs with beta(2)-ARs resulted in cAMP responses to the M71 ligand acetophenone. Finally, in situ hybridization studies showed extensive colocalization of M71 OR and beta(2)-AR expression in mouse olfactory epithelium. These data demonstrate the successful heterologous surface expression of a functional wild-type OR and reveal that persistent physical association with other GPCRs can control OR surface expression.

The inwardly rectifying potassium channel Kir2.1 is inhibited by a variety of G-protein-coupled receptors (GPCRs). However, the mechanisms underlying the inhibition have not been fully elucidated. In this study the role of the small GTPase, Rho, in mediating this inhibition was determined. Stimulation of the m1 muscarinic receptor inhibited Kir2.1, when both receptor and channel were coexpressed in tsA201 cells. The inhibition of Kir2.1 by carbachol was reversible and atropine-sensitive. Cotransfection with a dominant-negative mutant of the small GTPase Rho abolished the inhibition of Kir2.1 with current amplitudes remaining at control levels in the presence of carbachol. Conversely, cotransfection with the constitutively activated mutant of Rho resulted in a reduction in basal Kir2.1 current amplitudes, suggesting that Rho inhibits Kir2.1. To further confirm the involvement of Rho in the signal transduction pathway, cotransfection with C3 transferase (EFC3), a selective inhibitor of Rho, abolished the reduction in Kir2.1 currents noted upon application of carbachol under control conditions. Preincubation with the phosphatidylinositol 3-kinase inhibitor wortmannin or the Rho kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2 HCl (Y-27632) had no effect on agonist-induced inhibition of Kir2.1, precluding these kinases as downstream effectors of Rho in mediation of the signal. In addition, 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of mitogen-activated protein (MAP) kinase kinase (MEK), had no effect on the m1 receptor-induced inhibition of Kir2.1, suggesting that MAP kinases are not involved in the signaling pathway. In conclusion, these data indicate that the small GTPase, Rho, transduces the m1 muscarinic receptor-induced inhibition of Kir2.1 via an unidentified mechanism.

Fluorescence resonance energy transfer (FRET) was used to reveal aspects of the mechanism of signal transduction by epidermal growth factor receptors (EGFR). The superpositions of epidermal growth factor (EGF), transforming growth factor-alpha (TGFalpha) and an antibody fragment (29.1) to the carbohydrate extremity of the receptor's ectodomain as measured by FRET, show that 14% of EGFRs in A431 cells are oligomerized before growth factor binding. After binding growth factor and signaling, these oligomers dissociate before releasing growth factor. Time courses of the FRET-derived distances between constitutively oligomerized EGFRs during signal transduction show a transient structural change in the extracellular domain, which occurs simultaneously with the production of intracellular Ca2+ signals. The FRET measurements also show a slow increase in oligomerization of EGFR monomers after growth factor binding. The structural change found in the extracellular domain of oligomeric EGFRs is similar to that shown by others for EPO, Neu, Fas, and tumor necrosis factor receptors, and may therefore be a common property of the transduction of the receptor-mediated signals.

Dendritic cells (DCs) are key antigen-presenting cells (APCs) that act as central modulators of cellular immune responses. Genetic modification of DCs has considerable therapeutic potential in the treatment of a wide spectrum of diseases, including cancer and persistent viral infection. In this report, we show that pre-treatment of DCs with a recombinant adenovirus encoding the major adenovirus receptor, Coxsackie B and adenovirus receptor (CAR), significantly increased the uptake of recombinant adenoviruses (Ads) by primary immature monocyte-derived DCs. This could be correlated with CAR mRNA and surface protein expression. Transduction of DCs by recombinant adenoviruses did not significantly alter cellular viability. Therefore, we propose that pre-treatment of DCs with Ad5-CAR is one strategy to increase the susceptibility of DCs to transduction by recombinant Ads.

Unlabelled: Pregnancy is associated with immunological, endocrine, metabolic and vascular changes that may adversely affect the skin. The specific dermatoses of pregnancy are disease entities almost exclusively related to the pregnancy or the puerperium., Learning Objective: At the conclusion of this learning activity, participants should be aware of the main entities of the specific pregnancy dermatoses, their clinical presentations, the main diagnostic criteria and therapeutic options.

The inwardly rectifying potassium channel IRK1, a member of the Kir 2.0 family, is inhibited by m1 muscarinic receptor stimulation. In this study the mechanism of action underlying the muscarinic response was investigated by identification of the subtype of heterotrimeric G-protein involved in transduction of the signal. tsA201 cells were simultaneously transfected with cDNAs encoding IRK1, m1 and the Galpha subunit of either G(q), G(12) or G(13). The whole-cell patch-clamp technique was used to study the effects of G-protein transfection. Antibodies generated against the C-terminal regions of Galpha(q/11) and Galpha(12) were used to confirm G-protein expression by Western blot. When challenged with carbachol, IRK1 currents recorded from cells co-transfected with Galpha(q) were potently inhibited compared with controls. Conversely, co-transfection with Galpha(12) or Galpha(13) subunits had no effect on muscarinic-receptor-induced inhibition of IRK1. Concentration response curves revealed that carbachol was 16 times more potent at inhibiting IRK1 currents in cells co-transfected with Galpha(q) as compared with Galpha(12) co-transfected cells. Immunoblotting illustrated low levels of endogenous Galpha(q/11) and Galpha(12) in untransfected tsA cells. Transfection with Galpha(q) or Galpha(12) cDNAs greatly increased the levels of G-protein expression in both cell populations. G-protein expression did not interfere with m1 muscarinic receptor expression levels. These findings suggest that the m1 muscarinic-receptor-induced inhibition of IRK1 is mediated by the heterotrimeric G-protein, Galpha(q), in tsA cells.

The modulation of a transient T-type calcium current by the five muscarinic receptor subtypes, stably expressed in NIH 3T3 cells, was studied with the whole-cell patch-clamp technique. Voltage-step depolarizations applied to the NIH 3T3 cells revealed a low-voltage-activated (LVA) T-type calcium current that was inhibited by Ni2+ and unaffected by omega-conotoxin GVIA. In cells transfected with the m3 and m5 muscarinic receptors, application of acetylcholine (ACh) resulted in a pertussis-toxin-insensitive increase in peak T-type calcium current amplitude. The m3-induced atropine-sensitive increase in current amplitude was accompanied by a shift in the voltage dependence of activation to more hyperpolarized potentials. The increase in peak T-type calcium current amplitude and the shift in voltage dependence was mimicked by incubation with 500 microM 8-bromo-cAMP. Conversely, T-type calcium current amplitudes were reduced by incubation with 10 microM RpcAMPS, an inhibitor of cAMP-dependent protein kinase (PKA). Preincubation with 500 microM 8-bromo-cAMP or with 10 microM RpcAMPS abolished the increase in T-type calcium current amplitude previously noted on stimulation of the m3 muscarinic receptor by ACh. Application of ACh to NIH 3T3 cells stably transformed with the m1 muscarinic receptor resulted in no discernable change in T-type calcium current amplitude. However, on pre-incubation of the cells with calphostin C, an inhibitor of protein kinase C (PKC), application of ACh to the cells now resulted in a robust increase in T-type calcium current amplitude. Application of 500 nM PDBu, an activator of PKC, reduced the T-type calcium current amplitude. No significant changes in T-type calcium currents were observed on application of ACh to cells stably transfected with the m2 or m4 muscarinic receptors. However, after pre-incubation with forskolin, the m2 muscarinic receptor induced a decrease in T-type calcium current amplitude. Stimulation of the ml, m3 and m5 muscarinic receptors in the NIH 3T3 cell resulted in dose-dependent increases in the concentration of intracellular cAMP in comparison to control as determined by cAMP immunoassay. Conversely, stimulation of the m2 and m4 muscarinic receptors by carbachol resulted in a dose-dependent reduction in intracellular concentrations of cAMP, as compared with control basal levels. It is concluded that the m3 and m5 muscarinic receptors enhance T-type calcium channel activity. At least in the case of the m3 muscarinic receptor, the increased T-type channel activity appeared to be mediated via increased cAMP levels and subsequent activation of PKA. The lack of effect of the ml muscarinic receptor on the T-type calcium channel was probably due to the opposing actions of concomitant activation of both PKC and PKA. The physiological significance of these findings is discussed.

The modulation of a constitutively active IRK1-like inwardly rectifying potassium channel, that is endogenously expressed in the RBL-2H3 cell, was studied with the whole-cell patch-clamp technique. Activation of G-proteins by intracellular application of GTP gamma S revealed a dual modulation of the inward rectifier. An initial increase in inward current amplitude was induced by GTP gamma S, followed by a profound inhibition of the current. The stimulation of the inward rectifier by GTP gamma S was abolished by pretreatment with pertussis toxin. The inhibitory phase of the GTP gamma S-induced response was pertussis toxin-insensitive. Stimulation of the m1-muscarinic receptor expressed in the RBL cell after stable transfection, induced an inhibition of the inwardly rectifying currents. Application of protein kinase C activators such as phorbol 12-myristate 13-acetate and phorbol 12,13-dibutyrate, resulted in a strong inhibition of the currents. Application of the cAMP-dependent protein kinase activator 8-bromo cAMP also induced an inhibition of the inward rectifier. It is concluded that the inward rectifier of the RBL-2H3 cell may be inhibited both by activation of protein kinase C and by cAMP-dependent protein kinase. As this type of inward rectifier is widely expressed in the nervous system, these data imply that the channel can be inhibited by receptors that stimulate phospholipase C and/or stimulate adenylyl cyclase, and can be activated by receptors that inhibit adenylyl cyclase activity.

Modulation of L-type calcium channels by the five cloned muscarinic receptors was studied by expression of the receptors in NIH 3T3 cells. Application of acetylcholine (ACh) to cells transfected with m1-m5 resulted in a reduction in the L-type calcium current amplitude. Elevations in intracellular cAMP concentrations induced by 8-bromo-cAMP or forskolin resulted in no discernible change in the L-type calcium current. In addition, treatment with Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS), a protein kinase A (PKA) inhibitor, had no effect on the L-type currents. Conversely, application of phorbol dibutyrate, an activator of protein kinase C (PKC) or 8-bromo-cGMP, an activator of cGMP-dependent protein kinase (PKG), reduced the calcium currents. Incubation of the cells with KT5823, an inhibitor of PKG, resulted in a reduction of the response to 8-bromo-cGMP. The ACh-induced depression of L-type calcium current amplitude was sensitive to pertussis toxin (PTX) in cells transfected with the m2 or m4 receptor subtype. The m2-muscarinic-receptor-induced inhibition of the L-type calcium current was attenuated by preincubation of the cells with 8-bromo-cAMP and was unaffected by KT5823 or by calphostin C. The m1-muscarinic-receptor-induced inhibition of the L-type calcium conductance was insensitive to PTX treatment. However, the m1-induced response was blocked by preincubation of the cells with calphostin C. The present data indicate that the m2 (and possibly also the m4) muscarinic receptors inhibit the L-type calcium conductance by a reduction in cAMP concentration and that the m1 (and possibly also the m3 and m5) muscarinic receptors inhibit the L-type calcium channel via activation of PKC.

Modulation of the inwardly rectifying potassium channel (IRK1) by the m1 muscarinic receptor was studied with the whole-cell patch-clamp recording technique with the use of a mammalian expression system. After transfection with IRK1 and m1 muscarinic receptor genes, tsA cells expressed a cesium-sensitive inwardly rectifying potassium conductance that was reduced on application of the muscarinic receptor agonist carbachol. This reduction was reversible on washout of carbachol and could be completely inhibited by the muscarinic receptor antagonist atropine. Conversely, stimulation of the m2 muscarinic receptor, when coexpressed with IRK1, resulted in no change in IRK1 current amplitude. Phorbol-12, 13-dibutyrate, an activator of protein kinase c (PKC), mimicked the effect of m1 muscarinic receptor stimulation by inhibiting the IRK1 conductance. Preincubation with staurosporine or the specific PKC inhibitor calphostin C, before application of carbachol, fully prevented the inhibition of IRK1 by m1 muscarinic receptor stimulation. Administration of 8-bromo-cAMP, an activator of protein kinase A, and thapsigargin, a stimulator of intracellular calcium release, had no effect on IRK1, suggesting that these second messengers were not involved in the m1 muscarinic receptor-induced response. Therefore, the data indicate that the m1 muscarinic receptor inhibits IRK1, presumably via stimulation of PKC. As IRK1 is widely distributed throughout the central nervous system, it is possible that such an action on IRK1 underlies the inhibitory effects of muscarinic receptor stimulation on inwardly rectifying potassium conductances observed in the brain.

Objective: To investigate the effect of estrogen and progestogen on the resistance to blood flow in the uterine arteries of Turner's syndrome patients., Design: Prospective clinical study., Setting: A tertiary infertility clinic., Patients: Five Turner's syndrome patients, six patients who had surgical castration, and five patients with idiopathic primary ovarian failure., Interventions: The patients were treated with 2 mg E2 valerate to which 500 micrograms norgesterel was added for 10 days in a 28-day cycle. Transvaginal color Doppler was used to measure pulsatility index in the uterine arteries at eight regular intervals during a single cycle., Main Outcome Measure: Pulsatility index of the uterine arteries., Results: The administration of norgesterel to Turner's syndrome patients resulted in an increase in pulsatility index that was significantly higher than in patients who had surgical castration (confidence interval = 0.17 to 2.42)., Conclusion: The uterine arteries of Turner's syndrome patients are more sensitive to the tonic effect of progestogen. If manifest in cardiac arteries also this phenomenon may be partly responsible for the increased incidence of cardiovascular disease and shorter life expectancy in Turner's syndrome patients. To achieve optimal protection from cardiovascular disease, Turner's syndrome patients may benefit from hormone replacement treatment containing altered doses of estrogen and progestogen.

Spreading of Ca2+ signals in rat basophilic leukemia (RBL) cells occurs by release of ATP. Therefore we studied the effect of ATP on membrane currents. ATP (1-10 microM) activated large-conductance channels. Single channel events were resolved in the whole cell mode. Similar channel activity was observed in RBL cells transfected with the muscarinic M1 receptor after stimulation with carbachol as well as after intracellular infusion of aluminum fluoride. Activation was independent of internal Ca2+ (0-10 microM). The channels had a conductance of 250 pS in 135 mM Na+ and 70 pS in 100 mM Ca2+. The permeability (P) ratio was PCa/PNa/PCs/PMg = 16:1:0.6:0.6. These channels may contribute to secretory responses by allowing Ca2+ entry, leading to high Ca2+ concentrations in the vicinity of the channel pore.

Many couples with infertility will require a form of assisted reproduction to achieve a pregnancy. Assisted reproduction techniques, including intrauterine insemination, gamete intrafallopian transfer and in-vitro fertilisation, are considered in this review.

Activation of muscarinic receptors has been shown to inhibit L-type calcium conductances by mechanisms sensitive to pertussis toxin (PTX). In this study we show that agonist stimulation of the m4 muscarinic receptor leads to an increase in an L-type calcium conductance in the AtT-20 pituitary cell line, by a PTX-sensitive mechanism. The amplitude of the dihydropyridine (DHP)-sensitive or L-type calcium current was increased by acetylcholine (ACh), with no shift in the voltage dependence. This action of ACh was completely inhibited by PTX pre-treatment. Forskolin, cAMP and phorbol 12,13-dibutyrate reduced, while RpcAMPs, an inhibitor of cAMP-dependent protein kinase (PKA), increased the L-type calcium conductance. We propose that the m4 muscarinic receptor activates the L-type calcium channel by inhibition of adenylyl cyclase resulting in reduced cAMP levels and, hence, reduced PKA activity. This novel increase in calcium current via the m4 muscarinic receptor appears to reflect the coupling with an L-type channel of the D class, due to the sensitivity of the L-type calcium conductance to both DHPs and omega-conotoxin, and, thus, is distinct from the skeletal muscle and cardiac L-type channels of the C class previously studied.

This study demonstrates the stable expression of a functional ionotropic glutamate receptor in a mammalian cell line of non-neuronal origin. The kainate-selective glutamate receptor GluR6 was constitutively expressed under the control of a metallothionein promoter. Clones were isolated expressing approximately 3 pmol of receptor per mg of protein. Functionality of the recombinant GluR6 was demonstrated both by electrophysiology and by Ca2+ imaging. Application of kainate to the GluR6-transfected cells activated an inward current response at a holding potential of -60 mV. The kainate concentration needed to evoke 50% of the maximal response (EC50) was calculated to be 0.82 +/- 0.39 microM. The current-voltage relationship was found to be almost linear, with a reversal potential of -2.5 +/- 4.8 mV. Application of kainate also resulted in an increase in the intracellular Ca2+ concentration measured by Ca2+ imaging. The pharmacological profile of [3H]kainate binding to the recombinant GluR6 resembled the high-affinity [3H]kainate binding sites in rat brain, showing high affinity for domoate (Ki = 5.1 +/- 3.0 nM) and kainate (Kd = 12.9 +/- 2.4 nM). No decrease in GluR6 expression level was observed over > 75 passages of the transfected cells. When domoate, a slowly desensitizing GluR6 agonist, was included in the growth medium for 3 weeks, the number of GluR6 binding sites decreased by 30%, indicating the importance of complete channel closure for stable expression.

Ag-induced cross-linking of IgE bound to its high affinity receptor (Fc epsilon RI) at the surface of basophils or mast cells triggers a number of biochemical events culminating in the release of several inflammatory mediators. In rat basophilic leukemia (RBL-2H3) cells expressing the G protein-coupled m1 muscarinic receptor, Ag/IgE-induced cross-linking of Fc epsilon RI, calcium ionophore A23187, and carbachol through M1 receptors stimulated tyrosine phosphorylation of several proteins, including two of 42 and 44 kDa. Proteins of identical molecular masses were recognized by anti-MAP-kinase antibodies, and these immunoreactive proteins exhibited in part a slightly increased molecular mass on SDS polyacrylamide gels after incubation of cells with secretory stimuli. All stimuli led to the activation of MAP kinase, which co-purified on Mono Q chromatography with 42- and 44-kDa proteins, which were tyrosine phosphorylated in response to secretory stimuli and reacted with anti-(MAP kinase) antibodies. Finally, 42- and 44-kDa proteins immunoprecipitated by anti-MAP-kinase antibodies and anti-phosphotyrosine antibodies were recognized by anti-phosphotyrosine and anti-MAP-kinase antibodies, respectively. Primarily threonine and tyrosine residues were found to be phosphorylated in 42- and 44-kDa proteins immunoprecipitated from [32P]phosphate-labeled cells that had been treated with secretory stimuli. The dose dependence of secretagogue-induced MAP kinase activation correlated with that of increases in serotonin release from activated cells, and the maximum of MAP kinase activation coincided with the maximum rate of secretion. Down-regulation or inhibition of protein kinase C as well as incubation of cells with the tyrosine kinase inhibitor genistein markedly inhibited MAP kinase activation in parallel with serotonin release. Taken together, these findings demonstrate that 42- and 44-kDa MAP kinases are activated in response to secretory stimuli and provide some evidence for a functional link between MAP kinase activation and signaling events leading to mediator release in RBL cells.

Because of unresolved questions about the mechanism of Ag-stimulated Ca2+ influx, Ca2+ mobilization in response to carbachol and Ag was compared in transfected rat basophilic RBL-2H3(ml) cells that expressed both Fc epsilon and ml muscarinic receptors. Although the stimulants activated phospholipase C via different coupling mechanisms, a G protein for carbachol or a tyrosine kinase for Ag, they released Ca2+ from the same intracellular pool and used the same or very similar mechanisms for influx of Ca2+ as indicated by the similar patterns of inhibition of uptake of 45Ca2+ by various cations. With both stimulants, influx and sustained increases in free cytosolic Ca2+ ([Ca2+]i) were associated with relatively small increases in inositol 1,4,5-trisphosphate (IP3). Blockade of Ca2+ influx resulted in rapid decline in [Ca2+]i to basal levels; resumption of influx caused a substantial "spike" in [Ca2+]i before [Ca2+]i reequilibrated at the same former steady-state levels but without perturbing levels of IP3. Thus, the refilling and discharge of Ca2+ from IP3-sensitive stores might occur synchronously on resumption of influx, or asynchronously during sustained influx and elevation of [Ca2+]i. Together, the results suggested that influx of Ca2+ in response to stimulation via Fc epsilon receptors occurred through a pathway, analogous to that observed with other types of stimulants, in which Ca2+ influx follows emptying of intracellular Ca2+ stores by IP3. Also, secretion was highly dependent on this IP3-dependent pathway.

The discovery of five muscarinic receptor subtypes by molecular genetic techniques has resulted in new approaches to understanding their function. This involves the expression of the individual genes encoding each receptor subtype in isolation, such that their effects and mechanisms of action can be studied. The coupling of the receptors with G-proteins and ion channels is the subject of this review and emphasis is placed upon the assignment of genetically defined receptor subtypes with a given physiological function. Activation of inwardly rectifying potassium conductances by m2 and m4 and inhibition by m1, as well as stimulation of calcium-dependent conductances by m1, m3 and m5 are discussed.

Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The distributions of these subtypes and their potential physiological roles are discussed. By use of molecular genetic manipulation of cloned muscarinic receptor cDNAs, the regions of muscarinic receptors that specify G-protein coupling and ligand binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivities for different G-proteins and that multiple discontinuous epitopes contribute to their selectivities for different ligands. The residues that contribute to ligand binding and G-protein coupling are described, as well as the implied structures of these functional domains.

The modulation of an inwardly rectifying potassium conductance by muscarinic receptor stimulation was studied in the AtT-20 pituitary cell line, using the whole-cell patch-clamp technique. Only m4 mRNA was detected in these cells, thus, it is assumed that the actions of muscarinic receptor stimulation are mediated by the m4 receptor. AtT-20 cells express a slowly activating inwardly rectifying potassium conductance. Application of acetylcholine (ACh), resulted in an atropine sensitive, reversible increase in inwardly rectifying current. The ACh-induced current differed from the current recorded in control, in that it was fast activating, while the control current was slowly activating. Inclusion of GTP gamma S in the patch pipette activated an inward current with characteristics similar to the ACh-induced current, and the ACh-induced current response could be inhibited by pre-incubation with pertussis toxin (PTX). It is concluded that the m4 muscarinic receptor is coupled to an inwardly rectifying potassium conductance via a PTX sensitive G-protein.

The possibility that the ml muscarinic receptor subtype can induce release of intracellular granules and transmitters was studied by transfecting a cultured mast cell line. RBL-2H3 cells, with the ml receptor gene. Comparisons were made between carbachol- and antigen-induced activation of various secretory responses. Like antigen, carbachol stimulated inositol phospholipid hydrolysis and release of arachidonic acid with concomitant dose-dependent secretion of granular contents. Carbachol also stimulated a biphasic increase in intracellular calcium, as measured by single cell fura-2 measurements. Although the kinetics of the carbachol-induced rise in intracellular calcium differed from that induced by antigen, they both utilized the same intracellular pool of calcium, and the second phase of the rise in intracellular calcium was dependent on extracellular calcium in both cases. Thus, the ml muscarinic receptor activates release of granules by a mechanism ostensibly similar to that of antigen.

Electrophysiological recordings of responses to muscarinic receptor stimulation in cultured embryonic hippocampal neurons have been largely unsuccessful to date. In this study muscarinic receptor binding was demonstrated in 2-week-old embryonic rat hippocampal cultures. Using whole-cell patch-clamp recording we found that 1-5 microM carbachol produced multiple effects including depolarization, increased action potential firing rate, increased synaptic activity and a reduction in the amplitude of medium-duration afterhyperpolarizations. Voltage-clamp analysis revealed a time-dependent current relaxation with hyperpolarizing steps from a holding potential of about -40 mV which was inhibited by 10 microM muscarine or 50 microM carbachol and had characteristics similar to those of the m-current. Both atropine and pirenzepine inhibited all of these effects indicating that these cholinergic actions were mediated by muscarinic receptors. This study shows that muscarinic responses obtained classically in hippocampal brain slices can also be produced in cultured hippocampus.

The physiological responses to activation of the m5 muscarinic acetylcholine receptor were compared with those of m3 and m4 in transformed Chinese hamster ovary cells, using patch-clamp electrophysiological and biochemical techniques. Stimulation of the m5 receptor induced increases in both a calcium-dependent potassium conductance and phosphoinositide (PI) metabolism of similar magnitude to those activated by m3. Raising of intracellular calcium or injection of inositol-1,4,5-trisphosphate mimicked the activation of the calcium-dependent potassium conductance by both of these receptors. Although similar regarding these responses, the m3 and m5 receptors induced different cAMP responses. Stimulation of m5 receptors induced a 2-fold increase in cAMP levels, whereas m3 induced a 20-fold increase. These cAMP responses required greater than 100-fold more agonist than the PI responses, and both PI and cAMP responses were insensitive to pertussis toxin. Stimulation of m4 receptors caused little increase in PI metabolism and no electrophysiological effects. Stimulation of m4 receptors with low concentrations of agonist decreased cAMP levels, but at high agonist concentrations cAMP levels were elevated. After treatment with pertussis toxin, the decrease in cAMP levels induced by m4 was blocked and a marked increase in cAMP levels, comparable to those observed for m3 receptors, was uncovered at higher doses. The data indicate that each of the receptors has distinct functional properties.

1. The mechanism by which cloned m1 and m3 muscarinic receptor subtypes activate Ca2+-dependent channels was investigated with whole-cell and cell-attached patch-clamp recording techniques and with Fura-2 Ca2+ indicator dye measurements in cultured A9 L cells transfected with rat m1 and m3 cDNAs. 2. The Ca2+-dependent K+ and Cl- currents induced by muscarinic receptor stimulation were dependent on GTP. Responses were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. Intracellular GTP-gamma-S activated spontaneous fluctuations and permitted only one acetylcholine-(ACh) induced current response. These results implicate GTP-binding proteins (G protein) in the signal transduction pathway. This G protein is probably not pertussis toxin-sensitive as the ACh-induced electrical response was not abolished by pertussis toxin treatment. 3. Cell-attached single-channel recordings revealed activation of ion channels within the patch during application of ACh outside the patch, implying that second messengers might be involved in the ACh-induced response. Two types of K+ channel were activated, a discrete channel of 36 pS and channel activity calculated to be about 5 pS. 4. Application of 8-bromo cyclic AMP or 1-oleoyl-1,2-acetylglycerol (OAG) produced no electrical response and did not affect the ACh-induced responses. Phorbol myristic acetate (PMA) evoked no electrical response, but reduced the ACh-induced responses. 5. Inclusion of inositol 1,4,5-trisphosphate (IP3) in the intracellular pipette solution activated outward currents at -50 mV associated with an increase in conductance. The IP3-induced current response reversed polarity at -65 mV and showed a dependence on K+. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) from 20 nM to 1 microM also induced an outward current response associated with an increase in conductance. Inclusion of inositol 1,3,4,5-tetrakisphosphate (IP4) in the intracellular solution had no effect on the A9 L cells. 6. Fura-2 measurements revealed ACh-induced increases in Cai2+. The Ca2+ responses were abolished by atropine showing that they were muscarinic in nature. Removal of extracellular Ca2+ did not affect the initial ACh-induced increase in Cai2+ but subsequent Cai2+ responses to ACh were depressed, suggesting depletion of Ca2+ intracellular stores. Residual though small responses continued to be elicited by ACh. Barium (5 mM) had little effect and cobalt slightly reduced the ACh-induced Ca2+ response. 7. The ACh-induced currents recorded at -50 mV were unaffected by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of the antiarrhythmic agent disopyramide was studied on responses from voltage-clamped endplates at the neuromuscular junction of the garter snake. Disopyramide reduced endplate current amplitude and decay time constant in a concentration- and voltage-dependent manner. Endplate current decays remained monophasic in the presence of the drug. These results were interpreted in terms of the drug blocking the open form of the acetylcholine receptor-ion channel complex. Disopyramide produced a greater reduction of the amplitude of endplate currents than of miniature endplate currents. The reduction in miniature endplate current amplitude was not voltage-dependent. Analysis of endplate current driving functions showed that this was due to the rapid occurrence of channel block during the rising phase of the endplate current. The residual reduction, apart from that produced by channel block, is most probably due to receptor block. Disopyramide had a voltage-dependent blocking rate constant of about 10(7) M-1 S-1 at -90 mV. The unblocking rate constant was estimated from the results of experiments using paired ionophoretically applied pulses of acetylcholine. This value was again voltage-dependent and approximately 1 s-1. The actions of the (+)- and (-)-stereoisomers of disopyramide on endplate current decay were identical, indicating that the channel binding site at the neuromuscular junction is not stereoselective.

The electrophysiological responses to cholinergic stimulation of four cloned muscarinic receptor subtypes (m1-m4) were studied in A9 L cells transfected with the expression plasmids of each of the different subtypes, using the tight-seal whole-cell recording technique. Cells transfected with m1 and m3 muscarinic receptor subtypes were hyperpolarized by acetylcholine (ACh), whereas m2- and m4-transfected cells did not respond to ACh concentrations of up to 1 mM. Stimulation of both m1 and m3 muscarinic receptor subtypes evoked outward currents in cells voltage-clamped at -50 mV, associated with an increase in membrane conductance. These outward currents were blocked by atropine but not by tubocurarine. The ACh-induced currents of m1- and m3-transfected cells primarily involved potassium ions, although chloride ions also contributed to a minor extent. The potassium and chloride conductances were blocked by barium or cobalt and by buffering the intracellular calcium to low levels with 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid, showing a dependence of these conductances on calcium. Thus, m1- and m3-transfected cells respond to ACh in a manner that is qualitatively similar, evoking calcium-dependent potassium and chloride conductances, whereas m2- and m4-transfected cells are not coupled to electrically detectable responses in A9 L cells.

Using an oligonucleotide based on the sequence of a porcine brain muscarinic receptor cDNA, we recently cloned four distinct muscarinic receptors from the rat and human genomes. In the present study we transfected the rat homolog of the porcine brain muscarinic receptor cDNA into A9 L cells using a mammalian expression vector and a calcium phosphate precipitation procedure. Before transfection, A9 L cells do not bind muscarinic ligands and do not express muscarinic receptor mRNA. After transfection, A9 L cells expressed muscarinic receptor mRNA and saturable, high affinity binding sites for the muscarinic antagonists 3H-quinuclidinyl benzilate and 3H-pirenzepine. The muscarinic receptor antagonists AF DX-116 and pirenzepine displaced bound 3H-quinuclidinyl benzilate with inhibition curves suggestive of a single high affinity binding site. Competition of 3H-quinuclidinyl benzilate-labeled sites with the agonists acetylcholine and carbachol yielded broad inhibition curves, consistent with a heterogeneity of binding sites. In the presence of guanine nucleotide, the agonist inhibition curves were steeper, suggesting the presence of a single low affinity site. The effects of guanine nucleotides on agonist binding are consistent with coupling of these receptors to a guanine nucleotide-binding protein (G-protein) endogenous to A9 L cells. The electrical properties of the transfected A9 L cells were examined using the whole cell patch-clamp technique. Fifty microM acetylcholine induced a conductance which reversed in polarity at -60 mV. This conductance could be reversibly blocked by atropine. These data illustrate the utility of stable transfection of A9 L cells for the characterization of individual cloned muscarinic receptors, their G-protein coupling mechanisms, and resultant physiological responses.

The electrophysiological properties of A9 L cells stably transfected with m1 muscarinic receptor cDNA were examined by using the whole-cell patch-clamp technique. In current-clamp recordings, acetylcholine (AcCho) elicited a hyperpolarization of all transfected cells studied but had no effect on nontransfected A9 L cells. In voltage-clamp recordings, AcCho elicited an outward current at -50 mV accompanied by an increase in conductance. The onset of the current response was consistently delayed by several seconds with respect to the onset of the application of AcCho and could not be accounted for by diffusion. The AcCho-induced currents were reversibly inhibited by the muscarinic receptor antagonist atropine (1 microM) but were unaffected by the nicotinic receptor antagonist tubocurarine (50 microM). Ion-substitution experiments replacing K+ with N-methyl-D-glucamine and Cl- with methanesulfonate indicated that the current was carried mainly by K+, although a minor part appeared to be carried by Cl-. The AcCho-induced current could be blocked by the K+ channel blocking agents tetraethylammonium ion, 4-aminopyridine, apamin, and Ba2+ but not by Cs+. The AcCho-induced current was inhibited when 5 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was included in the patch pipette or when extracellular Cd2+ or Co2+ was applied, indicating a role for intracellular Ca2+ in the generation of the response. Thus, these results show that cloned m1 muscarinic receptors expressed in A9 L cells can activate a Ca2+-dependent K+ conductance, possibly via a second-messenger system.

The effects of disopyramide were studied at the neuromuscular junction in an attempt to elucidate the mechanism of its blocking action at this site. Disopyramide 5 X 10(-5) - 10(-3) mol litre-1 produced a concentration-dependent reduction of twitch amplitude in the indirectly stimulated chick biventer cervicis preparation, but greater concentrations were required to reduce twitches elicited directly in the presence of erabutoxin-b 1 microgram ml-1. Equieffective twitch blocking doses of either disopyramide or tubocurarine greatly reduced agonist responses to acetylcholine and carbachol, but the reduction was less for magnesium-blocked twitches. Neostigmine antagonized tubocurarine-induced, but not disopyramide-induced, blockade of twitches. Concentration-response profiles to acetylcholine and carbachol were shifted to the right in a non-parallel fashion and the maximal response was depressed by disopyramide 5 X 10(-5) - 10(-4) mol litre-1. Intracellular recording studies carried out in the cut, voltage-clamped costo-cutaneous muscle of the garter snake showed that disopyramide 5 X 10(-5) - 5 X 10(-4) mol litre-1 produced a concentration- and voltage-dependent reduction of the amplitude of neurally evoked endplate-currents (EPC) and of the time constant of decay (tau) of EPC. We conclude that disopyramide possesses a non-competitive blocking action at the neuromuscular junction, which is not reversible by anticholinesterase agents. The voltage-dependent nature of the block suggests that it is mediated via blockade of the open form of the acetylcholine-activated receptor-ion channel complex.

The majority of treated patients with myelomatosis had a significantly decreased number of T cells as measured by both lymphocyte stimulation to phytohaemagglutinin (PHA) and by their ability to form spontaneous rosettes with sheep red blood cells (SRBC). Seven untreated patients also had markedly depressed T cells. On the other hand, B-cell numbers' were significantly increased in the majority of the patients as judged by increased lymphocyte stimulation to pokeweed mitogen (PWM) and the markedly increased numbers of rosettes that were formed with antibody-coated complement activated SRBC, as well as by immunofluorescence cell markers to monospecific immunoglobulin antiserum.